U.S. patent application number 12/001025 was filed with the patent office on 2008-11-27 for genes and uses for plant improvement.
Invention is credited to Mark S. Abad, Jaishree M. Chittoor-Vijayanath, Marie Coffin, Barry S. Goldman, Ronald C. Rich.
Application Number | 20080295196 12/001025 |
Document ID | / |
Family ID | 39492875 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080295196 |
Kind Code |
A1 |
Abad; Mark S. ; et
al. |
November 27, 2008 |
Genes and uses for plant improvement
Abstract
Transgenic seed for crops with improved traits are provided by
trait-improving recombinant DNA in the nucleus of cells of the seed
where plants grown from such transgenic seed exhibit one or more
improved traits as compared to a control plant. Of particular
interest are transgenic plants that have increased yield. The
present invention also provides recombinant DNA molecules for
expression of a protein, and recombinant DNA molecules for
suppression of a protein.
Inventors: |
Abad; Mark S.; (Groves,
MO) ; Goldman; Barry S.; (St. Louis, MO) ;
Chittoor-Vijayanath; Jaishree M.; (Wildwood, MO) ;
Coffin; Marie; (Cary, NC) ; Rich; Ronald C.;
(Raleigh, NC) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD., ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
39492875 |
Appl. No.: |
12/001025 |
Filed: |
December 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60873247 |
Dec 6, 2006 |
|
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Current U.S.
Class: |
800/275 ;
435/419; 435/6.16; 800/298; 800/312; 800/314; 800/320; 800/320.1;
800/320.2; 800/320.3 |
Current CPC
Class: |
C12N 15/8282 20130101;
C12Q 2600/158 20130101; C12Q 2600/13 20130101; C12N 15/8283
20130101; C07K 14/415 20130101; C12N 15/8274 20130101; C12N 15/8271
20130101; C12N 15/8273 20130101; C12N 15/8261 20130101; G01N 33/68
20130101; C12N 15/8275 20130101; C12Q 1/6895 20130101; G01N
2333/415 20130101; C12N 15/8247 20130101; C12N 15/8241 20130101;
C12N 15/8251 20130101 |
Class at
Publication: |
800/275 ;
435/419; 800/298; 435/6; 800/312; 800/314; 800/320; 800/320.1;
800/320.2; 800/320.3 |
International
Class: |
A01H 1/04 20060101
A01H001/04; C12N 5/10 20060101 C12N005/10; A01H 5/00 20060101
A01H005/00; A01H 1/02 20060101 A01H001/02; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A plant cell nucleus with stably integrated, recombinant DNA
comprising a promoter that is functional in plant cells and that is
operably linked to DNA from a plant, bacteria or yeast that encodes
a protein having at least one domain of amino acids in a sequence
that exceeds the Pfam gathering cutoff for amino acid sequence
alignment with a protein domain family identified by a Pfam name
selected from the group of Pfam names consisting of L51_S25_CI-B8,
iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap,
Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RmaAD, NAF, BolA,
Pkinase, C2, FA_hydroxylase, p450, Complex1.sub.--30 kDa, Histone,
DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5,2-Hacid_dh,
Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank,
Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL,
ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk,
Ribonuclease_T2, Complex1.sub.--49 kDa, Gp_dh_C, Aldo_ket_red,
zf-AN1, TFIIS_C, MFS.sub.--1, Thioredoxin, DUF1005, LEA.sub.--3,
Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN.sub.--2, BPL_C, DUF26, Aa_trans,
ACT, ADH_N, NAD_binding.sub.--1, Auxin_inducible, B_lectin,
Anti-silence, Response_reg, 14-3-3, LRRNT.sub.--2, GDC-P, zf-CCHC,
NPH3, TPR.sub.--1, TFIIA, DHBP_synthase, UQ_con, TPR.sub.--2, TPT,
F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv,
TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N,
Methyltransf.sub.--1, CBM.sub.--20, Methyltransf.sub.--12,
Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf.sub.--8,
BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch.sub.--1, PGAM,
Aminotran.sub.--1.sub.--2, Kelch.sub.--2, UPF0261, CoA_binding,
DUF868, Peptidase_S10, Lung.sub.--7-TM_R, Oleosin, Sad1_UNC,
Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase,
Subtilisin_N, SAM.sub.--1, DUF298, ESCRT-III, DNA_pol_E_B,
Aminotran.sub.--3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588,
zf-CCCH, DnaJ, Pkinase_Tyr, Cupin.sub.--2, LRR.sub.--1,
Cupin.sub.--3, zf-CSL, FAR1, HD, FH2, APC8, PTR.sub.2,
MannoseP_isomer, Rib.sub.--5-P_isom_A, DUF1336, Phosphorylase,
DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh,
zf-C3HC4, PPR, PK, PurA, RMMBL, HTH.sub.--11, Tim17, and PBD
wherein said Pfam gathering cutoff for said protein domain families
are stated in Table 17; wherein said plant cell nucleus is selected
by screening a population of transgenic plants that have said
recombinant DNA and express said protein for an enhanced trait as
compared to control plants that do not have said recombinant DNA in
their nuclei; and wherein said enhanced trait is selected from
group of enhanced traits consisting of enhanced water use
efficiency, enhanced cold tolerance, enhanced heat tolerance,
enhanced resistance to salt exposure, enhanced shade tolerance,
increased yield, enhanced nitrogen use efficiency, enhanced seed
protein and enhanced seed oil.
2. The plant cell nucleus of claim 1 wherein said protein has an
amino acid sequence with at least 90% identity to a consensus amino
acid sequence in the group of consensus amino acid sequences
consisting of the consensus amino acid sequence constructed for SEQ
ID NO: 198 and homologs thereof listed in Table 2 through the
consensus amino acid sequence constructed for SEQ ID NO: 394 and
homologs thereof listed in Table 2.
3. The plant cell nucleus of claim 1 wherein said protein is
selected from the group of proteins identified in Table 1.
4. A plant cell nucleus with stably integrated, recombinant DNA to
suppress the level of an endogenous protein having at least one
domain of amino acids in a sequence that exceeds the Pfam gathering
cutoff for amino acid sequence alignment with a protein domain
family identified by Pfam name in the group of Pfam names
consisting of SNF5, LMBR1, TFIIS_M, TFIIS_C, and
Glyco_transf.sub.--8, wherein the Pfam gathering cutoff for said
protein domain families is stated in Table 17 wherein said plant
cell nucleus is selected by screening a population of transgenic
plants with said recombinant DNA and have the level of said
endogenous protein suppressed for an enhanced trait as compared to
control plants that do not have said recombinant DNA; and wherein
said enhanced trait is selected from the group of enhanced traits
consisting of enhanced water use efficiency, enhanced cold
tolerance, enhanced heat tolerance, enhanced resistance to salt
exposure, enhanced shade tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil.
5. The plant cell nucleus of claim 1 or 4 further comprising DNA
expressing a protein that provides tolerance from exposure to an
herbicide applied at levels that are lethal to a wild type of said
plant cell.
6. The plant cell nucleus of claim 5 wherein the agent of said
herbicide is a glyphosate, dicamba, or glufosinate compound.
7. A transgenic plant cell or plant comprising a plurality of plant
cells with a plant cell nucleus of claim 1 or 4.
8. The transgenic plant cell or plant of claim 7 which is
homozygous for said recombinant DNA.
9. A transgenic seed comprising a plurality of plant cells with a
plant cell nucleus of claim 1 or 4.
10. The transgenic seed of claim 9 from a corn, soybean, cotton,
canola, alfalfa, wheat or rice plant.
11. The transgenic corn seed of claim 10 wherein said seed can
produce corn plants that are resistant to disease from the Mal de
Rio Cuarto virus or the Puccina sorghi fungus or both.
12. A transgenic pollen grain comprising a haploid derivative of a
plant cell nucleus of claim 1 or 4.
13. A method for manufacturing non-natural, transgenic seed that
can be used to produce a crop of transgenic plants with an enhanced
trait resulting from expression of stably-integrated recombinant
DNA in a nucleus of claim 1 or 4, wherein said method for
manufacturing said transgenic seed comprising: (a) screening a
population of plants for said enhanced trait and said recombinant
DNA, wherein individual plants in said population can exhibit said
trait at a level less than, essentially the same as or greater than
the level that said trait is exhibited in control plants which do
not express the recombinant DNA, (b) selecting from said population
one or more plants that exhibit said trait at a level greater than
the level that said trait is exhibited in control plants, (c)
verifying that said recombinant DNA is stably integrated in said
selected plants, (d) analyzing tissue of said selected plant to
determine the production or suppression of a protein having the
function of a protein encoded by nucleotides having a sequence
selected from the group consisting of one of SEQ ID NO:198-394; and
(e) collecting seed from said selected plant.
14. The method of claim 14 wherein plants in said population
further comprise DNA expressing a protein that provides tolerance
to exposure to an herbicide applied at levels that are lethal to
wild type plant cells, and wherein said selecting is effected by
treating said population with said herbicide.
15. The method of claim 14 wherein said herbicide comprises a
glyphosate, dicamba, or glufosinate compound.
16. The method of claim 15 wherein said selecting is effected by
identifying plants with said enhanced trait.
17. The method of claim 16 wherein said seed is corn, soybean,
cotton, alfalfa, wheat or rice seed.
18. A method of producing hybrid corn seed comprising: (a)
acquiring hybrid corn seed from a herbicide tolerant corn plant
which also has stably-integrated, recombinant DNA in a nucleus of
claim 1 or 4; (b) producing corn plants from said hybrid corn seed,
wherein a fraction of the plants produced from said hybrid corn
seed is homozygous for said recombinant DNA, a fraction of the
plants produced from said hybrid corn seed is hemizygous for said
recombinant DNA, and a fraction of the plants produced from said
hybrid corn seed has none of said recombinant DNA; (c) selecting
corn plants which are homozygous and hemizygous for said
recombinant DNA by treating with an herbicide; (d) collecting seed
from herbicide-treated-surviving corn plants and planting said seed
to produce further progeny corn plants; (e) repeating steps (c) and
(d) at least once to produce an inbred corn line; (f) crossing said
inbred corn line with a second corn line to produce hybrid seed.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35USC .sctn. 119(e) of
U.S. provisional application Ser. No. 60/873,247, filed Dec. 6,
2006, herein incorporated by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Copy 1 and Copy 2) and a
computer readable form (CRF) of the sequence listing, all on CD-Rs,
each containing the file named 38-21(53949)B_seqListing.txt, which
is 67,317,760 bytes (measured in MS-WINDOWS) and was created on
Dec. 4, 2007, are incorporated herein by reference in their
entirety.
INCORPORATION OF COMPUTER PROGRAM LISTING
[0003] A Computer Program Listing with folders "hmmer-2.3.2" and
"161pfamDir" is contained on a CD-R and is incorporated herein by
reference in their entirety. Folder hmmer-2.3.2 contains the source
code and other associated file for implementing the HMMer software
for Pfam analysis. Folder 161pfamDir contains 161 profile Hidden
Markov Models. Both folders were created on the disk on Dec. 4,
2007 having a total size of 15,534,080 bytes when measured in
MS-WINDOWS.RTM. operating system.
FIELD OF THE INVENTION
[0004] Disclosed herein are transgenic plant cells, plants and
seeds comprising recombinant DNA and methods of making and using
such plant cells, plants and seeds
BACKGROUND OF THE INVENTION
[0005] Transgenic plants with enhanced traits such as improved
yield, environmental stress tolerance, pest resistance, herbicide
tolerance, modified seed compositions, and the like are desired by
both farmers and consumers. Although considerable efforts in plant
breeding have provided significant gains in desired traits, the
ability to introduce specific DNA into plant genomes provides
further opportunities for generation of plants with improved and/or
unique traits. The ability to develop transgenic plants with
improved traits depends in part on the identification of useful
recombinant DNA for production of transformed plants with improved
properties, e.g. by actually selecting a transgenic plant from a
screen for such improved property. An object of this invention is
to provide transgenic plant cell nuclei, plant cells, plants and
seeds by screening transgenic crop plants for one of more enhanced
agronomic traits where the nucleus in cells of the plant or seed
has recombinant DNA that was identified as imparting an improved
trait in a model plant, e.g. Arabidopsis thaliana. In some cases
the model plant may exhibit an improved trait that corresponds to
an enhanced agronomic trait, e.g. cold stress tolerance, water
deficit stress tolerance, low nitrogen stress tolerance and the
like. In other cases the model plant may exhibit an improved trait
that is a surrogate to an enhanced agronomic trait, e.g. salinity
stress tolerance being a surrogate to drought tolerance or
improvement in plant growth and development being a surrogate to
enhanced yield. A further object of the invention is to provide
screening methods requiring routine experimentation by which such
transgenic plant cell nuclei, cells, plants and seeds can be
identified by making a reasonable number of transgenic events and
engaging in screening identified in this specification and
illustrated in the examples.
SUMMARY OF THE INVENTION
[0006] This invention provides plant cell nuclei with recombinant
DNA that imparts enhanced agronomic traits in transgenic plants
having the nuclei in their cells. Recombinant DNA in this invention
is provided in a construct comprising a promoter that is functional
in plant cells and that is operably linked to DNA that encodes a
protein having at least one amino acid domain in a sequence that
exceeds the Pfam gathering cutoff for amino acid sequence alignment
with a protein domain family identified by a Pfam name in the group
of Pfam domain names identified in Table 17. In more specific
embodiments of the invention plant cells are provided which express
a protein having amino acid sequence with at least 90% identity to
a consensus amino acid sequence in the group of consensus amino
acid sequences consisting of the consensus amino acid sequence
constructed for SEQ ID NO: 198 and homologs thereof listed in Table
2 through the consensus amino acid sequence constructed for SEQ ID
NO: 394 and homologs thereof listed in Table 2. Amino acid
sequences of homologs are SEQ ID NO: 395 through 19,938. In even
more specific embodiments of the invention the protein expressed in
plant cells is a protein selected from the group of proteins
identified in Table 1 by annotation to a related protein in Genbank
and alternatively identified in Table 16 by identification of
protein domain family.
[0007] Other aspects of the invention are specifically directed to
transgenic plant cells, and transgenic plants comprising a
plurality of plant cells with such nuclei, progeny transgenic seed,
embryo and transgenic pollen from such plants. Such plant cell
nuclei are selected from a population of transgenic plants
regenerated from plant cells with a nucleus transformed with
recombinant DNA by screening the transgenic plants in the
population for an enhanced trait as compared to control plants that
do not have the recombinant DNA in their nucleus, where the
enhanced trait is enhanced water use efficiency, enhanced cold
tolerance, enhanced heat tolerance, enhanced shade tolerance,
enhanced tolerance to salt exposure, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein or enhanced seed
oil. In some aspects of the invention the recombinant DNA expresses
a protein that imparts the enhanced trait; in other aspects of the
invention the recombinant DNA expresses RNA for suppressing the
level of an endogenous protein. In yet another aspect of the
invention the nucleus of plant cells in plants, seeds, embryo and
pollen further comprise DNA expressing a protein that provides
tolerance from exposure to an herbicide applied at levels that are
lethal to a wild type plant cell. Such tolerance is especially
useful not only as an advantageous trait in such plants but is also
useful in a selection step in the methods of the invention. In
aspects of the invention the agent of such herbicide is a
glyphosate, dicamba, or glufosinate compound.
[0008] Yet other aspects of the invention provide nuclei in cells
of transgenic plants which are homozygous for the recombinant DNA
and transgenic seed of the invention from corn, soybean, cotton,
canola, alfalfa, wheat or rice plants.
[0009] In other embodiments for practice of various aspects of the
invention in Argentina the recombinant DNA in the nucleus is
provided in plant cells derived from corn lines that are and
maintain resistance to a virus such as the Mal de Rio Cuarto virus
or a fungus such as the Puccina sorghi fungus or to both.
[0010] This invention also provides methods for manufacturing
non-natural, transgenic seed that can be used to produce a crop of
transgenic plants with an enhanced trait resulting from expression
of stably-integrated, recombinant DNA in the nucleus of the plant
cells. In some aspects of the invention the recombinant DNA can
express a protein having at least one domain of amino acids in a
sequence that exceeds the Pfam gathering cutoff for amino acid
sequence alignment with a protein domain family identified by a
Pfam name in the group of Pfam names identified in Table 17; in
other aspects the recombinant DNA suppresses the level of such a
protein. More specifically the method comprises (a) screening a
population of plants for an enhanced trait and recombinant DNA,
where individual plants in the population can exhibit the trait at
a level less than, essentially the same as or greater than the
level that the trait is exhibited in control plants which do not
express the recombinant DNA; (b) selecting from the population one
or more plants that exhibit the trait at a level greater than the
level that said trait is exhibited in control plants; (c) verifying
that the recombinant DNA is stably integrated in said selected
plants; (d) analyzing tissue of a selected plant to determine the
production of a protein having the function of a protein encoded by
nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e)
collecting seed from a selected plant. In one aspect of the
invention the plants in the population further comprise DNA
expressing a protein that provides tolerance to exposure to an
herbicide applied at levels that are lethal to wild type plant
cells and where the selecting is effected by treating the
population with the herbicide, e.g. a glyphosate, dicamba, or
glufosinate compound. In another aspect of the invention the plants
are selected by identifying plants with the enhanced trait. The
methods are especially useful for manufacturing corn, soybean,
cotton, alfalfa, wheat or rice seed selected as having at least one
of the enhanced traits described above.
[0011] Another aspect of the invention provides a method of
producing hybrid corn seed comprising acquiring hybrid corn seed
from a herbicide tolerant corn plant which also has a nucleus of
this invention with stably-integrated, recombinant DNA The method
further comprises producing corn plants from said hybrid corn seed,
where a fraction of the plants produced from said hybrid corn seed
is homozygous for said recombinant DNA, a fraction of the plants
produced from said hybrid corn seed is hemizygous for said
recombinant DNA, and a fraction of the plants produced from said
hybrid corn seed has none of said recombinant DNA; selecting corn
plants which are homozygous and hemizygous for said recombinant DNA
by treating with an herbicide; collecting seed from
herbicide-treated-surviving corn plants and planting said seed to
produce further progeny corn plants; repeating the selecting and
collecting steps at least once to produce an inbred corn line; and
crossing the inbred corn line with a second corn line to produce
hybrid seed.
[0012] Another aspect of the invention provides a method of
selecting a plant comprising a nucleus of this invention in its
plant cells by using an immunoreactive antibody to detect the
presence of protein expressed by recombinant DNA in seed or plant
tissue. Another aspect of the invention provides anti-counterfeit
milled seed having, as an indication of origin, a nucleus of this
invention with unique recombinant DNA.
[0013] Aspects of the invention relating to nucleus in plant cells
having recombinant DNA for suppressing the expression of a protein
are identified in Table 1 and Table 16. More specific aspects of
the invention provide plant cells having recombinant DNA for
suppressing the expression of a protein having the function in a
plant of the protein with amino acid sequence of SEQ ID NO: 200,
201, 205, 207, 211 and 394 or the corresponding Pfam identified in
Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf.sub.--8,
respectively. Such suppression can be effected by any of a number
of ways known in the art, e.g. anti-sense suppression, RNAi or
mutation knockout and the like.
[0014] Another aspect of this invention relates to growing
transgenic plants with enhanced water use efficiency or enhanced
nitrogen use efficiency. For instance, this invention provides
methods of growing a corn, cotton or soybean crop without
irrigation water comprising planting seed having plant cells of the
invention which are selected for enhanced water use efficiency.
Alternatively methods comprise applying reduced irrigation water,
e.g. providing up to 300 millimeters of ground water during the
production of a corn crop. This invention also provides methods of
growing a corn, cotton or soybean crop without added nitrogen
fertilizer comprising planting seed having plant cells of the
invention which are selected for enhanced nitrogen use efficiency.
Alternatively methods comprise applying reduced amount of nitrogen
input as compared to the conventional input during the production
of a corn crop.
[0015] The various aspects of this invention are especially useful
for transgenic plant cells in seeds and transgenic plants having
any of the above-described enhanced traits in crop plants such as
corn (maize), soybean, cotton, canola (rape), wheat, sunflower,
sorghum, alfalfa, barley, millet, rice, tobacco, fruit and
vegetable crops, and turfgrass.
[0016] The invention also provides recombinant DNA constructs
comprising the DNA useful in the nuclei in plant cells for
imparting enhanced traits in plants having those cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates a consensus amino acid sequence of SEQ ID
NO: 227 and its homologs.
[0018] FIGS. 2, 3 and 4 illustrate plasmid maps.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In the attached sequence listing:
[0020] SEQ ID NO: 1-197 are nucleotide sequences of the coding
strand of DNA for "genes" used in the recombinant DNA imparting an
enhanced trait in plant cells, i.e. each represents a coding
sequence for a protein;
[0021] SEQ ID NO: 198-394 are amino acid sequences of the cognate
protein of the "genes" with nucleotide coding sequence 1-197;
[0022] SEQ ID NO: 395-19938 are amino acid sequences of homologous
proteins;
[0023] SEQ ID NO: 19939 is a consensus amino acid sequence.
[0024] SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base
vector useful for corn transformation; and
[0025] SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector
useful for soybean or canola transformation.
[0026] SEQ ID NO: 19942 is a DNA sequence of a plasmid base vector
useful for cotton transformation.
[0027] The nuclei of this invention are identified by screening
transgenic plants for one or more traits including improved drought
stress tolerance, improved heat stress tolerance, improved cold
stress tolerance, improved high salinity stress tolerance, improved
low nitrogen availability stress tolerance, improved shade stress
tolerance, improved plant growth and development at the stages of
seed imbibition through early vegetative phase, and improved plant
growth and development at the stages of leaf development, flower
production and seed maturity.
[0028] "Gene" refers to chromosomal DNA, plasmid DNA, cDNA,
synthetic DNA, or other DNA that encodes a peptide, polypeptide,
protein, or RNA molecule, and regions flanking the coding sequences
involved in the regulation of expression. In aspects of the
invention where an improved trait is provided by expression of a
protein, "gene" refers at least to coding nucleotide sequence for a
protein or a functional polypeptide fragment of a protein that
imparts the trait. In aspects of the invention where an improved
trait is provided by suppression of expression of an endogenous
protein, "gene" refers to any part of the gene that can be a target
for suppression.
[0029] "Transgenic seed" means a plant seed whose nucleus has been
altered by the incorporation of recombinant DNA, e.g., by
transformation as described herein. The term "transgenic plant" is
used to refer to the plant produced from an original transformation
event, or progeny from later generations or crosses of a plant to a
transformed plant, so long as the progeny contains a nucleus with
the recombinant DNA in its genome.
[0030] "Recombinant DNA" means a polynucleotide having a
genetically engineered modification introduced through combination
of endogenous and/or exogenous elements in a transcription unit,
manipulation via mutagenesis, restriction enzymes, and the like or
simply by inserting multiple copies of a native transcription unit.
Recombinant DNA may comprise DNA segments obtained from different
sources, or DNA segments obtained from the same source, but which
have been manipulated to join DNA segments which do not naturally
exist in the joined form. A recombinant polynucleotide may exist
outside of the cell, for example as a PCR fragment, or integrated
into a genome, such as a plant genome.
[0031] "Trait" means a physiological, morphological, biochemical,
or physical characteristic of a plant or particular plant material
or cell. In some instances, this characteristic is visible to the
human eye, such as seed or plant size, or can be measured by
biochemical techniques, such as detecting the protein, starch, or
oil content of seed or leaves, or by observation of a metabolic or
physiological process, e.g., by measuring uptake of carbon dioxide,
or by the observation of the expression level of a gene or genes,
e.g., by employing Northern analysis, RT-PCR, microarray gene
expression assays, or reporter gene expression systems, or by
agricultural observations such as stress tolerance, yield, or
pathogen tolerance.
[0032] A "control plant" is a plant without trait-improving
recombinant DNA in its nucleus. A control plant is used to measure
and compare trait improvement in a transgenic plant with such
trait-improving recombinant DNA. A suitable control plant may be a
non-transgenic plant of the parental line used to generate a
transgenic plant herein. Alternatively, a control plant may be a
transgenic plant that comprises an empty vector or marker gene, but
does not contain the recombinant DNA that produces the trait
improvement. A control plant may also be a negative segregant
progeny of hemizygous transgenic plant. In certain demonstrations
of trait improvement, the use of a limited number of control plants
can cause a wide variation in the control dataset. To minimize the
effect of the variation within the control dataset, a "reference"
is used. As use herein a "reference" is a trimmed mean of all data
from both transgenic and control plants grown under the same
conditions and at the same developmental stage. The trimmed mean is
calculated by eliminating a specific percentage, e.g., 20%, of the
smallest and largest observation from the data set and then
calculating the average of the remaining observation.
[0033] "Trait enhancement" means a detectable and desirable
difference in a characteristic in a transgenic plant relative to a
control plant or a reference. In some cases, the trait enhancement
can be measured quantitatively. For example, the trait improvement
can entail at least a 2% desirable difference in an observed trait,
at least a 5% desirable difference, at least about a 10% desirable
difference, at least about a 20% desirable difference, at least
about a 30% desirable difference, at least about a 50% desirable
difference, at least about a 70% desirable difference, or at least
about a 100% difference, or an even greater desirable difference.
In other cases, the trait enhancement is only measured
qualitatively. It is known that there can be a natural variation in
a trait. Therefore, the trait enhancement observed entails a change
of the normal distribution of the trait in the transgenic plant
compared with the trait distribution observed in a control plant or
a reference, which is evaluated by statistical methods provided
herein. Trait enhancement includes, but is not limited to, yield
increase, including increased yield under non-stress conditions and
increased yield under environmental stress conditions. Stress
conditions may include, for example, drought, shade, fungal
disease, viral disease, bacterial disease, insect infestation,
nematode infestation, cold temperature exposure, heat exposure,
osmotic stress, reduced nitrogen nutrient availability, reduced
phosphorus nutrient availability and high plant density.
[0034] Many agronomic traits can affect "yield", including without
limitation, plant height, pod number, pod position on the plant,
number of internodes, incidence of pod shatter, grain size,
efficiency of nodulation and nitrogen fixation, efficiency of
nutrient assimilation, resistance to biotic and abiotic stress,
carbon assimilation, plant architecture, resistance to lodging,
percent seed germination, seedling vigor, and juvenile traits.
Other traits that can affect yield include, efficiency of
germination (including germination in stressed conditions), growth
rate (including growth rate in stressed conditions), ear number,
seed number per ear, seed size, composition of seed (starch, oil,
protein) and characteristics of seed fill. Also of interest is the
generation of transgenic plants that demonstrate desirable
phenotypic properties that may or may not confer an increase in
overall plant yield. Such properties include enhanced plant
morphology, plant physiology or improved components of the mature
seed harvested from the transgenic plant.
[0035] "Yield-limiting environment" means the condition under which
a plant would have the limitation on yield including environmental
stress conditions.
[0036] "Stress condition" means a condition unfavorable for a
plant, which adversely affect plant metabolism, growth and/or
development. A plant under the stress condition typically shows
reduced germination rate, retarded growth and development, reduced
photosynthesis rate, and eventually leading to reduction in yield.
Specifically, "water deficit stress" used herein preferably refers
to the sub-optimal conditions for water and humidity needed for
normal growth of natural plants. Relative water content (RWC) can
be used as a physiological measure of plant water deficit. It
measures the effect of osmotic adjustment in plant water status,
when a plant is under stress conditions. Conditions that may result
in water deficit stress include heat, drought, high salinity and
PEG induced osmotic stress.
[0037] "Cold stress" means the exposure of a plant to a
temperatures below (two or more degrees Celsius below) those normal
for a particular species or particular strain of plant.
[0038] "Nitrogen nutrient" means any one or any mix of the nitrate
salts commonly used as plant nitrogen fertilizer, including, but
not limited to, potassium nitrate, calcium nitrate, sodium nitrate,
ammonium nitrate. The term ammonium as used herein means any one or
any mix of the ammonium salts commonly used as plant nitrogen
fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium
sulfate, etc.
[0039] "Low nitrogen availability stress" means a plant growth
condition that does not contain sufficient nitrogen nutrient to
maintain a healthy plant growth and/or for a plant to reach its
typical yield under a sufficient nitrogen growth condition. For
example, a low nitrogen condition can refers to a growth condition
with 50% or less of the conventional nitrogen inputs. "Sufficient
nitrogen growth condition" means a growth condition where the soil
or growth medium contains or receives optimal amounts of nitrogen
nutrient to sustain a healthy plant growth and/or for a plant to
reach its typical yield for a particular plant species or a
particular strain. One skilled in the art would recognize what
constitute such soil, media and fertilizer inputs for most plant
species.
[0040] "Shade stress" means a growth condition that has limited
light availability that triggers the shade avoidance response in
plant. Plants are subject to shade stress when localized at lower
part of the canopy, or in close proximity of neighboring
vegetation. Shade stress may become exacerbated when the planting
density exceeds the average prevailing density for a particular
plant species. The average prevailing densities per acre of a few
examples of crop plants in the USA in the year 2000 were: wheat
1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000,
canola 260,000-350,000, sunflower 17,000-23,000 and cotton
28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent
Application No. 20030101479).
[0041] "Increased yield" of a transgenic plant of the present
invention is evidenced and measured in a number of ways, including
test weight, seed number per plant, seed weight, seed number per
unit area (i.e., seeds, or weight of seeds, per acre), bushels per
acre, tons per acre, tons per acre, kilo per hectare. For example,
maize yield can be measured as production of shelled corn kernels
per unit of production area, e.g., in bushels per acre or metric
tons per hectare, often reported on a moisture adjusted basis,
e.g., at 15.5% moisture. Increased yield can result from improved
utilization of key biochemical compounds, such as nitrogen,
phosphorous and carbohydrate, or from improved tolerance to
environmental stresses, such as cold, heat, drought, salt, and
attack by pests or pathogens. Trait-improving recombinant DNA can
also be used to provide transgenic plants having improved growth
and development, and ultimately increased yield, as the result of
modified expression of plant growth regulators or modification of
cell cycle or photosynthesis pathways.
[0042] "Expression" means transcription of DNA to produce RNA. The
resulting RNA may be without limitation mRNA encoding a protein,
antisense RNA, or a double-stranded RNA for use in RNAi technology.
Expression also refers to production of encoded protein from
mRNA.
[0043] A "plant promoter" is a promoter capable of initiating
transcription in plant cells whether or not its origin is a plant
cell. Exemplary plant promoters include, but are not limited to,
those that are obtained from plants, plant viruses, and bacteria
which comprise genes expressed in plant cells such Agrobacterium or
Rhizobium. Examples of promoters under developmental control
include promoters that preferentially initiate transcription in
certain tissues, such as leaves, roots, or seeds. Such promoters
are referred to as "tissue preferred". Promoters which initiate
transcription only in certain tissues are referred to as "tissue
specific". A "cell type" specific promoter primarily drives
expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"repressible" promoter is a promoter which is under environmental
control. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions,
or certain chemicals, or the presence of light. Tissue specific,
tissue preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which is active under most
conditions. As used herein, "antisense orientation" includes
reference to a polynucleotide sequence that is operably linked to a
promoter in an orientation where the antisense strand is
transcribed. The antisense strand is sufficiently complementary to
an endogenous transcription product such that translation of the
endogenous transcription product is often inhibited.
[0044] As used herein, "operably linked" refers to the association
of two or more nucleic acid fragments on a single nucleic acid
fragment so that the function of one is affected by the other. For
example, a promoter is operably linked with a coding sequence when
it is capable of affecting the expression of that coding sequence
(i.e., that the coding sequence is under the transcriptional
control of the promoter). Coding sequences can be operably linked
to regulatory sequences in sense or antisense orientation.
[0045] A "consensus sequence" refers to an artificial, amino acid
sequence of conserved parts of the proteins encoded by homologous
genes, e.g., as determined by a CLUSTALW alignment of amino acid
sequence of homolog proteins.
[0046] Homologous genes are genes which encode proteins with the
same or similar biological function to the protein encoded by the
second gene. Homologous genes may be generated by the event of
speciation (see ortholog) or by the event of genetic duplication
(see paralog). "Orthologs" refer to a set of homologous genes in
different species that evolved from a common ancestral gene by
specification. Normally, orthologs retain the same function in the
course of evolution; and "paralogs" refer to a set of homologous
genes in the same species that have diverged from each other as a
consequence of genetic duplication. Thus, homologous genes can be
from the same or a different organism. As used herein, "homolog"
means a protein that performs the same biological function as a
second protein including those identified by sequence identity
search.
[0047] Percent identity refers to the extent to which two optimally
aligned DNA or protein segments are invariant throughout a window
of alignment of components, e.g., nucleotide sequence or amino acid
sequence. An "identity fraction" for aligned segments of a test
sequence and a reference sequence is the number of identical
components which are shared by sequences of the two aligned
segments divided by the total number of sequence components in the
reference segment over a window of alignment which is the smaller
of the full test sequence or the full reference sequence. "Percent
identity" ("% identity") is the identity fraction times 100. "%
identity to a consensus amino acid sequence" is 100 times the
identity fraction in a window of alignment of an amino acid
sequence of a test protein optimally aligned to consensus amino
acid sequence of this invention.
[0048] "Arabidopsis" means plants of Arabidopsis thaliana.
[0049] "Pfam" refers to a large collection of multiple sequence
alignments and hidden Markov models covering many common protein
families, e.g. Pfam version 18.0 (August 2005) contains alignments
and models for 7973 protein families and is based on the Swissprot
47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy,
"Profile Hidden Markov Models", Bioinformatics 14:755-763, 1998.
Pfam is currently maintained and updated by a Pfam Consortium. The
alignments represent some evolutionary conserved structure that has
implications for the protein's function. Profile hidden Markov
models (profile HMMs) built from the Pfam alignments are useful for
automatically recognizing that a new protein belongs to an existing
protein family even if the homology by alignment appears to be low.
Once one DNA is identified as encoding a protein which imparts an
enhanced trait when expressed in transgenic plants, other DNA
encoding proteins in the same protein family are identified by
querying the amino acid sequence of protein encoded by candidate
DNA against the Hidden Markov Model which characterizes the Pfam
domain using HMMER software, a current version of which is provided
in the appended computer listing. Candidate proteins meeting the
gathering cutoff for the alignment of a particular Pfam are in the
protein family and have cognate DNA that is useful in constructing
recombinant DNA for the use in the plant cells of this invention.
Hidden Markov Model databases for use with HMMER software in
identifying DNA expressing protein in a common Pfam for recombinant
DNA in the plant cells of this invention are also included in the
appended computer listing. The HMMER software and Pfam databases
are version 18.0 and were used to identify known domains in the
proteins corresponding to amino acid sequence of SEQ ID NO: 198
through SEQ ID NO: 394. All DNA encoding proteins that have scores
higher than the gathering cutoff disclosed in Table 17 by Pfam
analysis disclosed herein can be used in recombinant DNA of the
plant cells of this invention, e.g. for selecting transgenic plants
having enhanced agronomic traits. The relevant Pfams for use in
this invention, as more specifically disclosed below, are
L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA,
S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr,
LMBR1, RmaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450,
Complex1.sub.--30 kDa, Histone, DUF822, PEP-utilizers, PCI,
ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1,
FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N,
GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3,
SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1.sub.--49 kDa,
Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS.sub.--1, Thioredoxin,
DUF1005, LEA.sub.--3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN.sub.--2,
BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding.sub.--1,
Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3,
LRRNT.sub.--2, GDC-P, zf-CCHC, NPH3, TPR.sub.--1, TFIIA,
DHBP_synthase, UQ_con, TPR.sub.--2, TPT, F-box, adh_short,
Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK,
Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf.sub.--1,
CBM.sub.--20, Methyltransf.sub.--12, Rhodanese, Glycolytic, Actin,
Usp, eIF-4B, Glyco_transf.sub.--8, BURP, Alpha-amylase,
F420_oxidored, EGF_CA, Kelch.sub.--1, PGAM,
Aminotran.sub.--1.sub.--2, Kelch.sub.--2, UPF0261, CoA_binding,
DUF868, Peptidase_S10, Lung.sub.--7-TM_R, Oleosin, Sad1_UNC,
Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase,
Subtilisin_N, SAM.sub.--1, DUF298, ESCRT-III, DNA_pol_E_B,
Aminotran.sub.--3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588,
zf-CCCH, DnaJ, Pkinase_Tyr, Cupin.sub.--2, LRR.sub.--1,
Cupin.sub.--3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer,
Rib.sub.--5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp,
Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK,
PurA, RMMBL, HTH.sub.--11, Tim17, and PBD.
Recombinant DNA Constructs
[0050] The present invention provides recombinant DNA constructs
comprising one or more polynucleotides disclosed herein for
imparting one or more improved traits to transgenic plant when
incorporated into the nucleus of the plant cells. Such constructs
also typically comprise a promoter operatively linked to said
polynucleotide to provide for expression in the plant cells. Other
construct components may include additional regulatory elements,
such as 5' or 3' untranslated regions (such as polyadenylation
sites), intron regions, and transit or signal peptides. Such
recombinant DNA constructs can be assembled using methods known to
those of ordinary skill in the art.
[0051] In a preferred embodiment, a polynucleotide of the present
invention is operatively linked in a recombinant DNA construct to a
promoter functional in a plant to provide for expression of the
polynucleotide in the sense orientation such that a desired protein
or polypeptide fragment of a protein is produced. Also provided are
embodiments wherein a polynucleotide is operatively linked to a
promoter functional in a plant to provide for expression of gene
suppression RNA to suppress the level of an endogenous protein.
[0052] Recombinant constructs prepared in accordance with the
present invention also generally include a 3' untranslated DNA
region (UTR) that typically contains a polyadenylation sequence
following the polynucleotide coding region. Examples of useful 3'
UTRs include those from the nopaline synthase gene of Agrobacterium
tumefaciens (nos), a gene encoding the small subunit of a
ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7
transcript of Agrobacterium tumefaciens.
[0053] Constructs and vectors may also include a transit peptide
for targeting of a gene target to a plant organelle, particularly
to a chloroplast, leucoplast or other plastid organelle. For
descriptions of the use of chloroplast transit peptides, see U.S.
Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein
by reference.
[0054] Table 1 provides a list of genes that provided recombinant
DNA that was expressed in a model plant and identified from
screening as imparting an improved trait. When the stated
orientation is "sense", the expression of the gene or a homolog in
a crop plant provides the means to identify transgenic events that
provide an enhanced trait in the crop plant. When the stated
orientation is "antisense", the suppression of the native homolog
in a crop plant provides the means to identify transgenic events
that provide an enhanced trait in the crop plant. In some cases the
expression/suppression in the model plant exhibited an improved
trait that corresponds to an enhanced agronomic trait, e.g. cold
stress tolerance, water deficit stress tolerance, low nitrogen
stress tolerance and the like. In other cases the
expression/suppression in the model plant exhibited an improved
trait that is a surrogate to an enhance agronomic trait, e.g.
salinity stress tolerance being a surrogate to drought tolerance or
improvement in plant growth and development being a surrogate to
enhanced yield. Even when expression of a transgene or suppression
of a native gene imparts an improved trait in a model plant, not
every crop plant expressing the same transgene or suppressing the
same native gene will necessarily demonstrate an indicated enhanced
agronomic trait. For instance, it is well known that multiple
transgenic events are required to identify a transgenic plant that
can exhibit an enhanced agronomic trait. However, by with routine
experimentation a transgenic plant cell nuclei, cell, plant or seed
of this invention can be identified by making a reasonable number
of transgenic events and engaging in screening process identified
in this specification and illustrated in the examples. An
understanding of Table 1 is facilitated by the following
description of the headings:
[0055] "NUC SEQ ID NO" refers to a SEQ ID NO. for particular DNA
sequence in the Sequence Listing.
[0056] "PEP SEQ ID NO" refers to a SEQ ID NO. in the Sequence
Listing for the amino acid sequence of a protein cognate to a
particular DNA
[0057] "construct_id" refers to an arbitrary number used to
identify a particular recombinant DNA construct comprising the
particular DNA.
[0058] "Gene ID" refers to an arbitrary name used to identify the
particular DNA.
[0059] "orientation" refers to the orientation of the particular
DNA in a recombinant DNA construct relative to the promoter.
TABLE-US-00001 TABLE 1 NUC PEP Seq ID SEQ ID Construct No. No Gene
ID ID Orientation 1 198 CGPG106 11029 SENSE 2 199 CGPG1133 12223
SENSE 3 200 CGPG117 10422 ANTI-SENSE 4 201 CGPG1226 13485
ANTI-SENSE 5 202 CGPG1288 13235 SENSE 6 203 CGPG1301 13411 SENSE 7
204 CGPG1458 73944 SENSE 8 205 CGPG1542 13846 ANTI-SENSE 9 206
CGPG170 12602 SENSE 10 207 CGPG1828 74065 ANTI-SENSE 11 208
CGPG2206 72783 SENSE 12 209 CGPG2217 17210 SENSE 13 210 CGPG2292
72724 SENSE 14 211 CGPG2457 17805 ANTI-SENSE 15 212 CGPG2499 16610
SENSE 16 213 CGPG2653 76602 SENSE 17 214 CGPG2813 18456 SENSE 18
215 CGPG3002 18414 SENSE 19 216 CGPG3154 71538 SENSE 20 217
CGPG3235 76532 SENSE 21 218 CGPG3274 18231 SENSE 22 219 CGPG3275
18232 SENSE 23 220 CGPG3363 18256 SENSE 24 221 CGPG3367 18258 SENSE
25 222 CGPG3375 19193 SENSE 26 223 CGPG3528 71301 SENSE 27 224
CGPG3534 18354 SENSE 28 225 CGPG3638 77334 SENSE 29 226 CGPG3918
19767 SENSE 30 227 CGPG3920 19774 SENSE 31 228 CGPG3962 70992 SENSE
32 229 CGPG3972 19956 SENSE 33 230 CGPG3990 70948 SENSE 34 231
CGPG3994 70201 SENSE 35 232 CGPG4026 19973 SENSE 36 233 CGPG4048
70987 SENSE 37 234 CGPG4052 70950 SENSE 38 235 CGPG4057 70962 SENSE
39 236 CGPG4058 70915 SENSE 40 237 CGPG4069 19947 SENSE 41 238
CGPG4087 70969 SENSE 42 239 CGPG4088 70985 SENSE 43 240 CGPG4102
70971 SENSE 44 241 CGPG4121 70963 SENSE 45 242 CGPG4122 70994 SENSE
46 243 CGPG4140 70956 SENSE 47 244 CGPG4154 70995 SENSE 48 245
CGPG4311 73306 SENSE 49 246 CGPG4363 70657 SENSE 50 247 CGPG4369
70660 SENSE 51 248 CGPG442 74536 SENSE 52 249 CGPG4454 71328 SENSE
53 250 CGPG4456 71329 SENSE 54 251 CGPG4473 70755 SENSE 55 252
CGPG4588 70684 SENSE 56 253 CGPG4765 73330 SENSE 57 254 CGPG4788
76202 SENSE 58 255 CGPG4912 72807 SENSE 59 256 CGPG4926 72811 SENSE
60 257 CGPG4967 73235 SENSE 61 258 CGPG4977 72813 SENSE 62 259
CGPG5001 72825 SENSE 63 260 CGPG5025 73628 SENSE 64 261 CGPG5041
76105 SENSE 65 262 CGPG5116 73242 SENSE 66 263 CGPG5144 74217 SENSE
67 264 CGPG5171 73735 SENSE 68 265 CGPG5194 73256 SENSE 69 266
CGPG5200 73260 SENSE 70 267 CGPG5210 75822 SENSE 71 268 CGPG5221
72001 SENSE 72 269 CGPG5269 72056 SENSE 73 270 CGPG5404 77308 SENSE
74 271 CGPG5432 73766 SENSE 75 272 CGPG5518 72774 SENSE 76 273
CGPG5535 72788 SENSE 77 274 CGPG5540 72753 SENSE 78 275 CGPG5568
72709 SENSE 79 276 CGPG5577 73954 SENSE 80 277 CGPG5587 73137 SENSE
81 278 CGPG5594 73161 SENSE 82 279 CGPG5633 73057 SENSE 83 280
CGPG5640 73127 SENSE 84 281 CGPG5646 73033 SENSE 85 282 CGPG5656
73105 SENSE 86 283 CGPG5659 73141 SENSE 87 284 CGPG5661 73165 SENSE
88 285 CGPG5684 73155 SENSE 89 286 CGPG5694 73026 SENSE 90 287
CGPG5704 73120 SENSE 91 288 CGPG5714 73133 SENSE 92 289 CGPG5721
73134 SENSE 93 290 CGPG5728 73123 SENSE 94 291 CGPG5757 73981 SENSE
95 292 CGPG5764 73136 SENSE 96 293 CGPG5783 73172 SENSE 97 294
CGPG5791 73020 SENSE 98 295 CGPG5799 72946 SENSE 99 296 CGPG5856
74746 SENSE 100 297 CGPG5927 77312 SENSE 101 298 CGPG5941 75237
SENSE 102 299 CGPG5957 75240 SENSE 103 300 CGPG5967 74349 SENSE 104
301 CGPG6040 76422 SENSE 105 302 CGPG607 70812 SENSE 106 303
CGPG6178 77322 SENSE 107 304 CGPG6185 74662 SENSE 108 305 CGPG6306
76527 SENSE 109 306 CGPG6318 77020 SENSE 110 307 CGPG6326 77609
SENSE 111 308 CGPG6370 73485 SENSE 112 309 CGPG6429 73433 SENSE 113
310 CGPG6440 73411 SENSE 114 311 CGPG6516 73568 SENSE 115 312
CGPG6653 74688 SENSE 116 313 CGPG6712 74420 SENSE 117 314 CGPG6737
74435 SENSE 118 315 CGPG6747 74460 SENSE 119 316 CGPG6796 74566
SENSE 120 317 CGPG6805 77610 SENSE 121 318 CGPG6810 77618 SENSE 122
319 CGPG6952 77517 SENSE 123 320 CGPG6953 77518 SENSE 124 321
CGPG7121 76460 SENSE 125 322 CGPG7163 77069 SENSE 126 323 CGPG7168
76161 SENSE 127 324 CGPG7206 76171 SENSE 128 325 CGPG7225 76178
SENSE 129 326 CGPG7267 76467 SENSE 130 327 CGPG7272 77536 SENSE 131
328 CGPG7281 76576 SENSE 132 329 CGPG7308 74862 SENSE 133 330
CGPG7316 74863 SENSE 134 331 CGPG7371 74858 SENSE 135 332 CGPG7457
74933 SENSE 136 333 CGPG7520 75379 SENSE 137 334 CGPG7529 77816
SENSE 138 335 CGPG7636 75434 SENSE 139 336 CGPG7737 77821 SENSE 140
337 CGPG7767 75685 SENSE 141 338 CGPG7804 75654 SENSE 142 339
CGPG7823 75692 SENSE 143 340 CGPG7828 75657 SENSE 144 341 CGPG7833
75622 SENSE 145 342 CGPG7933 77549 SENSE 146 343 CGPG7986 77917
SENSE 147 344 CGPG8012 77568 SENSE 148 345 CGPG8015 77570 SENSE 149
346 CGPG8055 77338 SENSE 150 347 CGPG8062 77580 SENSE 151 348
CGPG8082 77928 SENSE 152 349 CGPG8083 77349 SENSE 153 350 CGPG8106
77357 SENSE 154 351 CGPG8107 77587 SENSE 155 352 CGPG8136 77933
SENSE 156 353 CGPG8152 77619 SENSE 157 354 CGPG8166 77621 SENSE 158
355 CGPG8377 77629 SENSE 159 356 CGPG8976 77832 SENSE 160 357
CGPG8987 76802 SENSE 161 358 CGPG9013 76829 SENSE 162 359 CGPG9080
76961 SENSE 163 360 CGPG9081 76973 SENSE 164 361 CGPG9130 77150
SENSE 165 362 CGPG9133 77186 SENSE 166 363 CGPG9134 77103 SENSE 167
364 CGPG9137 77139 SENSE 168 365 CGPG9141 77187 SENSE 169 366
CGPG9145 77140 SENSE 170 367 CGPG9147 77164 SENSE 171 368 CGPG9148
77176 SENSE 172 369 CGPG9155 77165 SENSE 173 370 CGPG9163 77166
SENSE 174 371 CGPG9170 77155 SENSE 175 372 CGPG9180 77180 SENSE 176
373 CGPG9183 77121 SENSE 177 374 CGPG9186 77157 SENSE 178 375
CGPG9205 77195 SENSE 179 376 CGPG9207 77124 SENSE 180 377 CGPG9219
77261 SENSE 181 378 CGPG9220 77273 SENSE 182 379 CGPG9230 77203
SENSE 183 380 CGPG9236 77275 SENSE 184 381 CGPG9238 77204 SENSE 185
382 CGPG9259 77266 SENSE 186 383 CGPG9271 77220 SENSE 187 384
CGPG9275 77268 SENSE 188 385 CGPG9278 77209 SENSE 189 386 CGPG9283
77269 SENSE 190 387 CGPG9309 77451 SENSE 191 388 CGPG9311 77452
SENSE 192 389 CGPG9322 77430 SENSE 193 390 CGPG9335 77432 SENSE 194
391 CGPG9341 77433 SENSE 195 392 CGPG9344 77444 SENSE 196 393
CGPG9345 77409 SENSE 197 394 CGPG976 12313 ANTI- SENSE 10 207
CGPG1828 16322 SENSE
Recombinant DNA
[0060] DNA for use in the present invention to improve traits in
plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID
NO:197, as well as the homologs of such DNA molecules. A subset of
the DNA for gene suppression aspects of the invention includes
fragments of the disclosed full polynucleotides consisting of
oligonucleotides of 21 or more consecutive nucleotides.
Oligonucleotides the larger molecules having a sequence selected
from the group consisting of SEQ ID NO:1 through SEQ ID NO:197 are
useful as probes and primers for detection of the polynucleotides
used in the invention. Also useful in this invention are variants
of the DNA. Such variants may be naturally occurring, including DNA
from homologous genes from the same or a different species, or may
be non-natural variants, for example DNA synthesized using chemical
synthesis methods, or generated using recombinant DNA techniques.
Degeneracy of the genetic code provides the possibility to
substitute at least one base of the protein encoding sequence of a
gene with a different base without causing the amino acid sequence
of the polypeptide produced from the gene to be changed. Hence, a
DNA useful in the present invention may have any base sequence that
has been changed from the sequences provided herein by substitution
in accordance with degeneracy of the genetic code.
[0061] Homologs of the genes providing DNA demonstrated as useful
in improving traits in model plants disclosed herein will generally
have significant identity with the DNA disclosed herein. DNA is
substantially identical to a reference DNA if, when the sequences
of the polynucleotides are optimally aligned there is about 60%
nucleotide equivalence; more preferably 70%; more preferably 80%
equivalence; more preferably 85% equivalence; more preferably 90%;
more preferably 95%; and/or more preferably 98% or 99% equivalence
over a comparison window. A comparison window is preferably at
least 50-100 nucleotides, and more preferably is the entire length
of the polynucleotide provided herein. Optimal alignment of
sequences for aligning a comparison window may be conducted by
algorithms; preferably by computerized implementations of these
algorithms (for example, the Wisconsin Genetics Software Package
Release 7.0-10.0, Genetics Computer Group, 575 Science Dr.,
Madison, Wis.). The reference polynucleotide may be a full-length
molecule or a portion of a longer molecule. Preferentially, the
window of comparison for determining polynucleotide identity of
protein encoding sequences is the entire coding region.
[0062] Proteins useful for imparting improved traits are entire
proteins or at least a sufficient portion of the entire protein to
impart the relevant biological activity of the protein. Proteins
useful for generation of transgenic plants having improved traits
include the proteins with an amino acid sequence provided herein as
SEQ ID NO: 198 through SEQ ID NO: 394, as well as homologs of such
proteins.
[0063] Homologs of the proteins useful in the invention are
identified by comparison of the amino acid sequence of the protein
to amino acid sequences of proteins from the same or different
plant sources, e.g., manually or by using known homology-based
search algorithms such as those commonly known and referred to as
BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a
protein from the same or a different organism that performs the
same biological function as the polypeptide to which it is
compared. An orthologous relation between two organisms is not
necessarily manifest as a one-to-one correspondence between two
genes, because a gene can be duplicated or deleted after organism
phylogenetic separation, such as speciation. For a given protein,
there may be no ortholog or more than one ortholog. Other
complicating factors include alternatively spliced transcripts from
the same gene, limited gene identification, redundant copies of the
same gene with different sequence lengths or corrected sequence. A
local sequence alignment program, e.g., BLAST, can be used to
search a database of sequences to find similar sequences, and the
summary Expectation value (E-value) used to measure the sequence
base similarity. As a protein hit with the best E-value for a
particular organism may not necessarily be an ortholog or the only
ortholog, a reciprocal BLAST search is used in the present
invention to filter hit sequences with significant E-values for
ortholog identification. The reciprocal BLAST entails search of the
significant hits against a database of amino acid sequences from
the base organism that are similar to the sequence of the query
protein. A hit is a likely ortholog, when the reciprocal BLAST's
best hit is the query protein itself or a protein encoded by a
duplicated gene after speciation. Thus, homolog is used herein to
describe proteins that are assumed to have functional similarity by
inference from sequence base similarity. The relationship of
homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO:
19,938 to the proteins with amino acid sequences of SEQ ID NO: to
198 to SEQ ID NO: 394 are found in the listing of Table 2.
[0064] Other functional homolog proteins differ in one or more
amino acids from those of a trait-improving protein disclosed
herein as the result of one or more of the well-known conservative
amino acid substitutions, e.g., valine is a conservative substitute
for alanine and threonine is a conservative substitute for serine.
Conservative substitutions for an amino acid within the native
sequence can be selected from other members of a class to which the
naturally occurring amino acid belongs. Representative amino acids
within these various classes include, but are not limited to: (1)
acidic (negatively charged) amino acids such as aspartic acid and
glutamic acid; (2) basic (positively charged) amino acids such as
arginine, histidine, and lysine; (3) neutral polar amino acids such
as glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such
as alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine. Conserved substitutes for an amino acid
within a native amino acid sequence can be selected from other
members of the group to which the naturally occurring amino acid
belongs. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Naturally conservative amino acids substitution groups
are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and
asparagine-glutamine. A further aspect of the invention comprises
proteins that differ in one or more amino acids from those of a
described protein sequence as the result of deletion or insertion
of one or more amino acids in a native sequence.
[0065] Homologs of the trait-improving proteins provided herein
will generally demonstrate significant sequence identity. Of
particular interest are proteins having at least 50% sequence
identity, more preferably at least about 70% sequence identity or
higher, e.g., at least about 80% sequence identity with an amino
acid sequence of SEQ ID NO:198 through SEQ ID NO: 394. Of course
useful proteins also include those with higher identity, e.g., 90%
to 99% identity. Identity of protein homologs is determined by
optimally aligning the amino acid sequence of a putative protein
homolog with a defined amino acid sequence and by calculating the
percentage of identical and conservatively substituted amino acids
over the window of comparison. The window of comparison for
determining identity can be the entire amino acid sequence
disclosed herein, e.g., the full sequence of any of SEQ ID NO: 198
through SEQ ID NO: 394.
[0066] Genes that are homologous to each other can be grouped into
families and included in multiple sequence alignments. Then a
consensus sequence for each group can be derived. This analysis
enables the derivation of conserved and class- (family) specific
residues or motifs that are functionally important. These conserved
residues and motifs can be further validated with 3D protein
structure if available. The consensus sequence can be used to
define the full scope of the invention, e.g., to identify proteins
with a homolog relationship. Thus, the present invention
contemplates that protein homologs include proteins with an amino
acid sequence that has at least 90% identity to such a consensus
amino acid sequence sequences.
Promoters
[0067] Numerous promoters that are active in plant cells have been
described in the literature. These include promoters present in
plant genomes as well as promoters from other sources, including
nopaline synthase (NOS) promoter and octopine synthase (OCS)
promoters carried on tumor-inducing plasmids of Agrobacterium
tumefaciens, caulimovirus promoters such as the cauliflower mosaic
virus or Figwort mosaic virus promoters. For instance, see U.S.
Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the
constitutive promoter derived from cauliflower mosaic virus
(CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic
Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a
maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice
actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize
RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize
PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3
promoter, U.S. Pat. No. 6,177,611 which discloses constitutive
maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3
oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice
actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which
discloses a root specific promoter, U.S. Pat. No. 6,084,089 which
discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which
discloses light inducible promoters, U.S. Pat. No. 6,140,078 which
discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which
discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060
which discloses phosphorus deficiency inducible promoters, U.S.
Patent Application Publication 2002/0192813A1 which discloses 5',
3' and intron elements useful in the design of effective plant
expression vectors, U.S. patent application Ser. No. 09/078,972
which discloses a coixin promoter, U.S. patent application Ser. No.
09/757,089 which discloses a maize chloroplast aldolase promoter,
and U.S. patent application Ser. No. 10/739,565 which discloses
water-deficit inducible promoters, all of which are incorporated
herein by reference. These and numerous other promoters that
function in plant cells are known to those skilled in the art and
available for use in recombinant polynucleotides of the present
invention to provide for expression of desired genes in transgenic
plant cells.
[0068] Furthermore, the promoters can include multiple "enhancer
sequences" to assist in elevating gene expression. Such enhancers
are known in the art. By including an enhancer sequence with such
constructs, the expression of the selected protein may be enhanced.
These enhancers often are found 5' to the start of transcription in
a promoter that functions in eukaryotic cells, but can often be
inserted in the forward or reverse orientation 5' or 3' to the
coding sequence. In some instances, these 5' enhancing elements are
introns. Deemed to be particularly useful as enhancers are the 5'
introns of the rice actin 1 and rice actin 2 genes. Examples of
other enhancers that can be used in accordance with the invention
include elements from the CaMV 35S promoter, octopine synthase
genes, the maize alcohol dehydrogenase gene, the maize shrunken 1
gene and promoters from non-plant eukaryotes.
[0069] In some aspects of the invention it is preferred that the
promoter element in the DNA construct be capable of causing
sufficient expression to result in the production of an effective
amount of a polypeptide in water deficit conditions. Such promoters
can be identified and isolated from the regulatory region of plant
genes that are over expressed in water deficit conditions. Specific
water-deficit-inducible promoters for use in this invention are
derived from the 5' regulatory region of genes identified as a heat
shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17
gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea
maize. Such water-deficit-inducible promoters are disclosed in U.S.
application Ser. No. 10/739,565, incorporated herein by
reference.
[0070] In some aspects of the invention, sufficient expression in
plant seed tissues is desired to effect improvements in seed
composition. Exemplary promoters for use for seed composition
modification include promoters from seed genes such as napin (U.S.
Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252),
zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166),
globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin
1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1)
(Stacy et al., (1996) Plant Mol. Biol. 31(6):1205-1216).
[0071] In some aspects of the invention, preferential expression in
plant green tissues is desired. Promoters of interest for such uses
include those from genes such as SSU (Fischhoff, et al., (1992)
Plant Mol. Biol. 20:81-93), aldolase and pyruvate orthophosphate
dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol.
41(1):42-48).
[0072] Gene suppression includes any of the well-known methods for
suppressing transcription of a gene or the accumulation of the mRNA
corresponding to that gene thereby preventing translation of the
transcript into protein. Posttranscriptional gene suppression is
mediated by transcription of RNA that forms double-stranded RNA
(dsRNA) having homology to a gene targeted for suppression.
Suppression can also be achieved by insertion mutations created by
transposable elements may also prevent gene function. For example,
in many dicot plants, transformation with the T-DNA of
Agrobacterium may be readily achieved and large numbers of
transformants can be rapidly obtained. Also, some species have
lines with active transposable elements that can efficiently be
used for the generation of large numbers of insertion mutations,
while some other species lack such options. Mutant plants produced
by Agrobacterium or transposon mutagenesis and having altered
expression of a polypeptide of interest can be identified using the
polynucleotides of the present invention. For example, a large
population of mutated plants may be screened with polynucleotides
encoding the polypeptide of interest to detect mutated plants
having an insertion in the gene encoding the polypeptide of
interest.
Gene Stacking
[0073] The present invention also contemplates that the
trait-improving recombinant DNA provided herein can be used in
combination with other recombinant DNA to create plants with
multiple desired traits or a further enhanced trait. The
combinations generated can include multiple copies of any one or
more of the recombinant DNA constructs. These stacked combinations
can be created by any method, including but not limited to cross
breeding of transgenic plants, or multiple genetic
transformation.
[0074] In particular embodiments, the inventors contemplate the use
of antibodies, either monoclonal or polyclonal which bind to the
proteins disclosed herein. Means for preparing and characterizing
antibodies are well known in the art (See, e.g., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference). The methods for generating
monoclonal antibodies (mAbs) generally begin along the same lines
as those for preparing polyclonal antibodies. Briefly, a polyclonal
antibody is prepared by immunizing an animal with an immunogenic
composition in accordance with the present invention and collecting
antisera from that immunized animal. A wide range of animal species
can be used for the production of antisera. Typically the animal
used for production of anti-antisera is a rabbit, a mouse, a rat, a
hamster, a guinea pig or a goat. Because of the relatively large
blood volume of rabbits, a rabbit is a preferred choice for
production of polyclonal antibodies.
[0075] As is well known in the art, a given composition may vary in
its immunogenicity. It is often necessary therefore to boost the
host immune system, as may be achieved by coupling a peptide or
polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as carriers. Means
for conjugating a polypeptide to a carrier protein are well known
in the art and include using glutaraldehyde,
m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and
bis-biazotized benzidine.
[0076] As is also well known in the art, the immunogenicity of a
particular immunogen composition can be enhanced by the use of
non-specific stimulators of the immune response, known as
adjuvants. Exemplary and preferred adjuvants include complete
Freund's adjuvant (a non-specific stimulator of the immune response
containing killed Mycobacterium tuberculosis), incomplete Freund's
adjuvants and aluminum hydroxide adjuvant.
[0077] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The production of
polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A
second, booster, injection may also be given. The process of
boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and stored,
and/or the animal can be used to generate mAbs.
[0078] mAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified antifungal
protein, polypeptide or peptide. The immunizing composition is
administered in a manner effective to stimulate antibody producing
cells. Rodents such as mice and rats are preferred animals,
however, the use of rabbit, sheep, or frog cells is also possible.
The use of rats may provide certain advantages (Goding, 1986, pp.
60-61), but mice are preferred, with the BALB/c mouse being most
preferred as this is most routinely used and generally gives a
higher percentage of stable fusions.
[0079] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are
selected for use in the mAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible. Often, a panel of animals will have been immunized and
the spleen of animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately 5.times.10.sup.7 to 2.times.10.sup.8
lymphocytes.
[0080] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma-producing fusion
procedures preferably are non-antibody-producing, have high fusion
efficiency, and enzyme deficiencies that render them incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0081] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, 1986, pp. 65-66;
Campbell, 1984, pp. 75-83). For example, where the immunized animal
is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1,
Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;
for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and
U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in
connection with human cell fusions.
[0082] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag-4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0 non-producer cell line.
[0083] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 ratio, though the ratio
may vary from about 20:1 to about 1:1, respectively, in the
presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Spend
virus have been described (Kohler and Milstein, 1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG,
(Gefter et al., 1977). The use of electrically induced fusion
methods is also appropriate (Goding, 1986, pp. 71-74).
[0084] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azasenne blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0085] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B-cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B-cells.
[0086] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immunobinding assays, and the like.
[0087] The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
can then be propagated indefinitely to provide mAbs. The cell lines
may be exploited for mAb production in two basic ways. A sample of
the hybridoma can be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific monoclonal antibody
produced by the fused cell hybrid. The body fluids of the animal,
such as serum or ascites fluid, can then be tapped to provide mAbs
in high concentration. The individual cell lines could also be
cultured in vitro, where the mAbs are naturally secreted into the
culture medium from which they can be readily obtained in high
concentrations. mAbs produced by either means may be further
purified, if desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
Transformation Methods
[0088] Numerous methods for producing plant cell nuclei with
recombinant DNA are known in the art and may be used in the present
invention. Two commonly used methods for plant transformation are
Agrobacterium-mediated transformation and microprojectile
bombardment. Microprojectile bombardment methods are illustrated in
U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880
(corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn) and
6,153,812 (wheat) and Agrobacterium-mediated transformation is
described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877
(soybean); 5,591,616 (corn); and 6,384,301 (soybean), all of which
are incorporated herein by reference. For Agrobacterium tumefaciens
based plant transformation system, additional elements present on
transformation constructs will include T-DNA left and right border
sequences to facilitate incorporation of the recombinant
polynucleotide into the plant genome.
[0089] In general it is preferred to introduce heterologous DNA
randomly, i.e., at a non-specific location, in the genome of a
target plant line. In special cases it may be useful to target
heterologous DNA insertion in order to achieve site-specific
integration, e.g., to replace an existing gene in the genome, to
use an existing promoter in the plant genome, or to insert a
recombinant polynucleotide at a predetermined site known to be
active for gene expression. Several site specific recombination
systems exist which are known to function implants include cre-lox
as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in
U.S. Pat. No. 5,527,695, both incorporated herein by reference.
[0090] Transformation methods of this invention are preferably
practiced in tissue culture on media and in a controlled
environment. "Media" refers to the numerous nutrient mixtures that
are used to grow cells in vitro, that is, outside of the intact
living organism. Recipient cell targets include, but are not
limited to, meristem cells, callus, immature embryos and gametic
cells such as microspores, pollen, sperm and egg cells. It is
contemplated that any cell from which a fertile plant may be
regenerated is useful as a recipient cell. Callus may be initiated
from tissue sources including, but not limited to, immature
embryos, seedling apical meristems, microspores and the like. Cells
capable of proliferating as callus are also recipient cells for
genetic transformation. Practical transformation methods and
materials for making transgenic plants of this invention, e.g.,
various media and recipient target cells, transformation of
immature embryos and subsequent regeneration of fertile transgenic
plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and
U.S. patent application Ser. No. 09/757,089, which are incorporated
herein by reference.
[0091] In practice DNA is introduced into only a small percentage
of target cell nuclei in any one experiment. Marker genes are used
to provide an efficient system for identification of those cells
with nuclei that are stably transformed by receiving and
integrating a transgenic DNA construct into their genomes.
Preferred marker genes provide selective markers that confer
resistance to a selective agent, such as an antibiotic or
herbicide. Potentially transformed cells with a nucleus of the
invention are exposed to the selective agent. In the population of
surviving cells will be those cells where, generally, the
resistance-conferring gene has been integrated and expressed at
sufficient levels to permit cell survival. Cells may be tested
further to confirm stable integration of the exogenous DNA in the
nucleus. Useful selective marker genes include those conferring
resistance to antibiotics such as kanamycin (nptII), hygromycin B
(aph IV) and gentamycin (aac3 and aacC4) or resistance to
herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS).
Examples of such selectable are illustrated in U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are
incorporated herein by reference. Screenable markers which provide
an ability to visually identify transformants can also be employed,
e.g., a gene expressing a colored or fluorescent protein such as a
luciferase or green fluorescent protein (GFP) or a gene expressing
a beta-glucuronidase or uidA gene (GUS) for which various
chromogenic substrates are known. It is also contemplated that
combinations of screenable and selectable markers will be useful
for identification of transformed cells. See PCT publication WO
99/61129 which discloses use of a gene fusion between a selectable
marker gene and a screenable marker gene, e.g., an NPTII gene and a
GFP gene.
[0092] Cells that survive exposure to the selective agent, or cells
that have been scored positive in a screening assay, may be
cultured in regeneration media and allowed to mature into plants.
Developing plantlets can be transferred to soil less plant growth
mix, and hardened off, e.g., in an environmentally controlled
chamber at about 85% relative humidity, 600 ppm CO.sub.2, and
25-250 microeinsteins m.sup.-2 s.sup.-1 of light, prior to transfer
to a greenhouse or growth chamber for maturation. Plants are
preferably matured either in a growth chamber or greenhouse. Plants
are regenerated from about 6 wk to 10 months after a transformant
is identified, depending on the initial tissue. During
regeneration, cells are grown to plants on solid media at about 19
to 28.degree. C. After regenerating plants have reached the stage
of shoot and root development, they may be transferred to a
greenhouse for further growth and testing. Plants may be pollinated
using conventional plant breeding methods known to those of skill
in the art and seed produced.
[0093] Progeny may be recovered from transformed plants and tested
for expression of the exogenous recombinant polynucleotide. Useful
assays include, for example, "molecular biological" assays, such as
Southern and Northern blotting and PCR; "biochemical" assays, such
as detecting the presence of RNA, e.g., double stranded RNA, or a
protein product, e.g., by immunological means (ELISAs and Western
blots) or by enzymatic function; plant part assays, such as leaf or
root assays; and also, by analyzing the phenotype of the whole
regenerated plant.
Discovery of Trait-Improving Recombinant DNA
[0094] To identify nuclei with recombinant DNA that confer improved
traits to plants, Arabidopsis thaliana was transformed with a
candidate recombinant DNA construct and screened for an improved
trait.
[0095] Arabidopsis thaliana is used a model for genetics and
metabolism in plants. Arabidopsis has a small genome, and
well-documented studies are available. It is easy to grow in large
numbers and mutants defining important genetically controlled
mechanisms are either available, or can readily be obtained.
Various methods to introduce and express isolated homologous genes
are available (see Koncz, e.g., Methods in Arabidopsis Research
e.g., (1992), World Scientific, New Jersey, New Jersey, in
"Preface").
[0096] A two-step screening process was employed which comprised
two passes of trait characterization to ensure that the trait
modification was dependent on expression of the recombinant DNA,
but not due to the chromosomal location of the integration of the
transgene. Twelve independent transgenic lines for each recombinant
DNA construct were established and assayed for the transgene
expression levels. Five transgenic lines with high transgene
expression levels were used in the first pass screen to evaluate
the transgene's function in T2 transgenic plants. Subsequently,
three transgenic events, which had been shown to have one or more
improved traits, were further evaluated in the second pass screen
to confirm the transgene's ability to impart an improved trait. The
following Table 3 summarizes the improved traits that have been
confirmed as provided by a recombinant DNA construct.
[0097] In particular, Table 3 reports:
[0098] "PEP SEQ ID" which is the amino acid sequence of the protein
cognate to the DNA in the recombinant DNA construct corresponding
to a protein sequence of a SEQ ID NO. in the Sequence Listing.
[0099] "construct_id" is an arbitrary name for the recombinant DNA
describe more particularly in Table 1.
[0100] "annotation" refers to a description of the top hit protein
obtained from an amino acid sequence query of each PEP SEQ ID NO to
GenBank database of the National Center for Biotechnology
Information (ncbi). More particularly, "gi" is the GenBank ID
number for the top BLAST hit.
[0101] "description" refers to the description of the top BLAST
hit.
[0102] "e-value" provides the expectation value for the BLAST
hit.
[0103] "% id" refers to the percentage of identically matched amino
acid residues along the length of the portion of the sequences
which is aligned by BLAST between the sequence of interest provided
herein and the hit sequence in GenBank.
[0104] "traits" identify by two letter codes the confirmed
improvement in a transgenic plant provided by the recombinant DNA.
The codes for improved traits are:
[0105] "CK" which indicates cold tolerance improvement identified
under a cold shock tolerance screen;
[0106] "CS" which indicates cold tolerance improvement identified
by a cold germination tolerance screen;
[0107] "DS" which indicates drought tolerance improvement
identified by a soil drought stress tolerance screen;
[0108] "PEG" which indicates osmotic stress tolerance improvement
identified by a PEG induced osmotic stress tolerance screen;
[0109] "HS" which indicates heat stress tolerance improvement
identified by a heat stress tolerance screen;
[0110] "SS" which indicates high salinity stress tolerance
improvement identified by a salt stress tolerance screen;
[0111] "LN" which indicates nitrogen use efficiency improvement
identified by a limited nitrogen tolerance screen;
[0112] "LL" which indicates attenuated shade avoidance response
identified by a shade tolerance screen under a low light
condition;
[0113] "PP" which indicates improved growth and development at
early stages identified by an early plant growth and development
screen;
[0114] "SP" which indicates improved growth and development at late
stages identified by a late plant growth and development screen
provided herein.
TABLE-US-00002 TABLE 3 PEP Annotation SEQ Construct e % ID ID value
id description trait 198 11029 1.00E-170 85
gb|AAD02882.1|gamma-tocopherol DS LN methyltransferase [Arabidopsis
thaliana] 199 12223 / / / CK PEG 200 10422 1.00E-133 100
gb|AAB47766.1|SNF5 homolog BSH LN [Arabidopsis thaliana] 201 13485
4.00E-39 55 emb|CAB79347.1|hypothetical LN protein [Arabidopsis
thaliana] 202 13235 7.00E-76 100 emb|CAB78153.1|putative protein LN
[Arabidopsis thaliana] 203 13411 3.00E-29 100
ref|NP_179638.1|unknown protein LN CK [Arabidopsis thaliana] 204
73944 0 95 ref|NP_189578.1|phosphorylase/ CS transferase,
transferring glycosyl groups [Arabidopsis thaliana] 205 13846 0 91
gb|AAN60291.1|unknown LN [Arabidopsis thaliana] 206 12602 0 100
ref|NP_565974.1|PP2A-4; hydrolase/ LN protein phosphatase type
2A/protein serine/threonine phosphatase [Arabidopsis thaliana] 207
74065 0 94 ref|NP_181390.1|DNA binding/ CS DS transcription factor
[Arabidopsis thaliana] gb|AAN13033.1|putative elongation 208 72783
0 83 ref|NP_564690.1|unknown protein CK PEG HS [Arabidopsis
thaliana] 209 17210 1.00E-92 100 ref|NP_563622.1|unknown protein CS
CK PEG [Arabidopsis thaliana] 210 72724 1.00E-128 90
ref|NP_190525.1|protein translocase/ LL protein transporter
[Arabidopsis thaliana 211 17805 1.00E-172 80
ref|NP_563761.1|unknown protein LN [Arabidopsis thaliana] 212 16610
0 100 gb|AAL44550.1|fructose bisphosphate LN HS aldolase
[Agrobacterium tumefaciens str. C58] 213 76602 0 78
ref|NP_566515.1|signal transducer CS HS [Arabidopsis thaliana] 214
18456 1.00E-135 81 ref|NP_176820.1|DNA binding/ CK SS PEG
transcription factor [Arabidopsis thaliana] 215 18414 1.00E-169 70
ref|NP_195583.1|translation initiation CK factor [Arabidopsis
thaliana] 216 71538 1.00E-137 90 emb|CAE85115.1|synaptotagmin SP
[Arabidopsis thaliana] 217 76532 0 99 ref|NP_564775.1|ATP binding/
CK CS carbohydrate binding/kinase/protein kinase/protein
serine/threonine kinase/protein-tyrosine kinase/sugar binding
[Arabidopsis thaliana] 218 18231 1.00E-142 100
emb|CAC83762.1|allene oxide CK LN cyclase [Arabidopsis thaliana]
219 18232 6.00E-85 89 ref|NP_191628.1|unknown protein CS
[Arabidopsis thaliana] 220 18256 1.00E-102 100
dbj|BAD44508.1|putative zinc finger SS CK HS protein (PMZ)
[Arabidopsis thaliana] 221 18258 1.00E-175 83
emb|CAA11525.1|transcription factor SP IIA large subunit
[Arabidopsis thaliana] 222 19193 1.00E-103 100
ref|NP_564682.1|lipid binding SP HS [Arabidopsis thaliana]
dbj|BAE73268.1|xylogen like protein 12 [Arabidopsis thaliana] 223
71301 0 84 ref|NP_568393.1|nucleic acid binding LN [Arabidopsis
thaliana] 224 18354 0 96 ref|NP_567568.1|ATP binding/ LN
kinase/protein kinase/protein serine/threonine kinase/protein-
tyrosine kinase [Arabidopsis thaliana] 225 77334 1.00E-117 100
ref|NP_178399.1|RNS1 LL (RIBONUCLEASE 1); endoribonuclease
[Arabidopsis thaliana] 226 19767 1.00E-180 95 gb|AAO69665.1|serine
threonine CS DS protein phosphatase [Phaseolus acutifolius] 227
19774 1.00E-100 57 ref|NP_974271.1|unknown protein CS PP
[Arabidopsis thaliana] 228 70992 1.00E-108 65
gb|ABE87200.1|hypothetical protein CS PEG MtrDRAFT_AC151668g4v1
[Medicago truncatula] 229 19956 1.00E-146 100
gb|AAF64040.1|14-3-3-like protein PP [Glycine max] 230 70948
3.00E-59 45 ref|NP_201297.1|CIP8 (COP1- PP HS INTERACTING PROTEIN
8); protein binding/zinc ion binding [Arabidopsis thaliana] 231
70201 0 68 ref|NP_567072.1|ATP binding/ CS SS kinase/protein
kinase/protein serine/threonine kinase/protein- tyrosine kinase
[Arabidopsis thaliana] 232 19973 0 96 gb|AAB04057.1|S-adenosyl-L-
PP SS methionine:delta24-sterol-C- methyltransferase 233 70987 0 93
gb|AAM83095.1|SOS2-like protein CK CS HS SS PEG kinase [Glycine
max] 234 70950 1.00E-116 77 dbj|BAD38167.1|putative leucine PP PEG
zipper protein [Oryza sativa (japonica cultivar-group)] 235 70962
1.00E-179 78 gb|ABE93200.1|conserved PP HS hypothetical protein
[Medicago truncatula] 236 70915 0 76 dbj|BAE71210.1|hypothetical
protein CS PP [Trifolium pratense] dbj|BAE71208.1| hypothetical
protein [Trifolium pratense] 237 19947 1.00E-116 65
gb|AAS38575.1|short-chain CS DS SS dehydrogenase Tic32 [Pisum
sativum] 238 70969 1.00E-144 76 ref|NP_174321.2|unknown protein SS
[Arabidopsis thaliana] 239 70985 0 69 dbj|BAB08296.1|unnamed
protein CS HS PEG product [Arabidopsis thaliana]
sp|Q9FMT4|Y5417_ARATH Protein At5g14170 240 70971 1.00E-92 63
gb|ABE80664.1|GNS1/SUR4 SS membrane protein [Medicago truncatula]
gb|ABE78502.1| GNS1/SUR4 membrane protein [Medicago truncatula] 241
70963 1.00E-96 56 ref|NP_177178.1|CYCD1; 1; cyclin- CS PP HS
dependent protein kinase regulator [Arabidopsis thaliana] 242 70994
2.00E-33 44 ref|NP_172962.1|RHA2A; protein PP DS HS SS
binding/ubiquitin-protein ligase/zinc ion binding [Arabidopsis
thaliana] 243 70956 0 85 gb|AAO23063.1|ent-kaurenoic acid CS SS HS
oxidase [Pisum sativum] 244 70995 1.00E-132 66
dbj|BAD73789.1|putative CK CS PP LL PEG uncharacterized
hypothalamus protein HT010 [Oryza sativa (japonica cultivar-group)]
245 73306 0 93 ref|NP_201243.1|phosphoric ester SS PEG hydrolase
[Arabidopsis thaliana] 246 70657 9.00E-31 73
emb|CAB78150.1|probable wound- SP LL induced protein [Arabidopsis
thaliana] 247 70660 2.00E-32 79 ref|NP_568217.1|transcription SP
regulator [Arabidopsis thaliana] 248 74536 0 91
ref|NP_001031061.1|PGDH; CS phosphoglycerate dehydrogenase
[Arabidopsis thaliana]] 249 71328 0 99 gb|AAD46022.1|Strong
simlarity to PP gb|286426 F10M6.190 cytochrome p450 homolog from
Arabidopsis thaliana BAC 250 71329 0 94 ref|NP_177477.1|heme
binding/iron LN ion binding/monooxygenase/oxygen binding
[Arabidopsis thaliana] 251 70755 1.00E-142 100
ref|NP_188925.1|unknown protein LL [Arabidopsis thaliana] 252 70684
3.00E-53 100 emb|CAB80194.1|putative protein CS LN HS PP
[Arabidopsis thaliana] 253 73330 1.00E-158 72
emb|CAB96855.1|putative protein CK CS SS [Arabidopsis thaliana] 254
76202 3.00E-83 67 emb|CAA16586.1|putative protein LL [Arabidopsis
thaliana] 255 72807 0 100 ref|NP_173478.2|nucleotide binding PEG CK
HS SS [Arabidopsis thaliana] 256 72811 1.00E-176 100
ref|NP_565782.1|nucleotide binding CK SS PEG [Arabidopsis thaliana]
257 73235 0 89 ref|NP_564867.1|pepsin A SS HS [Arabidopsis
thaliana] 258 72813 2.00E-79 93 ref|NP_850506.1|unknown protein LL
LN [Arabidopsis thaliana] 259 72825 1.00E-154 95
ref|NP_563635.1|oxidoreductase CK PEG HS [Arabidopsis thaliana] 260
73628 1.00E-163 92 ref|NP_849428.1|oxidoreductase CS DS LL HS PEG
[Arabidopsis thaliana] 261 76105 0 93 emb|CAB79123.1|receptor
kinase-like DS protein [Arabidopsis thaliana] 262 73242 0 100
ref|NP_567292.1|unknown protein SP PEG [Arabidopsis thaliana] 263
74217 0 100 emb|CAB93726.1|cytochrome P450- DS CK like protein
[Arabidopsis thaliana] 264 73735 0 97 gb|AAU94404.1|At3g48520 CK
[Arabidopsis thaliana] 265 73256 1.00E-114 77
ref|NP_566329.1|unknown protein CS PP PEG [Arabidopsis thaliana]
gb|AAL06975.1| 266 73260 4.00E-37 100 ref|NP_566488.1|unknown
protein CS [Arabidopsis thaliana] 267 75822 1.00E-90 88
emb|CAB63015.1|putative protein LL [Arabidopsis thaliana] 268 72001
0 96 ref|NP_200045.1|CYP96A4; heme CS PP HS binding/iron ion
binding/ monooxygenase/oxygen binding [Arabidopsis thaliana] 269
72056 0 92 ref|NP_010186.1|Essential, non- SP ATPase regulatory
subunit of the 26S proteasome lid required for the assembly and
activity of the 26S proteasome; 270 77308 0 100
ref|NP_178642.1|SCPL38; serine PEG carboxypeptidase [Arabidopsis
thaliana] 271 73766 1.00E-163 100 emb|CAB62363.1|MTN3-like protein
LL [Arabidopsis thaliana] 272 72774 1.00E-139 95
ref|NP_011052.1|Constituent of 66S LL CK HS pre-ribosomal
particles, involved in 60S ribosomal subunit biogenesis; 273 72788
0 81 ref|NP_010007.1|General repressor of CK CS SS transcription,
forms complex with Cyc8p, involved in the establishment of
repressive chromatin structure through interactions with histones
H3 and H4, appears to enhance expression of some genes 274 72753
1.00E-105 85 ref|NP_013125.1|One of four subunits CS PP HS PEG of
the endosomal sorting complex required for transport III (ESCRT-
III); 275 72709 0 94 ref|NP_011827.1|Low affinity LL methionine
permease, similar to Mup1p; 276 73954 0 98 ref|NP_200954.1|MIM; ATP
binding HS [Arabidopsis thaliana] 277 73137 1.00E-110 94
gb|AAD32800.1|putative thioredoxin CS H [Arabidopsis thaliana] 278
73161 1.00E-36 65 emb|CAB85993.1|putative protein CS PEG
[Arabidopsis thaliana] 279 73057 1.00E-133 95
ref|NP_012770.1|Tetrameric SP phosphoglycerate mutase, mediates the
conversion of 3-phosphoglycerate to 2-phosphoglycerate during
glycolysis and the reverse reaction during gluconeogenesis; 280
73127 0 99 dbj|BAB05414.1|aspartate SS HS aminotransferase
[Bacillus halodurans C-125] ref|NP_242561.1|aspartate
aminotransferase [Bacillus halodurans C-125] 281 73033 0 96
dbj|BAB07276.1|2,3- CK bisphosphoglycerate-independent
phosphoglycerate mutase [Bacillus halodurans C-125] 282 73105 0 99
emb|CAB13630.1|glutamine PP PEG synthetase [Bacillus subtilis
subsp. subtilis str. 168] 283 73141 0 95
ref|NP_414696.1|glutamate-1- CS semialdehyde aminotransferase
[Escherichia coli K12] 284 73165 0 99 ref|NP_288110.1|pyruvate
kinase DS PEG
[Escherichia coli O157:H7 EDL933] 285 73155 1.00E-173 92
emb|CAC47346.1|PROBABLE CS FRUCTOSE-BISPHOSPHATE ALDOLASE CLASS I
PROTEIN [Sinorhizobium meliloti]] 286 73026 1.00E-116 87
emb|CAG77169.1|triosephosphate PEG isomerase [Erwinia carotovora
subsp. atroseptica SCRI1043] 287 73120 8.00E-82 100
ref|NP_441918.1|nucleoside CS SS HS diphosphate kinase
[Synechocystis sp. PCC 6803] 288 73133 2.00E-63 90
gb|ABA76342.1|Nucleoside LL diphosphate kinase [Pseudomonas
fluorescens PfO-1] 289 73134 9.00E-98 81 emb|CAE15979.1|ribose
5-phosphate SS CK isomerase A (phosphoriboisomerase A)
[Photorhabdus luminescens subsp. laumondii TTO1] 290 73123 0 95
ref|NP_180412.2|nucleic acid binding CS PEG [Arabidopsis thaliana]
291 73981 0 93 ref|NP_014797.1|hypothetical protein; DS PEG Slp1p
[Saccharomyces cerevisiae] 292 73136 0 91 ref|NP_012454.1|Nuclear
actin- DS PP related protein involved in chromatin remodeling 293
73172 0 100 ref|NP_04952.1|Protein involved in CK PEG HS
ER-to-Golgi transport; Sly41p [Saccharomyces cerevisiae] 294 73020
0 93 ref|NP_014993.1|Proline permease, LL required for
high-affinity transport of proline 295 72946 0 95
ref|NP_012534.1|Vacuolar PEG LL transporter, imports large neutral
amino acids into the vacuole 296 74746 0 93 ref|NP_566298.1|ATP
binding/ CK kinase/protein kinase/protein serine/threonine
kinase/protein- tyrosine kinase [Arabidopsis thaliana] 297 77312 0
90 ref|NP_178463.1|CDC48B; ATP PP SS HS binding/ATP-dependent
peptidase/ ATPase/nucleoside-triphosphatase/ nucleotide
binding/serine-type endopeptidase [Arabidopsis thaliana] 298 75237
0 100 ref|NP_173390.1|kinase [Arabidopsis LN thaliana]
gb|AAF98405.1|Unknown protein [Arabidopsis thaliana] 299 75240 0
100 emb|CAB69854.1|putative protein CK [Arabidopsis thaliana] 300
74349 1.00E-148 79 ref|NP_565187.1|transcription LN regulator
[Arabidopsis thaliana] 301 76422 5.00E-44 100
ref|NP_171781.1|unknown protein LN [Arabidopsis thaliana] 302 70812
1.00E-152 92 ref|NP_850182.1|PUR ALPHA-1; PP PEG nucleic acid
binding [Arabidopsis thaliana] 303 77322 1.00E-172 100
gb|AAW38983.1|At5g10750 LN LL [Arabidopsis thaliana] 304 74662
4.00E-89 100 ref|NP_001031939.1|ubiquitin PP HS conjugating
enzyme/ubiquitin-like activating enzyme [Arabidopsis thaliana] 305
76527 0 96 emb|CAB51062.1|cell division cycle CS protein 23 homolog
[Arabidopsis thaliana] 306 77020 0 98 emb|CAB75781.1|putative
transporter CS protein [Arabidopsis thaliana]
ref|NP_190154.1|transporter [Arabidopsis thaliana] 307 77609 0 96
gb|AAU95452.1|At5g04420 LL SS [Arabidopsis thaliana] 308 73485 0
100 emb|CAD84238.1|Glyceraldehyde 3- PEG phosphate dehydrogenase
309 73433 1.00E-132 65 ref|ZP_00819153.1|putative alcohol SP LN PP
dehydrogenase [Marinobacter aquaeolei VT8] 310 73411 0 99
emb|CAB14878.1|pyruvate kinase CS HS PP PEG [Bacillus subtilis
subsp. subtilis str. 168] 311 73568 0 97 emb|CAC41401.1|PROBABLE SP
SUCCINATE-SEMIALDEHYDE DEHYDROGENASE [NADP+] PROTEIN [Sinorhizobium
meliloti] 312 74688 0 98 emb|CAA16688.1|receptor protein CS PP SS
PEG kinase - like protein [Arabidopsis thaliana] 313 74420
1.00E-142 99 emb|CAD85691.1|Phosphoglycerate PP LN SS mutase family
[Nitrosomonas europaea ATCC 19718] 314 74435 1.00E-117 100
emb|CAC41549.1|PROBABLE LL PHOSPHOGLYCERATE MUTASE 1 PROTEIN
[Sinorhizobium meliloti] 315 74460 0 95
ref|NP_441738.1|fructose-1,6- CK bisphosphatase [Synechocystis sp.
PCC 6803] 316 74566 0 93 emb|CAC48499.1|Putative trehalose SP PP SS
PEG HS synthase protein [Sinorhizobium meliloti 1021] 317 77610 0
94 gb|AAC23406.1|hypothetical protein CS HS [Arabidopsis thaliana]
318 77618 0 100 ref|NP_850453.1|JAR1 PP HS (JASMONATE RESISTANT 1)
[Arabidopsis thaliana] 319 77517 7.00E-77 100
emb|CAB75802.1|putative protein CS LL [Arabidopsis thaliana] 320
77518 1.00E-49 100 ref|NP_199600.1|oxidoreductase, SS LL HS acting
on NADH or NADPH, quinone or similar compound as acceptor
[Arabidopsis thaliana] 321 76460 / / / CS PEG 322 77069 0 85
ref|NP_201196.1|unknown protein PP [Arabidopsis thaliana] 323 76161
1.00E-161 100 ref|NP_851282.1|unknown protein CK [Arabidopsis
thaliana] 324 76171 1.00E-178 95 ref|NP_176204.1|oxidoreductase LL
[Arabidopsis thaliana] 325 76178 5.00E-60 86
ref|NP_565029.1|unknown protein CS PP HS LL PEG [Arabidopsis
thaliana] 326 76467 6.00E-86 89 ref|NP_565671.1|unknown protein LL
[Arabidopsis thaliana] 327 77536 1.00E-119 83
gb|AAS99692.1|At1g10020 SP CS DS PP LN [Arabidopsis thaliana] 328
76576 1.00E-145 100 ref|NP_181023.1|FAH1 (FATTY DS ACID HYDROXYLASE
1); catalytic [Arabidopsis thaliana] 329 74862 1.00E-145 100
gb|ABA77057.1|Delta 1-pyrroline-5- PP carboxylate reductase
[Pseudomonas fluorescens PfO-1] 330 74863 1.00E-93 100
ref|NP_012420.1|Nucleosome LL PEG CK SS assembly factor, involved
in chromatin assembly after DNA replication] 331 74858 5.00E-50 68
gb|AAK14395.1|response regulator LL protein [Dianthus caryophyllus]
332 74933 1.00E-177 95 gb|AAK85899.1|AGR_C_118p LL [Agrobacterium
tumefaciens str. C58] 333 75379 1.00E-105 76 dbj|BAD73205.1|unknown
protein LN [Oryza sativa (japonica cultivar- group)] 334 77816
4.00E-41 89 gb|ABA98984.1|expressed protein SP PP HS [Oryza sativa
(japonica cultivar- group)] 335 75434 3.00E-52 79
ref|XP_472650.1|OSJNBa0027P08.15 LN [Oryza sativa (japonica
cultivar- group)] 336 77821 0 71 gb|ABE84883.1|conserved PP SS HS
PEG hypothetical protein [Medicago truncatula] 337 75685 0 82
ref|XP_475937.1|unknown protein LL [Oryza sativa (japonica
cultivar- group)] 338 75654 0 100 ref|NP_563865.1|unknown protein
SP CK HS [Arabidopsis thaliana] 339 75692 1.00E-119 87
dbj|BAD30296.1|peptidyl-prolyl cis- CK LN trans isomerase-like
protein [Oryza sativa (japonica cultivar-group)] 340 75657 6.00E-57
67 dbj|BAD38392.1|DNAJ heat shock N- CS LL HS terminal
domain-containing protein- like [Oryza sativa (japonica cultivar-
group)] 341 75622 1.00E-30 78 dbj|BAD45825.1|unknown protein LN
[Oryza sativa (japonica cultivar- group)] 342 77549 1.00E-132 89
emb|CAB80891.1|AT4g00820 PP HS [Arabidopsis thaliana]] 343 77917 0
91 ref|NP_197917.1|EBF2 (EIN3- PP PEG BINDING F BOX PROTEIN 2)
[Arabidopsis thaliana] 344 77568 9.00E-99 76
ref|NP_849792.1|nucleic acid binding PP [Arabidopsis thaliana] 345
77570 7.00E-54 100 gb|AAS75309.1|multidomain CK SS cyclophilin type
peptidyl-prolyl cis- trans isomerase [Arabidopsis thaliana]] 346
77338 4.00E-24 65 gb|AAM61454.1|unknown PEG [Arabidopsis thaliana]
347 77580 3.00E-45 100 dbj|BAB01457.1|unnamed protein PEG CS HS SS
product [Arabidopsis thaliana] 348 77928 4.00E-38 100
ref|NP_196244.1|unknown protein HS [Arabidopsis thaliana] 349 77349
1.00E-81 93 emb|CAC05463.1|putative lipid LL SS transfer protein
[Arabidopsis thaliana] 350 77357 1.00E-155 95
ref|NP_175357.1|unknown protein PEG [Arabidopsis thaliana] 351
77587 / / / CS PP HS SS 352 77933 / / / SS 353 77619 3.00E-40 98
dbj|BAB09403.1|unnamed protein CK SS PEG HS product [Arabidopsis
thaliana] 354 77621 4.00E-58 74 ref|NP_197632.1|Rac GTPase CK
activator [Arabidopsis thaliana] [Arabidopsis thaliana] 355 77629
5.00E-36 100 ref|NP_196372.1|GRP19 [Arabidopsis SS HS thaliana] 356
77832 0 83 ref|NP_917762.1|P0501G01.24 CS SS PEG HS LN [Oryza
sativa (japonica cultivar- group)] 357 76802 0 76
ref|NP_181908.1|actin binding CK [Arabidopsis thaliana]
gb|AAB64026.1|unknown protein [Arabidopsis thaliana] 358 76829 0 89
ref|NP_909912.1|ferredoxin-NADP+ CK reductase [Oryza sativa] 359
76961 0 65 ref|XP_473189.1|OSJNBa0073E02.11 LL LN [Oryza sativa
(japonica cultivar- group)] 360 76973 1.00E-151 76
ref|XP_480055.1|unknown protein LL [Oryza sativa (japonica
cultivar- group)] 361 77150 0 76 dbj|BAD61385.1|putative SP HS PEG
nucleostemin [Oryza sativa (japonica cultivar-group)] 362 77186 0
85 dbj|BAD27898.1|putative PP pentatricopeptide (PPR) repeat-
containing protein [Oryza sativa (japonica cultivar-group)] 363
77103 1.00E-174 66 ref|NP_568580.1|catalytic LL DS [Arabidopsis
thaliana] 364 77139 2.00E-93 58 ref|NP_171690.1|PFC1 (PALEFACE CS
SS 1) [Arabidopsis thaliana] 365 77187 1.00E-160 83
ref|NP_913437.1|3-methyl-2- CS PP LL PEG oxobutanoate
hydroxy-methyl- transferase-like protein [Oryza sativa (japonica
cultivar-group)] 366 77140 0 77 dbj|BAD36145.1|membrane protein CK
PEG SS PTM1-like [Oryza sativa (japonica cultivar-group)] 367 77164
0 91 gb|ABF93778.1|DNA polymerase PP HS delta small subunit,
putative, expressed [Oryza sativa (japonica cultivar-group)] 368
77176 0 76 ref|NP_914476.1|putative LL phytochrome P450 [Oryza
sativa (japonica cultivar-group)] 369 77165 1.00E-139 75
ref|NP_914949.1|serine/threonine CK LL LN protein kinase-like
protein [Oryza sativa (japonica cultivar-group)] 370 77166 0 85
dbj|BAB75233.1|all3534 [Nostoc sp. CK PP PCC 7120] 371 77155 0 97
emb|CAA35550.1|hycE [Escherichia PP coli] 372 77180 2.00E-62 61
gb|ABA99663.1|expressed protein CS PP HS PEG [Oryza sativa
(japonica cultivar- group)] 373 77121 1.00E-166 93
dbj|BAB05404.1|transcriptional CK repressor of the biotin operon
[Bacillus halodurans C-125] 374 77157 0 100
ref|NP_531453.1|3,4-dihydroxy-2- CK LL butanone-4-phoshate
synthase/GTP cyclohydrolase II [Agrobacterium tumefaciens str. C58]
375 77195 0 94 gb|AAM71555.1|mannose-6- PP SS PEG LL phosphate
isomerase/mannose-1-
phosphate guanylyl transferase [Chlorobium tepidum TLS] 376 77124
1.00E-180 100 ref|NP_418404.1|biotin--protein PP SS PEG ligase
[Escherichia coli K12] 377 77261 0 100 ref|NP_416982.1|hydrogenase
4, PEG subunit [Escherichia coli K12] 378 77273 0 93
dbj|BAB06534.1|glycine PP PEG dehydrogenase subunit 1 [Bacillus
halodurans C-125] 379 77203 1.00E-176 60 dbj|BAD33942.1|putative
serine PEG CK carboxypeptidase precursor [Oryza sativa (japonica
cultivar-group)] 380 77275 4.00E-37 55 gb|ABA91490.1|expressed
protein SS HS PEG [Oryza sativa (japonica cultivar- group)] 381
77204 2.00E-27 44 ref|XP_469963.1|putative protease CK CS inhibitor
[Oryza sativa (japonica cultivar-group)] 382 77266 7.00E-25 73
gb|AAT93978.1|unknown protein CS PP PEG [Oryza sativa (japonica
cultivar- group)] 383 77220 1.00E-169 78 dbj|BAD52854.1|putative
non- LL phototropic hypocotyl 3 [Oryza sativa (japonica
cultivar-group)] 384 77268 4.00E-71 85 gb|ABF95596.1|ETC complex I
PP SS subunit conserved region family protein, expressed [Oryza
sativa (japonica cultivar-group)] 385 77209 9.00E-86 85
ref|XP_479456.1|putative 60S PP LL ribosome subunit biogenesis
protein [Oryza sativa (japonica cultivar- group)] 386 77269
2.00E-56 70 dbj|BAD32031.1|unknown protein SS [Oryza sativa
(japonica cultivar- group)] 387 77451 0 71 ref|XP_475231.1|putative
PP SS microtubule-associated protein [Oryza sativa (japonica
cultivar-group)] 388 77452 1.00E-106 69 dbj|BAD69045.1|unknown
protein PP [Oryza sativa (japonica cultivar- group)] 389 77430
1.00E-160 83 dbj|BAD33328.1|putative protein CS PP SS
serine/threonine kinase [Oryza sativa (japonica cultivar-group)]
390 77432 0 86 ref|NP_849565.1|carbohydrate PP HS PEG
transporter/nucleoside transporter/ sugar porter [Arabidopsis
thaliana] gb|AAM19835.1| AT4g35300/F23E12_140 [Arabidopsis
thaliana] 391 77433 0 100 gb|AAK59487.1|putative cleavage CK PP and
polyadenylation specificity factor [Arabidopsis thaliana] 392 77444
0 96 ref|NP_199947.1|unknown protein CS PP [Arabidopsis thaliana]
393 77409 0 91 ref|NP_001032163.1|unknown protein PP [Arabidopsis
thaliana] 394 12313 0 94 ref|NP_189150.1|QUA1 SP (QUASIMODO1);
transferase, transferring glycosyl groups/ transferase,
transferring hexosyl groups [Arabidopsis thaliana]
Trait Improvement Screens
[0115] DS-Improvement of drought tolerance identified by a soil
drought stress tolerance screen: Drought or water deficit
conditions impose mainly osmotic stress on plants. Plants are
particularly vulnerable to drought during the flowering stage. The
drought condition in the screening process disclosed in Example 1B
started from the flowering time and was sustained to the end of
harvesting. The present invention provides recombinant DNA that can
improve the plant survival rate under such sustained drought
condition. Exemplary recombinant DNA for conferring such drought
tolerance are identified as such in Table 3. Such recombinant DNA
may find particular use in generating transgenic plants that are
tolerant to the drought condition imposed during flowering time and
in other stages of the plant life cycle. As demonstrated from the
model plant screen, in some embodiments of transgenic plants with
trait-improving recombinant DNA grown under such sustained drought
condition can also have increased total seed weight per plant in
addition to the increased survival rate within a transgenic
population, providing a higher yield potential as compared to
control plants.
[0116] PEG-Improvement of drought tolerance identified by PEG
induced osmotic stress tolerance screen: Various drought levels can
be artificially induced by using various concentrations of
polyethylene glycol (PEG) to produce different osmotic potentials
(Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130). Several
physiological characteristics have been reported as being reliable
indications for selection of plants possessing drought tolerance.
These characteristics include the rate of seed germination and
seedling growth. The traits can be assayed relatively easily by
measuring the growth rate of seedling in PEG solution. Thus, a
PEG-induced osmotic stress tolerance screen is a useful surrogate
for drought tolerance screen. As demonstrated from the model plant
screen, embodiments of transgenic plants with trait-improving
recombinant DNA identified in the PEG-induced osmotic stress
tolerance screen can survive better drought conditions providing a
higher yield potential as compared to control plants.
[0117] SS-Improvement of drought tolerance identified by high
salinity stress tolerance screen: Three different factors are
responsible for salt damages: (1) osmotic effects, (2) disturbances
in the mineralization process, and (3) toxic effects caused by the
salt ions, e.g., inactivation of enzymes. While the first factor of
salt stress results in the wilting of the plants that is similar to
drought effect, the ionic aspect of salt stress is clearly distinct
from drought. The present invention provides genes that help plants
maintain biomass, root growth, and/or plant development in high
salinity conditions, which are identified as such in Table 3. Since
osmotic effect is one of the major components of salt stress, which
is common to the drought stress, trait-improving recombinant DNA
identified in a high salinity stress tolerance screen can also
provide transgenic crops with improved drought tolerance. As
demonstrated from the model plant screen, embodiments of transgenic
plants with trait-improving recombinant DNA identified in a high
salinity stress tolerance screen can survive better drought
conditions and/or high salinity conditions providing a higher yield
potential as compared to control plants.
[0118] HS-Improvement of drought tolerance identified by heat
stress tolerance screen: Heat and drought stress often occur
simultaneously, limiting plant growth. Heat stress can cause the
reduction in photosynthesis rate, inhibition of leaf growth and
osmotic potential in plants. Thus, genes identified by the present
invention as heat stress tolerance conferring genes may also impart
improved drought tolerance to plants. As demonstrated from the
model plant screen, embodiments of transgenic plants with
trait-improving recombinant DNA identified in a heat stress
tolerance screen can survive better heat stress conditions and/or
drought conditions providing a higher yield potential as compared
to control plants.
[0119] CK and CS-Improvement of tolerance to cold stress: Low
temperature may immediately result in mechanical constraints,
changes in activities of macromolecules, and reduced osmotic
potential. In the present invention, two screening conditions,
i.e., cold shock tolerance screen (CK) and cold germination
tolerance screen (CS), were set up to look for transgenic plants
that display visual growth advantage at lower temperature. In cold
germination tolerance screen, the transgenic Arabidopsis plants
were exposed to a constant temperature of 8.degree. C. from
planting until day 28 post plating. The trait-improving recombinant
DNA identified by such screen are particular useful for the
production of transgenic plant that can germinate more robustly in
a cold temperature as compared to the wild type plants. In cold
shock tolerance screen, the transgenic plants were first grown
under the normal growth temperature of 22.degree. C. until day 8
post plating, and subsequently were placed under 8.degree. C. until
day 28 post plating. As demonstrated from the model plant screen,
embodiments of transgenic plants with trait-improving recombinant
DNA identified in a cold shock stress tolerance screen and/or a
cold germination stress tolerance screen can survive better cold
conditions providing a higher yield potential as compared to
control plants.
[0120] Improvement of tolerance to multiple stresses: Different
kinds of stresses often lead to identical or similar reaction in
the plants. Genes that are activated or inactivated as a reaction
to stress can either act directly in a way the genetic product
reduces a specific stress, or they can act indirectly by activating
other specific stress genes. By manipulating the activity of such
regulatory genes, i.e., multiple stress tolerance genes, the plant
can be enabled to react to different kinds of stresses. For
examples, PEP SEQ ID NO: 231 can be used to improve both salt
stress tolerance and cold stress tolerance in plants. Of particular
interest, plants transformed with PEP SEQ ID NO: 233 can resist
heat stress, salt stress and cold stress. In addition to these
multiple stress tolerance genes, the stress tolerance conferring
genes provided by the present invention may be used in combinations
to generate transgenic plants that can resist multiple stress
conditions.
[0121] PP-Improvement of early plant growth and development: It has
been known in the art that to minimize the impact of disease on
crop profitability, it is important to start the season with
healthy and vigorous plants. This means avoiding seed and seedling
diseases, leading to increased nutrient uptake and increased yield
potential. Traditionally early planting and applying fertilizer are
the methods used for promoting early seedling vigor. In early
development stage, plant embryos establish only the basic
root-shoot axis, a cotyledon storage organ(s), and stem cell
populations, called the root and shoot apical meristems, that
continuously generate new organs throughout post-embryonic
development. "Early growth and development" used herein encompasses
the stages of seed imbibition through the early vegetative phase.
The present invention provides genes that are useful to produce
transgenic plants that have advantages in one or more processes
including, but not limited to, germination, seedling vigor, root
growth and root morphology under non-stressed conditions. The
transgenic plants starting from a more robust seedling are less
susceptible to the fungal and bacterial pathogens that attach
germinating seeds and seedling. Furthermore, seedlings with
advantage in root growth are more resistant to drought stress due
to extensive and deeper root architecture. Therefore, it can be
recognized by those skilled in the art that genes conferring the
growth advantage in early stages to plants may also be used to
generate transgenic plants that are more resistant to various
stress conditions due to improved early plant development. The
present invention provides such exemplary recombinant DNA that
confer both the stress tolerance and growth advantages to plants,
identified as such in Table 3, e.g., PEP SEQ ID NO: 268 which can
improve the plant early growth and development, and impart heat and
cold tolerance to plants. As demonstrated from the model plant
screen, embodiments of transgenic plants with trait-improving
recombinant DNA identified in the early plant development screen
can grow better under non-stress conditions and/or stress
conditions providing a higher yield potential as compared to
control plants.
[0122] SP-Improvement of late plant growth and development: "Late
growth and development" used herein encompasses the stages of leaf
development, flower production, and seed maturity. In certain
embodiments, transgenic plants produced using genes that confer
growth advantages to plants provided by the present invention,
identified as such in Table 3, exhibit at least one phenotypic
characteristics including, but not limited to, increased rosette
radius, increased rosette dry weight, seed dry weight, silique dry
weight, and silique length. On one hand, the rosette radius and
rosette dry weight are used as the indexes of photosynthesis
capacity, and thereby plant source strength and yield potential of
a plant. On the other hand, the seed dry weight, silique dry weight
and silique length are used as the indexes for plant sink strength,
which are considered as the direct determinants of yield. As
demonstrated from the model plant screen, embodiments of transgenic
plants with trait-improving recombinant DNA identified in the late
development screen can grow better and/or have improved development
during leaf development and seed maturation providing a higher
yield potential as compared to control plants.
[0123] LL-Improvement of tolerance to shade stress identified in a
low light screen: The effects of light on plant development are
especially prominent at the seedling stage. Under normal light
conditions with unobstructed direct light, a plant seeding develops
according to a characteristic photomorphogenic pattern, in which
plants have open and expanded cotyledons and short hypocotyls. Then
the plant's energy is devoted to cotyledon and leaf development
while longitudinal extension growth is minimized. Under low light
condition where light quality and intensity are reduced by shading,
obstruction or high population density, a seedling displays a
shade-avoidance pattern, in which the seedling displays a reduced
cotyledon expansion, and hypocotyls extension is greatly increased.
As the result, a plant under low light condition increases
significantly its stem length at the expanse of leaf, seed or fruit
and storage organ development, thereby adversely affecting of
yield. The present invention provides recombinant DNA that enable
plants to have an attenuated shade avoidance response so that the
source of plant can be contributed to reproductive growth
efficiently, resulting higher yield as compared to the wild type
plants. As demonstrated from the model plant screen, embodiments of
transgenic plants with trait-improving recombinant DNA identified
in a shade stress tolerance screen can have attenuated shade
response under shade conditions providing a higher yield potential
as compared to control plants. The transgenic plants generated by
the present invention may be suitable for a higher density
planting, thereby resulting increased yield per unit area.
[0124] LN-Improvement of Tolerance to Low Nitrogen Availability
Stress
[0125] Nitrogen is a key factor in plant growth and crop yield. The
metabolism, growth and development of plants are profoundly
affected by their nitrogen supply. Restricted nitrogen supply
alters shoot to root ratio, root development, activity of enzymes
of primary metabolism and the rate of senescence (death) of older
leaves. All field crops have a fundamental dependence on inorganic
nitrogenous fertilizer. Since fertilizer is rapidly depleted from
most soil types, it must be supplied to growing crops two or three
times during the growing season. Enhanced nitrogen use efficiency
by plants should enable crops cultivated under low nitrogen
availability stress condition resulted from low fertilizer input or
poor soil quality.
[0126] According to the present invention, transgenic plants
generated using the recombinant nucleotides, which confer enhanced
nitrogen use efficiency, identified as such in Table 3, exhibit one
or more desirable traits including, but not limited to, increased
seedling weight, greener leaves, increased number of rosette
leaves, increased or decreased root length. One skilled in the art
may recognize that the transgenic plants provided by the present
invention with enhanced nitrogen use efficiency may also have
altered amino acid or protein compositions, increased yield and/or
better seed quality. The transgenic plants of the present invention
may be productively cultivated under low nitrogen growth
conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer
inputs, which would cause the growth of wild type plants to cease
or to be so diminished as to make the wild type plants practically
useless. The transgenic plants also may be advantageously used to
achieve earlier maturing, faster growing, and/or higher yielding
crops and/or produce more nutritious foods and animal feedstocks
when cultivated using nitrogen non-limiting growth conditions.
[0127] Stacked Traits: The present invention also encompasses
transgenic plants with stacked engineered traits, e.g., a crop
having an improved phenotype resulting from expression of a
trait-improving recombinant DNA, in combination with herbicide
and/or pest resistance traits. For example, genes of the current
invention can be stacked with other traits of agronomic interest,
such as a trait providing herbicide resistance, for example a
RoundUp Ready.RTM. trait, or insect resistance, such as using a
gene from Bacillus thuringensis to provide resistance against
lepidopteran, coliopteran, homopteran, hemiopteran, and other
insects. Herbicides for which resistance is useful in a plant
include glyphosate herbicides, phosphinothricin herbicides, oxynil
herbicides, imidazolinone herbicides, dinitroaniline herbicides,
pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides,
sulfonamide herbicides and gluphosinate herbicides. To illustrate
that the production of transgenic plants with herbicide resistance
is a capability of those of ordinary skill in the art, reference is
made to U.S. patent application publications 2003/0106096A1 and
2002/0112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549
and 6,376,754, all of which are incorporated herein by reference.
To illustrate that the production of transgenic plants with pest
resistance is a capability of those of ordinary skill in the art
reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which
disclose plants expressing an endotoxin of Bacillus thuringiensis
bacteria, to U.S. Pat. No. 6,506,599 which discloses control of
invertebrates which feed on transgenic plants which express dsRNA
for suppressing a target gene in the invertebrate, to U.S. Pat. No.
5,986,175 which discloses the control of viral pests by transgenic
plants which express viral replicase, and to U.S. Patent
Application Publication 2003/0150017 A1 which discloses control of
pests by a transgenic plant which express a dsRNA targeted to
suppressing a gene in the pest, all of which are incorporated
herein by reference.
[0128] Once one recombinant DNA has been identified as conferring
an improved trait of interest in transgenic Arabidopsis plants,
several methods are available for using the sequence of that
recombinant DNA and knowledge about the protein it encodes to
identify homologs of that sequence from the same plant or different
plant species or other organisms, e.g., bacteria and yeast. Thus,
in one aspect, the invention provides methods for identifying a
homologous gene with a DNA sequence homologous to any of SEQ ID NO:
1 through SEQ ID NO: 197, or a homologous protein with an amino
acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID
NO: 394. In another aspect, the present invention provides the
protein sequences of identified homologs for a sequence listed as
SEQ ID NO: 395 through SEQ ID NO: 19938. In yet another aspect, the
present invention also includes linking or associating one or more
desired traits, or gene function with a homolog sequence provided
herein.
[0129] The trait-improving recombinant DNA and methods of using
such trait-improving recombinant DNA for generating transgenic
plants with improved traits provided by the present invention are
not limited to any particular plant species. Indeed, the plants
according to the present invention may be of any plant species,
i.e., may be monocotyledonous or dicotyledonous. Preferably, they
will be agricultural useful plants, i.e., plants cultivated by man
for purposes of food production or technical, particularly
industrial applications. Of particular interest in the present
invention are corn and soybean plants. The recombinant DNA
constructs optimized for soybean transformation and recombinant DNA
constructs optimized for corn transformation are provided by the
present invention. Other plants of interest in the present
invention for production of transgenic plants having improved
traits include, without limitation, cotton, canola, wheat,
sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit
and vegetable crops, and turfgrass.
[0130] In certain embodiments, the present invention contemplates
to use an orthologous gene in generating the transgenic plants with
similarly improved traits as the transgenic Arabidopsis
counterpart. Improved physiological properties in transgenic plants
of the present invention may be confirmed in responses to stress
conditions, for example in assays using imposed stress conditions
to detect improved responses to drought stress, nitrogen
deficiency, cold growing conditions, or alternatively, under
naturally present stress conditions, for example under field
conditions. Biomass measures may be made on greenhouse or field
grown plants and may include such measurements as plant height,
stem diameter, root and shoot dry weights, and, for corn plants,
ear length and diameter.
[0131] Trait data on morphological changes may be collected by
visual observation during the process of plant regeneration as well
as in regenerated plants transferred to soil. Such trait data
includes characteristics such as normal plants, bushy plants,
taller plants, thicker stalks, narrow leaves, striped leaves,
knotted phenotype, chlorosis, albino, anthocyanin production, or
altered tassels, ears or roots. Other enhanced traits may be
identified by measurements taken under field conditions, such as
days to pollen shed, days to silking, leaf extension rate,
chlorophyll content, leaf temperature, stand, seedling vigor,
internode length, plant height, leaf number, leaf area, tillering,
brace roots, stay green, stalk lodging, root lodging, plant health,
barreness/prolificacy, green snap, and pest resistance. In
addition, trait characteristics of harvested grain may be
confirmed, including number of kernels per row on the ear, number
of rows of kernels on the ear, kernel abortion, kernel weight,
kernel size, kernel density and physical grain quality.
[0132] To confirm hybrid yield in transgenic corn plants expressing
genes of the present invention, it may be desirable to test hybrids
over multiple years at multiple locations in a geographical
location where maize is conventionally grown, e.g., in Iowa,
Illinois or other locations in the Midwestern United States, under
"normal" field conditions as well as under stress conditions, e.g.,
under drought or population density stress.
[0133] Transgenic plants can be used to provide plant parts
according to the invention for regeneration or tissue culture of
cells or tissues containing the constructs described herein. Plant
parts for these purposes can include leaves, stems, roots, flowers,
tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen,
ovaries, cells and protoplasts, or any other portion of the plant
which can be used to regenerate additional transgenic plants,
cells, protoplasts or tissue culture. Seeds of transgenic plants
are provided by this invention can be used to propagate more plants
containing the trait-improving recombinant DNA constructs of this
invention. These descendants are intended to be included in the
scope of this invention if they contain a trait-improving
recombinant DNA construct of this invention, whether or not these
plants are selfed or crossed with different varieties of
plants.
[0134] The various aspects of the invention are illustrated by
means of the following examples which are in no way intended to
limit the full breath and scope of claims.
EXAMPLES
Example 1
Identification of Recombinant DNA that Confers Improved Trait(s) to
Plants
A. Plant Expression Constructs for Arabidopsis Transformation
[0135] Each gene of interest was amplified from a genomic or cDNA
library using primers specific to sequences upstream and downstream
of the coding region. Transformation vectors were prepared to
constitutively transcribe DNA in either sense orientation (for
enhanced protein expression) or anti-sense orientation (for
endogenous gene suppression) under the control of an enhanced
Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142)
directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer,
e.g., Genetics 149: 633-639, 1998; International patent application
NO. PCT/EP98/07577). The transformation vectors also contain a bar
gene as a selectable marker for resistance to glufosinate
herbicide. The transformation of Arabidopsis plants was carried out
using the vacuum infiltration method known in the art (Bethtold,
e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the
plants, named as T1 seeds, were subsequently grown in a
glufosinate-containing selective medium to select for plants which
were actually transformed and which produced T2 transgenic
seed.
B. Soil Drought Tolerance Screen
[0136] This example describes a soil drought tolerance screen to
identify Arabidopsis plants transformed with recombinant DNA that
wilt less rapidly and/or produce higher seed yield when grown in
soil under drought conditions
[0137] T2 seeds were sown in flats filled with Metro/Mix.RTM. 200
(The Scotts.RTM. Company, USA). Humidity domes were added to each
flat and flats were assigned locations and placed in
climate-controlled growth chambers. Plants were grown under a
temperature regime of 22.degree. C. at day and 20.degree. C. at
night, with a photoperiod of 16 hours and average light intensity
of 170 .mu.mol/m.sup.2/s. After the first true leaves appeared,
humidity domes were removed. The plants were sprayed with
glufosinate herbicide and put back in the growth chamber for 3
additional days. Flats were watered for 1 hour the week following
the herbicide treatment. Watering was continued every seven days
until the flower bud primordia became apparent, at which time
plants were watered for the last time.
[0138] To identify drought tolerant plants, plants were evaluated
for wilting response and seed yield. Beginning ten days after the
last watering, plants were examined daily until 4 plants/line had
wilted. In the next six days, plants were monitored for wilting
response. Five drought scores were assigned according to the visual
inspection of the phenotypes: 1 for healthy, 2 for dark green, 3
for wilting, 4 severe wilting, and 5 for dead. A score of 3 or
higher was considered as wilted.
[0139] At the end of this assay, seed yield measured as seed weight
per plant under the drought condition was characterized for the
transgenic plants and their controls and analyzed as a quantitative
response according to example 1M.
[0140] Two approaches were used for statistical analysis on the
wilting response. First, the risk score was analyzed for wilting
phenotype and treated as a qualitative response according to the
example 1L. Alternatively, the survival analysis was carried out in
which the proportions of wilted and non-wilted transgenic and
control plants were compared over each of the six days under
scoring and an overall log rank test was performed to compare the
two survival curves using S-PLUS statistical software (S-PLUS 6,
Guide to statistics, Insightful, Seattle, Wash., USA). A list of
recombinant DNA constructs which improve drought tolerance in
transgenic plants is illustrated in Table 4.
TABLE-US-00003 TABLE 4 Drought Seed Time to wilting PEP score yield
Risk SEQ Construct Nomination Delta P- Delta P- score P- ID NO ID
ID Orientation mean value mean value mean value 198 11029 CGPG106
SENSE 0.109 0.381 0.072 0.717 0.119 1.000 292 73136 CGPG5764 SENSE
0.030 0.090 0.136 0.067 0.104 1.000 284 73165 CGPG5661 SENSE -0.031
0.504 0.526 0.018 -0.075 1.000 260 73628 CGPG5025 SENSE 0.342 0.038
-0.474 0.022 0.208 1.000 291 73981 CGPG5757 SENSE 0.517 0.026
-0.096 0.000 0.573 1.000 207 74065 CGPG1828 ANTI- -0.078 0.164
0.646 0.027 0.048 1.000 SENSE 263 74217 CGPG5144 SENSE -0.026 0.414
0.418 0.032 0.071 1.000 261 76105 CGPG5041 SENSE 0.288 0.029 -1.780
0.027 0.173 1.000 328 76576 CGPG7281 SENSE 0.241 0.032 -0.555 0.442
0.212 1.000 327 77536 CGPG7272 SENSE 0.089 0.194 1.059 0.001 0.042
1.000 226 19767 CGPG3918 SENSE 0.117 0.038 0.164 0.296 / / 237
19947 CGPG4069 SENSE -0.009 0.852 -0.104 0.504 / / 242 70994
CGPG4122 SENSE 0.040 0.021 -0.131 0.447 / / 363 77103 CGPG9134
SENSE 0.145 0.023 -0.499 0.096 / /
[0141] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference (p value, of the delta of
a quantitative response or of the risk score of a qualitative
response, is the probability that the observed difference between
the transgenic plants and the reference occur by chance)
If p<0.2 and delta or risk score mean >0, the transgenic
plants showed a trend of trait improvement as compared to the
reference.
[0142] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed
improved drought tolerance evidenced by the second criteria as
illustrated in Example 1L and 1M.
C. Heat Stress Tolerance Screen
[0143] Under high temperatures, Arabidopsis seedlings become
chlorotic and root growth is inhibited. This example sets forth the
heat stress tolerance screen to identify Arabidopsis plants
transformed with the gene of interest that are more resistant to
heat stress based on primarily their seedling weight and root
growth under high temperature. T2 seeds were plated on 1/2.times.MS
salts, 1% phytagel, with 10 .mu.g/ml BASTA (7 per plate with 2
control seeds; 9 seeds total per plate). Plates were placed at
4.degree. C. for 3 days to stratify seeds. Plates were then
incubated at room temperature for 3 hours and then held vertically
for 11 additional days at temperature of 34.degree. C. at day and
20.degree. C. at night. Photoperiod was 16 h. Average light
intensity was .about.140 .mu.mol/m.sup.2/s. After 14 days of
growth, plants were scored for glufosinate resistance, root length,
final growth stage, visual color, and seedling fresh weight. A
photograph of the whole plate was taken on day 14.
[0144] The seedling weight and root length were analyzed as
quantitative responses according to example 1M. The final grow
stage at day 14 was scored as success if 50% of the plants had
reached 3 rosette leaves and size of leaves are greater than 1 mm
(Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth
stage data was analyzed as a qualitative response according to
example 1L. A list of recombinant DNA constructs that improve heat
tolerance in transgenic plants illustrated in Table 5.
TABLE-US-00004 TABLE 5 Seedling Root length Growth stage weight at
at day 14 at day 14 day 14 PEP Construct Nomination Delta Risk
score Delta SEQ ID ID ID Orientation mean P-value mean P-value mean
P-value 239 70985 CGPG4088 SENSE 0.480 0.036 1.176 0.196 1.333
0.018 233 70987 CGPG4048 SENSE 0.228 0.021 -0.036 0.670 1.214 0.002
268 72001 CGPG5221 SENSE 0.273 0.004 0.210 0.330 1.393 0.009 274
72753 CGPG5540 SENSE 0.434 0.006 0.414 0.258 1.100 0.006 276 73954
CGPG5577 SENSE 0.406 0.002 0.455 0.282 1.274 0.000 325 76178
CGPG7225 SENSE 0.714 0.001 1.093 0.145 1.537 0.000 361 77150
CGPG9130 SENSE 0.219 0.032 0.178 0.237 1.159 0.002 367 77164
CGPG9147 SENSE 0.317 0.026 0.651 0.099 1.152 0.002 390 77432
CGPG9335 SENSE 0.581 0.001 0.457 0.124 1.551 0.000 351 77587
CGPG8107 SENSE 0.488 0.006 0.628 0.072 1.328 0.001 348 77928
CGPG8082 SENSE 0.462 0.032 0.191 0.301 1.498 0.004 212 16610
CGPG2499 SENSE 0.055 0.640 0.109 0.609 0.983 0.020 220 18256
CGPG3363 SENSE 0.070 0.138 0.071 0.541 1.021 0.004 222 19193
CGPG3375 SENSE 0.255 0.094 0.267 0.368 1.023 0.003 252 70684
CGPG4588 SENSE 0.010 0.781 0.003 0.979 1.002 0.045 230 70948
CGPG3990 SENSE 0.282 0.095 0.351 0.335 1.045 0.023 243 70956
CGPG4140 SENSE 0.155 0.313 0.091 0.320 1.129 0.018 235 70962
CGPG4057 SENSE 0.469 0.085 0.267 0.508 1.410 0.007 241 70963
CGPG4121 SENSE -0.002 0.979 -0.131 0.311 0.976 0.005 242 70994
CGPG4122 SENSE 0.140 0.574 1.204 0.096 1.207 0.030 244 70995
CGPG4154 SENSE 0.374 0.112 1.640 0.171 1.328 0.013 208 72783
CGPG2206 SENSE 0.387 0.057 0.053 0.509 1.013 0.019 255 72807
CGPG4912 SENSE 0.357 0.071 0.443 0.020 1.099 0.009 259 72825
CGPG5001 SENSE 0.171 0.349 0.213 0.234 1.118 0.012 287 73120
CGPG5704 SENSE 0.096 0.490 0.193 0.364 1.080 0.033 280 73127
CGPG5640 SENSE -0.220 0.077 -0.194 0.030 0.643 0.014 293 73172
CGPG5783 SENSE 0.283 0.197 0.887 0.162 1.154 0.007 310 73411
CGPG6440 SENSE 0.198 0.389 0.631 0.345 1.320 0.003 260 73628
CGPG5025 SENSE 0.406 0.149 1.475 0.365 1.169 0.016 316 74566
CGPG6796 SENSE 0.627 0.092 0.777 0.466 1.714 0.014 338 75654
CGPG7804 SENSE 0.292 0.237 0.804 0.315 0.940 0.017 340 75657
CGPG7828 SENSE 0.390 0.067 1.222 0.307 1.521 0.011 213 76602
CGPG2653 SENSE 0.036 0.748 0.092 0.670 0.752 0.025 372 77180
CGPG9180 SENSE 0.068 0.555 -0.045 0.177 0.871 0.022 380 77275
CGPG9236 SENSE 0.282 0.125 0.971 0.285 1.027 0.013 297 77312
CGPG5927 SENSE 0.201 0.344 0.710 0.372 1.039 0.032 320 77518
CGPG6953 SENSE 0.238 0.344 0.069 0.655 1.188 0.015 342 77549
CGPG7933 SENSE 0.184 0.180 -0.012 / 0.978 0.007 347 77580 CGPG8062
SENSE 0.157 0.143 -0.074 / 0.891 0.049 317 77610 CGPG6805 SENSE
0.023 0.937 -0.063 / 1.207 0.029 318 77618 CGPG6810 SENSE 0.002
0.942 -0.074 / 1.015 0.006 353 77619 CGPG8152 SENSE 0.213 0.316
-0.076 0.457 0.956 0.019 355 77629 CGPG8377 SENSE 0.095 0.124
-0.063 / 1.133 0.005 334 77816 CGPG7529 SENSE 0.033 0.705 0.856
0.385 0.611 0.038
[0145] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta or
risk score mean >0, the transgenic plants showed a trend of
trait improvement as compared to the reference.
[0146] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349,
366, or 387 showed improved heat stress tolerance evidenced by the
second criteria as illustrated in Example 1L and 1M.
D. Salt Stress Tolerance Screen
[0147] This example sets forth the high salinity stress screen to
identify Arabidopsis plants transformed with the gene of interest
that are tolerant to high levels of salt based on their rate of
development, root growth and chlorophyll accumulation under high
salt conditions.
[0148] T2 seeds were plated on glufosinate selection plates
containing 90 mM NaCl and grown under standard light and
temperature conditions. All seedlings used in the experiment were
grown at a temperature of 22.degree. C. at day and 20.degree. C. at
night, a 16-hour photoperiod, an average light intensity of
approximately 120 umol/m.sup.2. On day 11, plants were measured for
primary root length. After 3 more days of growth (day 14), plants
were scored for transgenic status, primary root length, growth
stage, visual color, and the seedlings were pooled for fresh weight
measurement. A photograph of the whole plate was also taken on day
14.
[0149] The seedling weight and root length were analyzed as
quantitative responses according to example 1M. The final growth
stage at day 14 was scored as success if 50% of the plants reached
3 rosette leaves and size of leaves are greater than 1 mm (Boyes,
D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth
stage data was analyzed as a qualitative response according to
example 1L. A list of recombinant DNA constructs that improve high
salinity tolerance in transgenic plants illustrated in Table 6.
TABLE-US-00005 TABLE 6 Growth Seedling Root length Root length
stage at weight at at day 11 at day 14 day 14 day 14 PEP Construct
Delta Delta Delta Delta SEQ ID ID Orientation mean P-value mean
P-value mean P-value mean P-value 220 18256 SENSE 0.192 0.232 0.236
0.080 1.291 0.151 0.598 0.039 214 18456 SENSE 0.213 0.089 0.209
0.197 2.062 0.197 0.476 0.046 232 19973 SENSE 0.289 0.012 0.299
0.002 0.976 0.054 1.002 0.013 231 70201 SENSE 0.363 0.030 0.403
0.052 2.083 0.229 1.057 0.014 243 70956 SENSE 0.006 0.923 0.128
0.016 -0.045 NA -0.364 0.371 238 70969 SENSE 0.435 0.091 0.471
0.027 0.653 0.473 0.994 0.058 240 70971 SENSE 0.337 0.063 0.257
0.041 1.418 0.226 0.593 0.030 233 70987 SENSE 0.363 0.030 0.284
0.044 1.434 0.380 0.865 0.012 273 72788 SENSE 0.484 0.019 0.456
0.016 1.813 0.314 0.929 0.008 256 72811 SENSE 0.385 0.024 0.409
0.002 2.968 0.031 0.805 0.004 287 73120 SENSE 0.341 0.001 0.244
0.019 0.613 0.103 0.755 0.015 280 73127 SENSE 0.501 0.028 0.395
0.055 0.212 0.175 0.739 0.023 289 73134 SENSE 0.226 0.134 0.266
0.045 0.152 0.487 0.378 0.056 257 73235 SENSE 0.431 0.020 0.360
0.018 1.618 0.327 0.650 0.009 245 73306 SENSE 0.228 0.046 0.219
0.018 0.249 0.217 0.708 0.008 253 73330 SENSE 0.196 0.225 0.186
0.029 0.000 NA 0.543 0.045 316 74566 SENSE 0.331 0.001 0.076 0.422
0.597 0.031 0.716 0.003 312 74688 SENSE 0.480 0.076 0.415 0.031
0.000 NA 0.808 0.062 376 77124 SENSE 0.340 0.034 0.322 0.015 2.306
0.137 0.411 0.168 364 77139 SENSE 0.339 0.096 0.287 0.041 1.683
0.294 0.602 0.039 375 77195 SENSE 0.445 0.009 0.345 0.010 0.367
0.185 0.582 0.022 384 77268 SENSE 0.495 0.047 0.468 0.102 1.906
0.079 0.992 0.042 386 77269 SENSE 0.454 0.038 0.426 0.004 1.996
0.065 1.136 0.001 380 77275 SENSE 0.457 0.008 0.487 0.040 2.119
0.154 1.070 0.010 297 77312 SENSE 0.335 0.086 0.361 0.013 0.960
0.208 0.723 0.063 389 77430 SENSE 0.303 0.030 0.360 0.004 0.131
0.495 0.894 0.022 320 77518 SENSE 0.171 0.017 0.185 0.026 -0.134
0.443 0.339 0.109 345 77570 SENSE 0.326 0.091 0.241 0.134 1.396
0.090 0.862 0.026 351 77587 SENSE 0.178 0.083 0.096 0.665 0.995
0.426 0.632 0.036 353 77619 SENSE 0.389 0.058 0.398 0.011 1.913
0.257 0.857 0.040 355 77629 SENSE 0.404 0.043 0.389 0.019 2.523
0.083 0.826 0.040 336 77821 SENSE 0.393 0.063 0.444 0.033 0.196
0.228 0.619 0.015 356 77832 SENSE 0.134 0.238 0.304 0.011 0.914
0.156 0.413 0.050 352 77933 SENSE 0.112 0.044 0.165 0.009 0.749
0.482 0.517 0.171 347 77580 SENSE -0.019 0.814 0.087 0.018 -0.158
0.151 0.124 0.204
[0150] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta or
risk score mean >0, the transgenic plants showed a trend of
trait improvement as compared to the reference.
[0151] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327,
330, 349, 366, or 387 showed improved salt stress tolerance
evidenced by the second criteria as illustrated in Example 1L.
E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance
Screen
[0152] There are numerous factors, which can influence seed
germination and subsequent seedling growth, one being the
availability of water. Genes, which can directly affect the success
rate of germination and early seedling growth, are potentially
useful agronomic traits for improving the germination and growth of
crop plants under drought stress. In this assay, PEG was used to
induce osmotic stress on germinating transgenic lines of
Arabidopsis thaliana seeds in order to screen for osmotically
resistant seed lines.
[0153] T2 seeds were plated on BASTA selection plates containing 3%
PEG and grown under standard light and temperature conditions.
Seeds were plated on each plate containing 3% PEG, 1/2.times.MS
salts, 1% phytagel, and 10 .mu.g/ml glufosinate. Plates were placed
at 4.degree. C. for 3 days to stratify seeds. On day 11, plants
were measured for primary root length. After 3 more days of growth,
i.e., at day 14, plants were scored for transgenic status, primary
root length, growth stage, visual color, and the seedlings were
pooled for fresh weight measurement. A photograph of the whole
plate was taken on day 14.
[0154] Seedling weight and root length were analyzed as
quantitative responses according to example 1M. The final growth
stage at day 14 was scored as success or failure based on whether
the plants reached 3 rosette leaves and size of leaves are greater
than 1 mm. The growth stage data was analyzed as a qualitative
response according to example 1L. A list of recombinant DNA
constructs that improve osmotic stress tolerance in transgenic
plants illustrated in Table 7.
TABLE-US-00006 TABLE 7 Growth Seedling Root length Root length
stage at weight at at day 11 at day 14 day 14 day 14 PEP Construct
Delta P- Delta P- Delta P- Delta P- SEQ ID ID Orientation mean
value mean value mean value mean value 199 12223 SENSE / / / / / /
0.353 0.024 214 18456 SENSE 0.320 0.018 0.274 0.033 3.109 0.073
0.553 0.006 302 70812 SENSE 0.195 0.078 0.078 0.296 4 0.000 0.363
0.016 234 70950 SENSE 0.322 0.056 0.427 0.016 4 0.000 0.381 0.127
239 70985 SENSE 0.270 0.066 0.221 0.005 4 0.000 0.372 0.007 233
70987 SENSE 0.227 0.205 0.128 0.470 4 0.000 0.495 0.000 228 70992
SENSE 0.369 0.002 0.370 0.013 4 0.000 0.574 0.027 244 70995 SENSE
0.379 0.049 0.320 0.065 4 0.000 0.491 0.003 208 72783 SENSE 0.154
0.084 0.043 0.553 4 0.000 0.264 0.044 255 72807 SENSE 0.165 0.080
0.158 0.150 4 0.000 0.333 0.047 256 72811 SENSE 0.489 0.052 0.491
0.050 4 0.000 0.784 0.016 259 72825 SENSE 0.423 0.055 0.388 0.080
2.741 0.161 0.608 0.020 295 72946 SENSE 0.345 0.053 0.473 0.050
0.898 0.632 0.415 0.181 286 73026 SENSE 0.636 0.021 0.619 0.028
2.741 0.161 0.752 0.045 290 73123 SENSE 0.460 0.048 0.445 0.007 NA
NA 0.609 0.069 278 73161 SENSE 0.510 0.024 0.506 0.006 2.624 0.246
0.626 0.073 284 73165 SENSE 0.304 0.101 0.285 0.044 2.499 0.270
0.643 0.077 293 73172 SENSE 0.197 0.056 0.137 0.204 2.374 0.293
0.660 0.081 262 73242 SENSE 0.305 0.029 0.332 0.022 2.249 0.317
0.677 0.085 265 73256 SENSE 0.325 0.084 0.182 0.178 2.124 0.341
0.694 0.089 308 73485 SENSE 0.133 0.070 0.039 0.583 1.999 0.365
0.711 0.093 291 73981 SENSE 0.239 0.147 0.238 0.281 1.874 0.389
0.728 0.096 316 74566 SENSE 0.451 0.004 0.248 0.118 1.749 0.412
0.745 0.100 312 74688 SENSE 0.190 0.191 0.102 0.520 1.624 0.436
0.762 0.104 330 74863 SENSE 0.174 0.164 0.216 0.109 1.499 0.460
0.779 0.108 366 77140 SENSE 0.644 0.005 0.572 0.015 1.374 0.484
0.796 0.112 361 77150 SENSE 0.313 0.047 0.359 0.044 1.249 0.508
0.813 0.116 365 77187 SENSE 0.273 0.084 0.032 0.763 1.124 0.532
0.830 0.120 375 77195 SENSE 0.319 0.021 0.256 0.041 NA NA 0.847
0.124 379 77203 SENSE 0.113 0.485 0.019 0.829 0.999 0.555 0.864
0.127 377 77261 SENSE 0.115 0.230 0.085 0.315 0.874 0.579 0.881
0.131 270 77308 SENSE 0.174 0.321 0.074 0.700 0.749 0.603 0.898
0.135 346 77338 SENSE 0.227 0.158 0.196 0.272 0.624 0.627 0.915
0.139 350 77357 SENSE 0.169 0.300 0.259 0.066 2.715 0.169 0.303
0.020 390 77432 SENSE 0.306 0.043 0.122 0.270 2.570 0.214 0.734
0.019 347 77580 SENSE 0.300 0.107 0.240 0.009 2.288 0.119 0.500
0.179 353 77619 SENSE 0.142 0.216 0.139 0.002 4 0.000 0.129 0.582
356 77832 SENSE 0.236 0.006 0.266 0.001 1.333 0.435 0.286 0.037 343
77917 SENSE 0.174 0.163 0.160 0.006 4 0.000 0.330 0.009
[0155] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference.
If p<0.2 and delta or risk score mean >0, the transgenic
plants showed a trend of trait improvement as compared to the
reference.
[0156] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310,
321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic
stress tolerance evidenced by the second criteria as illustrated in
Example 1L and 1M.
F. Cold Shock Tolerance Screen
[0157] This example set forth a screen to identify Arabidopsis
plants transformed with the genes of interest that are more
tolerant to cold stress subjected during day 8 to day 28 after seed
planting. During these crucial early stages, seedling growth and
leaf area increase were measured to assess tolerance when
Arabidopsis seedlings were exposed to low temperatures. Using this
screen, genetic alterations can be found that enable plants to
germinate and grow better than wild type plants under sudden
exposure to low temperatures.
[0158] Eleven seedlings from T2 seeds of each transgenic line plus
one control line were plated together on a plate containing
1/2.times. Gamborg Salts with 0.8 Phytagel.TM., 1% Phytagel, and
0.3% Sucrose. Plates were then oriented horizontally and stratified
for three days at 4.degree. C. At day three, plates were removed
from stratification and exposed to standard conditions (16 hr
photoperiod, 22.degree. C. at day and 20.degree. C. at night) until
day 8. At day eight, plates were removed from standard conditions
and exposed to cold shock conditions (24 hr photoperiod, 8.degree.
C. at both day and night) until the final day of the assay, i.e.,
day 28. Rosette areas were measured at day 8 and day 28, which were
analyzed as quantitative responses according to example 1M. A list
of recombinant nucleotides that improve cold shock stress tolerance
in plants illustrated in Table 8.
TABLE-US-00007 TABLE 8 Rosette area Rosette area Rosette area at
day 8 at day 28 difference PEP Construct Nomination Delta Risk
score Delta SEQ ID ID ID Orientation mean P-alue mean P-value mean
P-value 199 12223 CGPG1133 SENSE 0.112 0.617 0.334 0.014 0.273
0.143 218 18231 CGPG3274 SENSE -0.131 0.269 1.144 0.002 1.179 0.003
215 18414 CGPG3002 SENSE 0.997 0.034 1.665 0.001 1.895 0.001 214
18456 CGPG2813 SENSE 0.099 0.757 0.799 0.001 0.684 0.001 233 70987
CGPG4048 SENSE 0.864 0.136 1.213 0.010 1.146 0.002 244 70995
CGPG4154 SENSE 1.088 0.008 1.262 0.023 1.407 0.025 208 72783
CGPG2206 SENSE 0.814 0.023 0.957 0.018 0.899 0.052 273 72788
CGPG5535 SENSE 0.262 0.058 1.090 0.016 1.212 0.021 256 72811
CGPG4926 SENSE 0.880 0.058 0.669 0.002 0.715 0.018 259 72825
CGPG5001 SENSE 0.032 0.887 0.338 0.071 0.357 0.027 281 73033
CGPG5646 SENSE 0.322 0.394 1.311 0.012 1.480 0.017 293 73172
CGPG5783 SENSE 0.395 0.209 1.065 0.030 1.099 0.074 253 73330
CGPG4765 SENSE 0.347 0.515 0.835 0.065 1.043 0.007 264 73735
CGPG5171 SENSE 0.624 0.076 0.655 0.001 0.718 0.005 315 74460
CGPG6747 SENSE 0.452 0.005 0.542 0.043 0.455 0.154 296 74746
CGPG5856 SENSE 0.304 0.346 0.978 0.001 1.061 0.007 299 75240
CGPG5957 SENSE 0.479 0.153 0.652 0.143 0.875 0.039 338 75654
CGPG7804 SENSE 0.531 0.140 1.712 0.010 1.979 0.015 339 75692
CGPG7823 SENSE 0.526 0.063 2.027 0.001 2.246 0.001 323 76161
CGPG7168 SENSE 0.768 0.000 1.810 0.004 2.146 0.003 217 76532
CGPG3235 SENSE 0.921 0.008 1.216 0.001 1.324 0.002 357 76802
CGPG8987 SENSE 0.468 0.097 1.578 0.000 1.872 0.000 358 76829
CGPG9013 SENSE -0.434 0.329 0.709 0.010 0.314 0.118 373 77121
CGPG9183 SENSE 0.081 0.385 0.757 0.023 0.698 0.014 366 77140
CGPG9145 SENSE -0.221 0.165 0.896 0.007 1.043 0.005 374 77157
CGPG9186 SENSE 0.190 0.566 1.110 0.016 1.176 0.015 369 77165
CGPG9155 SENSE 1.573 0.043 0.683 0.038 0.699 0.022 370 77166
CGPG9163 SENSE 0.777 0.011 1.432 0.016 1.570 0.025 381 77204
CGPG9238 SENSE 0.545 0.139 1.313 0.003 1.436 0.006 391 77433
CGPG9341 SENSE 1.054 0.103 0.895 0.027 0.659 0.084 345 77570
CGPG8015 SENSE 0.538 0.215 0.866 0.025 0.898 0.023 353 77619
CGPG8152 SENSE -0.589 0.322 0.503 0.068 0.558 0.049 354 77621
CGPG8166 SENSE 0.053 0.863 0.716 0.044 0.792 0.040 203 13411
CGPG1301 SENSE 0.183 0.427 0.577 0.033 0.573 0.031 209 17210
CGPG2217 SENSE -0.244 0.685 0.162 0.038 0.156 0.104 220 18256
CGPG3363 SENSE 0.272 0.400 0.639 0.008 0.719 0.020 289 73134
CGPG5721 SENSE 0.014 0.947 1.053 0.000 1.062 0.025 263 74217
CGPG5144 SENSE 0.168 0.390 0.261 0.043 0.206 0.075 379 77203
CGPG9230 SENSE 0.606 0.051 0.620 0.032 0.778 0.039
[0159] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference (p value, of the delta of
a quantitative response or of the risk score of a qualitative
response, is the probability that the observed difference between
the transgenic plants and the reference occur by chance)
If p<0.2 and delta or risk score mean >0, the transgenic
plants showed a trend of trait improvement as compared to the
reference.
[0160] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved
cold stress tolerance evidenced by the second criterial as
illustrated in Example 1L.
G. Cold Germination Tolerance Screen
[0161] This example sets forth a screen to identify Arabidopsis
plants transformed with the genes of interests are resistant to
cold stress based on their rate of development, root growth and
chlorophyll accumulation under low temperature conditions.
T2 seeds were plated and all seedlings used in the experiment were
grown at 8.degree. C. Seeds were first surface disinfested using
chlorine gas and then seeded on assay plates containing an aqueous
solution of 1/2.times. Gamborg's B/5 Basal Salt Mixture
(Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel.TM.
(Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH
adjusted to 5.8 using KOH. Test plates were held vertically for 28
days at a constant temperature of 8.degree. C., a photoperiod of 16
hr, and average light intensity of approximately 100
umol/m.sup.2/s. At 28 days post plating, root length was measured,
growth stage was observed, the visual color was assessed, and a
whole plate photograph was taken.
[0162] The root length at day 28 was analyzed as a quantitative
response according to example 1M. The growth stage at day 7 was
analyzed as a qualitative response according to example 1L. A list
of recombinant DNA constructs that improve cold stress tolerance in
transgenic plants illustrated in Table 9.
TABLE-US-00008 TABLE 9 Root length Growth stage at day 28 at day 28
PEP Construct Nomination Delta Delta SEQ ID ID ID Orientation mean
P-value mean P-value 209 17210 CGPG2217 SENSE 0.164 0.089 4.000
0.000 219 18232 CGPG3275 SENSE -0.081 0.613 4.000 0.000 226 19767
CGPG3918 SENSE 0.212 0.007 4.000 0.000 227 19774 CGPG3920 SENSE
0.140 0.011 4.000 0.000 237 19947 CGPG4069 SENSE / / 4.000 0.000
231 70201 CGPG3994 SENSE 0.311 0.010 4.000 0.000 252 70684 CGPG4588
SENSE 0.335 0.209 4.000 0.000 236 70915 CGPG4058 SENSE 0.231 0.258
4.000 0.000 243 70956 CGPG4140 SENSE 0.113 0.525 4.000 0.000 241
70963 CGPG4121 SENSE 0.210 0.252 4.000 0.000 239 70985 CGPG4088
SENSE 0.283 0.063 4.000 0.000 233 70987 CGPG4048 SENSE 0.392 0.202
4.000 0.000 228 70992 CGPG3962 SENSE 0.008 0.959 4.000 0.000 244
70995 CGPG4154 SENSE 0.426 0.002 4.000 0.000 268 72001 CGPG5221
SENSE 0.070 0.728 4.000 0.000 274 72753 CGPG5540 SENSE 0.189 0.010
2.889 0.121 273 72788 CGPG5535 SENSE 0.548 0.009 4.000 0.000 287
73120 CGPG5704 SENSE 0.401 0.146 4.000 0.000 290 73123 CGPG5728
SENSE 0.171 0.166 4.000 0.000 277 73137 CGPG5587 SENSE 0.382 0.019
4.000 0.000 283 73141 CGPG5659 SENSE 0.348 0.029 1.891 0.219 285
73155 CGPG5684 SENSE 0.194 0.105 4.000 0.000 278 73161 CGPG5594
SENSE 0.327 0.018 2.889 0.121 265 73256 CGPG5194 SENSE 0.176 0.473
4.000 0.000 266 73260 CGPG5200 SENSE 0.256 0.047 4.000 0.000 253
73330 CGPG4765 SENSE 0.229 0.016 4.000 0.000 310 73411 CGPG6440
SENSE 0.232 0.213 4.000 0.000 260 73628 CGPG5025 SENSE 0.304 0.040
1.330 0.467 204 73944 CGPG1458 SENSE 0.426 0.026 4.000 0.000 207
74065 CGPG1828 ANTI-SENSE 0.215 0.026 2.599 0.205 248 74536 CGPG442
SENSE -0.054 0.431 4.000 0.000 312 74688 CGPG6653 SENSE 0.645 0.006
4.000 0.000 340 75657 CGPG7828 SENSE 0.394 0.017 2.680 0.180 325
76178 CGPG7225 SENSE 0.334 0.107 4.000 0.000 321 76460 CGPG7121
SENSE 0.136 0.381 4.000 0.000 305 76527 CGPG6306 SENSE 0.238 0.001
0.335 0.421 217 76532 CGPG3235 SENSE 0.250 0.331 4.000 0.000 213
76602 CGPG2653 SENSE 0.283 0.387 4.000 0.000 306 77020 CGPG6318
SENSE 0.507 0.038 0.000 0.000 364 77139 CGPG9137 SENSE 0.445 0.005
0.000 0.000 372 77180 CGPG9180 SENSE 0.370 0.046 4.000 0.000 365
77187 CGPG9141 SENSE 0.456 0.036 4.000 0.000 381 77204 CGPG9238
SENSE 0.314 0.002 4.000 0.000 382 77266 CGPG9259 SENSE 0.017 0.948
4.000 0.000 389 77430 CGPG9322 SENSE 0.095 0.233 4.000 0.000 392
77444 CGPG9344 SENSE 0.171 0.292 4.000 0.000 319 77517 CGPG6952
SENSE 0.132 0.001 2.507 0.235 327 77536 CGPG7272 SENSE -0.084 0.675
4.000 0.000 351 77587 CGPG8107 SENSE 0.415 0.112 4.000 0.000 317
77610 CGPG6805 SENSE 0.267 0.180 4.000 0.000 356 77832 CGPG8976
SENSE 0.153 0.579 4.000 0.000
[0163] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta or
risk score mean >0, the transgenic plants showed a trend of
trait improvement as compared to the reference.
[0164] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 347 showed improved cold stress
tolerance evidenced by the second criteria as illustrated in
Example 1L and 1M.
H. Shade Tolerance Screen
[0165] Plants undergo a characteristic morphological response in
shade that includes the elongation of the petiole, a change in the
leaf angle, and a reduction in chlorophyll content. While these
changes may confer a competitive advantage to individuals, in a
monoculture the shade avoidance response is thought to reduce the
overall biomass of the population. Thus, genetic alterations that
prevent the shade avoidance response may be associated with higher
yields. Genes that favor growth under low light conditions may also
promote yield, as inadequate light levels frequently limit yield.
This protocol describes a screen to look for Arabidopsis plants
that show an attenuated shade avoidance response and/or grow better
than control plants under low light intensity. Of particular
interest, we were looking for plants that didn't extend their
petiole length, had an increase in seedling weight relative to the
reference and had leaves that were more close to parallel with the
plate surface.
[0166] T2 seeds were plated on glufosinate selection plates with
1/2MS medium. Seeds were sown on 1/2.times.MS salts, 1% Phytagel,
10 ug/ml BASTA. Plants were grown on vertical plates at a
temperature of 22.degree. C. at day, 20.degree. C. at night and
under low light (approximately 30 uE/m.sup.2/s, far/red ratio
(655/665/725/735) .about.0.35 using PLAQ lights with GAM color
filter #680). Twenty-three days after seedlings were sown,
measurements were recorded including seedling status, number of
rosette leaves, status of flower bud, petiole leaf angle, petiole
length, and pooled fresh weights. A digital image of the whole
plate was taken on the measurement day. Seedling weight and petiole
length were analyzed as quantitative responses according to example
1M. The number of rosette leaves, flowering bud formation and leaf
angel were analyzed as qualitative responses according to example
1L.
[0167] A list of recombinant DNA constructs that improve shade
tolerance in plants illustrated in Table 10.
TABLE-US-00009 TABLE 10 Seedling Petiole Leaf angle weight at
length at at day 23 day 23 day 23 PEP Construct Nomination RS Delta
Delta SEQ ID ID ID Orientation mean P-value mean P-value mean
P-value 251 70755 CGPG4473 SENSE NA NA -0.751 0.017 -0.307 0.059
244 70995 CGPG4154 SENSE NA NA 0.251 0.070 0.008 0.944 275 72709
CGPG5568 SENSE NA NA -0.903 0.070 -0.608 0.019 210 72724 CGPG2292
SENSE NA NA -0.707 0.098 -0.638 0.020 272 72774 CGPG5518 SENSE NA
NA -0.268 0.110 -0.195 0.087 258 72813 CGPG4977 SENSE NA NA -0.469
0.092 -0.186 0.054 294 73020 CGPG5791 SENSE NA NA -1.443 0.010
-1.265 0.010 288 73133 CGPG5714 SENSE NA NA -0.852 0.009 -0.896
0.012 260 73628 CGPG5025 SENSE NA NA 0.311 0.011 0.317 0.146 271
73766 CGPG5432 SENSE NA NA -0.988 0.048 -0.671 0.088 314 74435
CGPG6737 SENSE NA NA -0.735 0.042 -0.755 0.049 331 74858 CGPG7371
SENSE NA NA -0.667 0.010 -0.808 0.008 330 74863 CGPG7316 SENSE NA
NA 0.176 0.060 0.168 0.515 332 74933 CGPG7457 SENSE NA NA -0.100
0.801 -0.533 0.094 340 75657 CGPG7828 SENSE NA NA 0.193 0.059 0.244
0.085 337 75685 CGPG7767 SENSE NA NA -0.917 0.075 -0.974 0.049 267
75822 CGPG5210 SENSE NA NA -0.267 0.194 -0.297 0.090 324 76171
CGPG7206 SENSE NA NA -0.350 0.065 -0.570 0.049 254 76202 CGPG4788
SENSE NA NA -1.153 0.009 -1.713 0.019 326 76467 CGPG7267 SENSE NA
NA -0.484 0.177 -0.743 0.088 359 76961 CGPG9080 SENSE NA NA -0.425
0.098 -0.265 0.051 360 76973 CGPG9081 SENSE NA NA 0.333 0.052 0.248
0.003 363 77103 CGPG9134 SENSE NA NA 0.363 0.093 0.198 0.072 374
77157 CGPG9186 SENSE NA NA 0.287 0.044 0.107 0.086 369 77165
CGPG9155 SENSE NA NA -0.081 0.413 -0.572 0.012 368 77176 CGPG9148
SENSE NA NA -0.577 0.025 -0.651 0.085 365 77187 CGPG9141 SENSE NA
NA 0.335 0.002 0.263 0.046 385 77209 CGPG9278 SENSE NA NA 0.671
0.027 0.451 0.011 383 77220 CGPG9271 SENSE NA NA 0.463 0.008 0.231
0.228 225 77334 CGPG3638 SENSE NA NA 0.531 0.067 0.336 0.031 349
77349 CGPG8083 SENSE NA NA 0.133 0.001 0.035 0.781 319 77517
CGPG6952 SENSE NA NA -0.843 0.142 -1.458 0.062 320 77518 CGPG6953
SENSE NA NA 0.354 0.007 0.380 0.010 307 77609 CGPG6326 SENSE NA NA
0.252 0.087 0.211 0.145
[0168] For "seeding weight" and "leaf angle", if p<0.05 and
delta or risk score mean >0, the transgenic plants showed
statistically significant trait improvement as compared to the
reference. If p<0.2 and delta or risk score mean >0, the
transgenic plants showed a trend of trait improvement as compared
to the reference with p<0.2.
[0169] For "petiole length", if p<0.05 and delta <0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta
<0, the transgenic plants showed a trend of trait improvement as
compared to the reference.
[0170] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 246, 295, 303, 325, or 375
showed enhanced shade tolerance by the second criteria as
illustrated in Example 1L and 1M.
I. Early Plant Growth and Development Screen
[0171] This example sets forth a plate based phenotypic analysis
platform for the rapid detection of phenotypes that are evident
during the first two weeks of growth. In this screen, we were
looking for genes that confer advantages in the processes of
germination, seedling vigor, root growth and root morphology under
non-stressed growth conditions to plants. The transgenic plants
with advantages in seedling growth and development were determined
by the seedling weight and root length at day 14 after seed
planting.
[0172] T2 seeds were plated on glufosinate selection plates and
grown under standard conditions (.about.100 uE/m.sup.2/s, 16 h
photoperiod, 22.degree. C. at day, 20.degree. C. at night). Seeds
were stratified for 3 days at 4.degree. C. Seedlings were grown
vertically (at a temperature of 22.degree. C. at day 20.degree. C.
at night). Observations were taken on day 10 and day 14. Both
seedling weight and root length at day 14 were analyzed as
quantitative responses according to example 1M.
[0173] A list recombinant DNA constructs that improve early plant
growth and development illustrated in Table 11.
TABLE-US-00010 TABLE 11 Root Root Seedling length at length at
weight at day 10 day 14 day 14 PEP Construct Nomination Delta P-
Delta P- Delta P- SEQ ID ID ID Orientation mean value mean value
mean value 227 19774 CGPG3920 SENSE 0.488 0.097 0.307 0.100 0.550
0.163 229 19956 CGPG3972 SENSE 0.248 0.062 0.154 0.214 0.284 0.001
232 19973 CGPG4026 SENSE 0.326 0.047 0.066 0.577 0.444 0.012 302
70812 CGPG607 SENSE 0.704 0.037 0.546 0.031 0.740 0.033 236 70915
CGPG4058 SENSE 0.292 0.005 0.278 0.008 0.331 0.114 230 70948
CGPG3990 SENSE 0.255 0.269 0.093 0.229 0.375 0.026 234 70950
CGPG4052 SENSE 0.106 0.577 0.124 0.374 0.239 0.041 235 70962
CGPG4057 SENSE 0.171 0.075 0.144 0.226 0.283 0.015 241 70963
CGPG4121 SENSE 0.149 0.057 0.112 0.074 0.062 0.513 242 70994
CGPG4122 SENSE 0.198 0.063 0.121 0.114 0.240 0.124 244 70995
CGPG4154 SENSE 0.132 0.052 0.101 0.006 0.198 0.045 249 71328
CGPG4454 SENSE 0.209 0.136 0.251 0.122 0.445 0.093 268 72001
CGPG5221 SENSE 0.183 0.060 0.142 0.042 0.195 0.149 274 72753
CGPG5540 SENSE 0.113 0.045 0.053 0.348 0.081 0.730 282 73105
CGPG5656 SENSE 0.046 0.577 0.138 0.057 0.298 0.142 292 73136
CGPG5764 SENSE 0.212 0.077 0.133 0.027 0.330 0.037 265 73256
CGPG5194 SENSE 0.123 0.503 0.371 0.135 0.623 0.069 313 74420
CGPG6712 SENSE 0.249 0.085 0.093 0.358 0.363 0.042 316 74566
CGPG6796 SENSE 0.169 0.003 0.147 0.114 0.173 0.171 304 74662
CGPG6185 SENSE -0.205 0.337 0.009 0.878 -0.009 0.958 312 74688
CGPG6653 SENSE 0.221 0.083 0.059 0.358 0.295 0.163 329 74862
CGPG7308 SENSE 0.201 0.122 0.044 0.515 0.383 0.006 325 76178
CGPG7225 SENSE 0.217 0.018 0.093 0.364 0.240 0.234 322 77069
CGPG7163 SENSE / / / / 0.641 0.074 376 77124 CGPG9207 SENSE 0.337
0.121 0.256 0.045 0.762 0.034 371 77155 CGPG9170 SENSE 0.181 0.262
0.265 0.035 0.419 0.000 367 77164 CGPG9147 SENSE 0.589 0.068 0.369
0.066 0.899 0.051 370 77166 CGPG9163 SENSE 0.383 0.025 0.391 0.006
0.787 0.019 372 77180 CGPG9180 SENSE 0.442 0.058 0.267 0.031 0.402
0.339 362 77186 CGPG9133 SENSE 0.448 0.022 0.383 0.079 0.654 0.057
365 77187 CGPG9141 SENSE 0.453 0.043 0.227 0.127 0.660 0.013 375
77195 CGPG9205 SENSE 0.221 0.114 0.188 0.160 0.259 0.064 385 77209
CGPG9278 SENSE 0.360 0.092 0.181 0.203 0.594 0.047 382 77266
CGPG9259 SENSE / / / / 0.470 0.020 384 77268 CGPG9275 SENSE 0.353
0.111 0.170 0.244 0.386 0.041 378 77273 CGPG9220 SENSE 0.255 0.070
0.023 0.586 0.510 0.141 297 77312 CGPG5927 SENSE / / / / 1.006
0.065 393 77409 CGPG9345 SENSE 0.410 0.027 0.396 0.011 0.461 0.040
389 77430 CGPG9322 SENSE 0.209 0.183 0.211 0.247 0.399 0.092 390
77432 CGPG9335 SENSE 0.342 0.113 0.285 0.122 0.425 0.055 391 77433
CGPG9341 SENSE 0.204 0.120 0.222 0.041 0.382 0.085 392 77444
CGPG9344 SENSE 0.185 0.068 0.227 0.003 0.276 0.002 387 77451
CGPG9309 SENSE 0.266 0.097 0.208 0.042 0.014 0.981 388 77452
CGPG9311 SENSE 0.286 0.021 0.182 0.123 0.284 0.201 327 77536
CGPG7272 SENSE 0.121 0.389 0.147 0.026 0.384 0.027 342 77549
CGPG7933 SENSE 0.349 0.006 0.207 0.032 0.383 0.022 344 77568
CGPG8012 SENSE 0.312 0.020 0.157 0.015 0.359 0.071 351 77587
CGPG8107 SENSE 0.151 0.128 0.223 0.014 0.370 0.030 318 77618
CGPG6810 SENSE 0.179 0.018 0.104 0.276 0.154 0.388 334 77816
CGPG7529 SENSE 0.145 0.400 0.171 0.142 0.312 0.181 336 77821
CGPG7737 SENSE 0.114 0.406 0.126 0.208 0.311 0.079 343 77917
CGPG7986 SENSE 0.136 0.083 0.162 0.011 -0.242 0.458
[0174] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta or
risk score mean >0, the transgenic plants showed a trend of
trait improvement as compared to the reference.
[0175] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved
early plant growth and development evidenced by the second criteria
as illustrated in Example 1L and 1M.
J. Late Plant Growth and Development Screen
[0176] This example sets forth a soil based phenotypic platform to
identify genes that confer advantages in the processes of leaf
development, flowering production and seed maturity to plants.
[0177] Arabidopsis plants were grown on a commercial potting
mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of
30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat
moss, 10-20% processed bark ash, 1-15% pine bark and a starter
nutrient charge. Soil was supplemented with Osmocote time-release
fertilizer at a rate of 30 mg/ft.sup.3. T2 seeds were imbibed in 1%
agarose solution for 3 days at 4.degree. C. and then sown at a
density of .about.5 per 21/2'' pot. Thirty-two pots were ordered in
a 4 by 8 grid in standard greenhouse flat. Plants were grown in
environmentally controlled rooms under a 16 h day length with an
average light intensity of .about.200 .mu.moles/m.sup.2/s. Day and
night temperature set points were 22.degree. C. and 20.degree. C.,
respectively. Humidity was maintained at 65%. Plants were watered
by sub-irrigation every two days on average until mid-flowering, at
which point the plants were watered daily until flowering was
complete.
[0178] Application of the herbicide glufosinate was performed to
select T2 individuals containing the target transgene. A single
application of glufosinate was applied when the first true leaves
were visible. Each pot was thinned to leave a single
glufosinate-resistant seedling .about.3 days after the selection
was applied.
[0179] The rosette radius was measured at day 25. The silique
length was measured at day 40. The plant parts were harvested at
day 49 for dry weight measurements if flowering production was
stopped. Otherwise, the dry weights of rosette and silique were
carried out at day 53. The seeds were harvested at day 58. All
measurements were analyzed as quantitative responses according to
example 1M.
[0180] A list of recombinant DNA constructs that improve late plant
growth and development illustrated in Table 12.
TABLE-US-00011 TABLE 12 Rosette dry Rosette Seed net dry Silique
dry Silique weight radius at weight weight length at at day 53 day
25 at day 62 at day 53 day 40 PEP Construct Delta Delta Delta Delta
Delta SEQ ID ID mean P-value mean P-value mean P-value mean P-value
mean P-value 394 12313 -0.075 0.489 0.654 0.000 0.157 0.174 -0.024
0.275 221 18258 -0.494 0.017 -0.183 0.083 0.533 0.006 -0.269 0.234
0.056 0.009 246 70657 0.373 0.040 0.187 0.037 0.355 0.081 -0.187
0.222 -0.003 0.972 247 70660 -0.010 0.936 -0.072 0.431 0.445 0.026
0.116 0.369 0.145 0.045 216 71538 -0.167 0.168 0.017 0.686 0.521
0.005 -0.196 0.113 0.023 0.557 269 72056 0.563 0.008 NA NA -0.251
0.159 -0.289 0.314 -0.046 0.314 279 73057 -0.266 0.035 0.167 0.114
0.396 0.034 -0.304 0.085 0.023 0.543 311 73568 -0.140 0.115 -0.085
0.340 0.553 0.019 0.344 0.010 0.040 0.458 222 19193 -0.296 0.110
0.081 0.385 1.076 0.011 NA NA 0.073 0.001 262 73242 0.159 0.132
0.133 0.050 1.074 0.009 0.652 0.002 0.045 0.048 309 73433 0.477
0.026 0.113 0.120 1.140 0.006 0.552 0.016 -0.009 0.872 316 74566
-0.387 0.023 0.104 0.047 1.213 0.003 -0.169 0.130 0.012 0.598 338
75654 0.604 0.012 -0.006 0.899 -1.193 0.004 -0.258 0.057 -0.004
0.962 361 77150 0.611 0.003 -0.098 0.115 -0.363 0.011 -0.398 0.203
0.001 0.990 327 77536 0.043 0.692 0.089 0.040 0.725 0.015 0.419
0.016 0.075 0.105 334 77816 -0.324 0.021 0.005 0.954 1.207 0.000
-0.038 0.679 0.039 0.378
[0181] If p<0.05 and delta or risk score mean >0, the
transgenic plants showed statistically significant trait
improvement as compared to the reference. If p<0.2 and delta or
risk score mean >0, the transgenic plants showed a trend of
trait improvement as compared to the reference.
K. Low Nitrogen Tolerance Screen
[0182] Under low nitrogen conditions, Arabidopsis seedlings become
chlorotic and have less biomass. This example sets forth the
limited nitrogen tolerance screen to identify Arabidopsis plants
transformed with the gene of interest that are altered in their
ability to accumulate biomass and/or retain chlorophyll under low
nitrogen condition.
[0183] T2 seeds were plated on glufosinate selection plates
containing 0.5.times.N-Free Hoagland's T 0.1 mM NH.sub.4NO.sub.3 T
0.1% sucrose T 1% phytagel media and grown under standard light and
temperature conditions. At 12 days of growth, plants were scored
for seedling status (i.e., viable or non-viable) and root length.
After 21 days of growth, plants were scored for BASTA resistance,
visual color, seedling weight, number of green leaves, number of
rosette leaves, root length and formation of flowering buds. A
photograph of each plant was also taken at this time point.
[0184] The seedling weight and root length were analyzed as
quantitative responses according to example 1M. The number green
leaves, the number of rosette leaves and the flowerbud formation
were analyzed as qualitative responses according to example 1L. The
leaf color raw data were collected on each plant as the percentages
of five color elements (Green, DarkGreen, LightGreen, RedPurple,
YellowChlorotic) using a computer imaging system. A statistical
logistic regression model was developed to predict an overall value
based on five colors for each plant.
[0185] A list of recombinant DNA constructs that improve low
nitrogen availability tolerance in plants illustrated in Table
13.
TABLE-US-00012 TABLE 13 Root length Leaf color Rosette weight PEP
Construct Nomination Delta Risk score Delta SEQ ID ID ID
Orientation mean P-value mean P-value mean P-value 200 10422
CGPG117 ANTI- -0.435 0.008 1.311 0.021 0.050 0.396 SENSE 206 12602
CGPG170 SENSE -0.004 0.941 -0.052 0.895 0.115 0.048 202 13235
CGPG1288 SENSE -0.047 0.267 -0.085 0.864 0.128 0.068 203 13411
CGPG1301 SENSE 0.323 0.202 -3.233 0.126 0.174 0.044 201 13485
CGPG1226 ANTI- 0.311 0.096 1.162 0.038 0.241 0.019 SENSE 205 13846
CGPG1542 ANTI- -0.267 0.190 1.506 0.004 0.176 0.281 SENSE 212 16610
CGPG2499 SENSE -0.037 0.677 1.325 0.004 -0.004 0.918 211 17805
CGPG2457 ANTI- -0.348 0.016 3.523 0.067 -0.105 0.116 SENSE 218
18231 CGPG3274 SENSE -0.207 0.117 0.925 0.062 -0.093 0.094 224
18354 CGPG3534 SENSE 0.104 0.070 -0.407 0.124 0.189 0.048 252 70684
CGPG4588 SENSE 0.379 0.001 -3.676 0.003 0.328 0.030 223 71301
CGPG3528 SENSE -0.394 0.027 1.269 0.042 -0.128 0.026 250 71329
CGPG4456 SENSE -0.169 0.230 0.475 0.074 -0.365 0.337 258 72813
CGPG4977 SENSE -0.361 0.193 0.521 0.076 0.013 0.120 309 73433
CGPG6429 SENSE 0.129 0.201 -0.091 0.794 0.186 0.002 300 74349
CGPG5967 SENSE -0.139 0.019 0.592 0.080 0.045 0.080 313 74420
CGPG6712 SENSE 0.612 0.043 -3.086 0.063 0.173 0.002 298 75237
CGPG5941 SENSE -0.097 0.237 0.948 0.031 0.074 0.136 333 75379
CGPG7520 SENSE 0.000 0.998 0.529 0.048 -0.017 0.755 335 75434
CGPG7636 SENSE -0.336 0.039 1.208 0.076 -0.109 0.107 341 75622
CGPG7833 SENSE -0.503 0.001 1.776 0.007 0.096 0.497 339 75692
CGPG7823 SENSE -0.055 0.165 -0.377 0.328 0.132 0.083 301 76422
CGPG6040 SENSE 0.197 0.178 -0.263 0.734 0.162 0.094 359 76961
CGPG9080 SENSE -0.361 0.136 1.472 0.013 -0.190 0.066 369 77165
CGPG9155 SENSE 0.366 0.041 -1.982 0.143 0.253 0.099 303 77322
CGPG6178 SENSE / / 1.691 0.060 -0.185 0.052 207 16322 CGPG1828
SENSE / / -0.021 0.982 0.116 0.043 356 77832 CGPG8976 SENSE / /
2.472 0.011 -0.028 0.445
[0186] For leaf color and rosette weight, if p<0.05 and delta or
risk score mean >0, the transgenic plants showed statistically
significant trait improvement as compared to the reference. If
p<0.2 and delta or risk score mean >0, the transgenic plants
showed a trend of trait improvement as compared to the reference
with p<0.2. For root length, if p<0.05, the transgenic plants
showed statistically significant trait improvement as compared to
the reference. If p<0.2, the transgenic plants showed a trend of
trait improvement as compared to the reference.
[0187] Transgenic plants comprising recombinant DNA expressing a
protein as set forth in SEQ ID NO: 198 or 327 showed improved
tolerance to low nitrogen condition evidenced by the second
criteria as illustrated in Example 1L and 1M.
L. Statistic Analysis for Qualitative Responses
[0188] A list of responses that were analyzed as qualitative
responses illustrated in Table 14.
TABLE-US-00013 TABLE 14 response screen categories (success vs.
failure) Wilting response Risk Soil drought tolerance screen
non-wilted vs. wilted Score growth stage at day 14 heat stress
tolerance screen 50% of plants reach stage1.03 vs. not growth stage
at day 14 salt stress tolerance screen 50% of plants reach
stage1.03 vs. not growth stage at day 14 PEG induced osmotic stress
tolerance 50% of plants reach stage1.03 vs. not screen growth stage
at day 7 cold germination tolerance screen 50% of plants reach
stage 0.5 vs. not number of rosette leaves Shade tolerance screen 5
leaves appeared vs. not at day 23 Flower bud formation at Shade
tolerance screen flower buds appear vs. not day 23 leaf angle at
day 23 Shade tolerance screen >60 degree vs. <60 degree
number of green leaves at limited nitrogen tolerance screen 6 or 7
leaves appeared vs. not day 21 number of rosette leaves limited
nitrogen tolerance screen 6 or 7 leaves appeared vs. not at day 21
Flower bud formation at limited nitrogen tolerance screen flower
buds appear vs. not day 21
Plants were grouped into transgenic and reference groups and were
scored as success or failure according to Table 14. First, the risk
(R) was calculated, which is the proportion of plants that were
scored as of failure plants within the group. Then the relative
risk (RR) was calculated as the ratio of R (transgenic) to R
(reference). Risk score (RS) was calculated as -log.sub.2.sup.RR.
Two criteria were used to determine a transgenic with enhanced
trait(s). Transgenic plants comprising recombinant DNA disclosed
herein showed trait enhancement according to either or both of the
two criteria.
[0189] For the first criteria, the risk scores from multiple events
of the transgene of interest were evaluated for statistical
significance by t-test using SAS statistical software (SAS 9,
SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). RS with
a value greater than 0 indicates that the transgenic plants perform
better than the reference. RS with a value less than 0 indicates
that the transgenic plants perform worse than the reference. The RS
with a value equal to 0 indicates that the performance of the
transgenic plants and the reference don't show any difference. If
p<0.05 and risk score mean >0, the transgenic plants showed
statistically significant trait enhancement as compared to the
reference. If p<0.2 and risk score mean >0, the transgenic
plants showed a trend of trait enhancement as compared to the
reference.
[0190] For the second criteria, the RS from each event was
evaluated for statistical significance by t-test using SAS
statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute
Inc, Cary, N.C., USA). The RS with a value greater than 0 indicates
that the transgenic plants from this events perform better than the
reference. The RS with a value less than 0 indicates that the
transgenic plants from this event perform worse than the reference.
The RS with a value equal to 0 indicates that the performance of
the transgenic plants from this event and the reference don't show
any difference. If p<0.05 and risk score mean >0, the
transgenic plants from this event showed statistically significant
trait enhancement as compared to the reference. If p<0.2 and
risk score mean >0, the transgenic plants showed a trend of
trait enhancement as compared to the reference. If two or more
events of the transgene of interest showed improvement in the same
response, the transgene was deemed to show trait enhancement.
M. Statistic Analysis for Quantitative Responses
[0191] A list of responses that were analyzed as quantitative
responses illustrated in Table 15.
TABLE-US-00014 TABLE 15 response screen seed yield Soil drought
stress tolerance screen seedling weight at day 14 heat stress
tolerance screen root length at day 14 heat stress tolerance screen
seedling weight at day 14 salt stress tolerance screen root length
at day 14 salt stress tolerance screen root length at day 11 salt
stress tolerance screen seedling weight at day 14 PEG induced
osmotic stress tolerance screen root length at day 11 PEG induced
osmotic stress tolerance screen root length at day 14 PEG induced
osmotic stress tolerance screen rosette area at day 8 cold shock
tolerance screen rosette area at day28 cold shock tolerance screen
difference in rosette area cold shock tolerance screen from day 8
to day 28 root length at day 28 cold germination tolerance screen
seedling weight at day 23 Shade tolerance screen petiole length at
day 23 Shade tolerance screen root length at day 14 Early plant
growth and development screen Seedling weight at day14 Early plant
growth and development screen Rosette dry weight at Late plant
growth and development screen day 53 rosette radius at day 25 Late
plant growth and development screen seed dry weight at day 58 Late
plant growth and development screen silique dry weight at day 53
Late plant growth and development screen silique length at day 40
Late plant growth and development screen Seedling weight at day 21
Limited nitrogen tolerance screen Root length at day 21 Limited
nitrogen tolerance screen
[0192] The measurements (M) of each plant were transformed by
log.sub.2 calculation. The Delta was calculated as
log.sub.2M(transgenic)-log.sub.2M(reference). Two criteria were
used to determine trait enhancement. A transgene of interest could
show trait enhancement according to either or both of the two
criteria. The measurements (M) of each plant were transformed by
log.sub.2 calculation. The Delta was calculated as
log.sub.2M(transgenic)-log.sub.2M(reference). If the measured
response was Petiole Length for the Low Light assay, Delta was
subsequently multiplied by -1, to account for the fact that a
shorter petiole length is considered an indication of trait
enhancement.
[0193] For the first criteria, the Deltas from multiple events of
the transgene of interest were evaluated for statistical
significance by t-test using SAS statistical software (SAS 9,
SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). Delta
with a value greater than 0 indicates that the transgenic plants
perform better than the reference. Delta with a value less than 0
indicates that the transgenic plants perform worse than the
reference. The Delta with a value equal to 0 indicates that the
performance of the transgenic plants and the reference don't show
any difference. If p<0.05 and risk score mean >0, the
transgenic plants showed statistically significant trait
enhancement as compared to the reference. If p<0.2 and risk
score mean >0, the transgenic plants showed a trend of trait
enhancement as compared to the reference.
[0194] For the second criteria, the delta from each event was
evaluated for statistical significance by t-test using SAS
statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute
Inc, Cary, N.C., USA). The Delta with a value greater than 0
indicates that the transgenic plants from this event performs
better than the reference. The Delta with a value less than 0
indicates that the transgenic plants from this event perform worse
than the reference. The Delta with a value equal to 0 indicates
that the performance of the transgenic plants from this event and
the reference don't show any difference. If p<0.05 and delta
mean >0, the transgenic plants from this event showed
statistically significant trait improvement as compared to the
reference. If p<0.2 and delta mean >0, the transgenic plants
showed a trend of trait enhancement as compared to the reference.
If two or more events of the transgene of interest showed
enhancement in the same response, the transgene was deemed to show
trait improvement.
Example 2
Identification of Homologs
[0195] A BLAST searchable "All Protein Database" is constructed of
known protein sequences using a proprietary sequence database and
the National Center for Biotechnology Information (NCBI)
non-redundant amino acid database (nr.aa). For each organism from
which a DNA sequence provided herein was obtained, an "Organism
Protein Database" is constructed of known protein sequences of the
organism; the Organism Protein Database is a subset of the All
Protein Database based on the NCBI taxonomy ID for the
organism.
[0196] The All Protein Database is queried using amino acid
sequence of cognate protein for gene DNA used in trait-improving
recombinant DNA, i.e., sequences of SEQ ID NO: 198 through SEQ ID
NO: 394 using "blastp" with E-value cutoff of 1e-8. Up to 1000 top
hits were kept, and separated by organism names. For each organism
other than that of the query sequence, a list is kept for hits from
the query organism itself with a more significant E-value than the
best hit of the organism. The list contains likely duplicated
genes, and is referred to as the Core List. Another list was kept
for all the hits from each organism, sorted by E-value, and
referred to as the Hit List.
[0197] The Organism Protein Database is queried using amino acid
sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using "blastp"
with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST
searchable database is constructed based on these hits, and is
referred to as "SubDB". SubDB was queried with each sequence in the
Hit List using "blastp" with E-value cutoff of 1e-8. The hit with
the best E-value is compared with the Core List from the
corresponding organism. The hit is deemed a likely ortholog if it
belongs to the Core List, otherwise it is deemed not a likely
ortholog and there is no further search of sequences in the Hit
List for the same organism. Likely orthologs from a large number of
distinct organisms were identified and are reported by amino acid
sequences of SEQ ID NO: 395 to SEQ ID NO: 19938. These orthologs
are reported in Tables 2 as homologs to the proteins cognate to
genes used in trait-improving recombinant DNA.
TABLE-US-00015 TABLE 2 SEQ ID NO: homolog SEQ ID NOs 198: 3331 2914
8408 6836 10845 14799 13562 8848 2509 13003 875 10945 4176 7318
4820 19156 19137 14942 17166 13809 3506 3410 1343 8019 7882 6490
3550 4999 3130 12635 8774 10864 17857 18089 16263 7295 10882 18252
3728 14833 11485 15463 18826 2924 18723 1446 2812 7847 3395 4209
15768 10679 18379 14040 3974 1087 11574 6329 5482 5609 6441 5537
17042 4716 4516 12643 11384 12736 8137 1344 19473 8452 4094 2460
18071 16891 5620 2364 7018 5200 11131 18133 12170 2731 1687 5014
18511 9343 19283 6682 8123 8124 10597 10622 10608 10600 16221 4280
11494 4867 9035 9507 7969 19159 19425 5248 17367 8135 8773 1287
2671 9365 18729 13828 8754 7170 14529 16604 8341 1135 6224 9739
11212 12962 4331 13555 14356 14357 14783 7509 2032 4692 5243 199:
15320 200: 7710 14519 16336 7019 7872 7719 18299 10453 4650 15008
10089 699 15645 10533 11751 12924 10526 5313 17532 8825 6941 7743
4155 14541 9797 16905 8304 12080 19803 3087 1397 7534 5547 2423
201: 11777 6444 1416 4353 15071 3413 14526 202: 19073 6651 15740
7399 15791 2977 7789 9248 14697 15923 12131 5470 965 11100 11069
12624 12892 3580 5270 18663 2916 1684 8107 4240 19240 18582 19054
15775 7301 17416 10566 16351 13846 16386 18431 4439 4056 8159 8292
12484 7316 3763 4783 18427 15501 16164 7762 4015 4556 18955 12061
8636 9665 9713 10212 17555 5330 12850 16436 19045 16034 16692 2571
11676 12666 11853 5348 8771 12218 3157 10749 203: 5608 2382 17993
16598 7505 6976 204: 11369 13527 10897 6893 6962 11592 837 5116
19669 1655 7244 2623 2591 16504 17797 4636 15133 4141 7112 7939
6898 15965 16706 16979 11784 16115 9620 15750 14652 3008 7838 14787
4563 1873 15811 7271 4317 16603 1715 14288 11341 6188 1546 5683
5225 3033 6069 11268 15981 15490 4181 8633 6058 7993 6763 9671
14892 17296 990 8184 2935 8536 11232 12598 12638 5112 10787 1107
13858 5864 16398 16380 17071 16520 1973 5455 13852 19228 1695 17402
2865 17120 2507 5601 14952 1462 6616 6973 3092 1382 7755 3870 9765
5711 1257 6119 15310 3792 15970 16032 14584 7282 15140 7273 3013
15249 13253 13241 2265 17700 18976 4009 17354 2223 17559 3151 6320
17010 8939 11258 734 573 15015 3050 16344 3931 11238 7691 11499
1112 4825 8344 13519 11669 5640 13588 5473 15108 7210 17617 5994
5980 16627 17906 5876 6079 18894 6956 12887 14694 10968 1061 2778
6096 10570 680 974 3120 16300 5885 7626 13881 4244 4233 15039 4486
3103 3115 7654 11890 12359 10800 9409 12657 8977 9182 17818 2942
9780 9899 18496 4118 18188 10437 13907 17312 15730 13906 2603 12770
4837 12953 16663 9237 9546 16293 18238 6022 13908 10266 9180 5538
15536 10370 15958 5217 8154 13095 15972 14841 15262 12702 10766
15960 18726 19395 10345 5964 13064 3946 17225 17262 17220 17266
17224 17252 5465 5495 12800 12814 12758 3021 12400 12492 16461
13638 5616 17449 8397 17258 5669 5675 6852 9796 176011 7569 9775
17664 205: 14023 6117 12539 6696 18601 1463 19893 6621 9577 9166
17890 17889 528 5413 18313 2784 8102 3988 890 19106 571 6791 2402
912 206: 17912 8799 6566 12137 4356 5803 5253 6078 9123 17523 12392
17806 6917 19039 7350 17767 11311 17082 15535 19392 11233 470 8318
14963 16736 8867 14135 5246 4674 2124 9901 13302 15459 18997 2954
1026 667 7561 16841 8092 9084 4065 1836 13713 13697 17640 7422
19580 4565 15193 526 13108 7065 11703 11362 3633 19430 6221 17433
15616 10082 10189 3089 15238 6633 8106 16180 15317 16364 207: 14575
8831 11211 14985 17619 18829 2521 5219 15413 15835 1204 6569 4661
4457 6066 14578 932 13694 7541 4813 15707 19563 1326 16107 5009
7518 14825 13777 6175 15925 13617 5765 14838 9091 10776 15679 5909
19373 756 208: 19581 18655 16310 7497 6382 8261 936 7358 8883 4866
2146 8601 2932 1626 10354 15192 11509 9909 13503 488 17477 14483
16941 2824 1072 4989 14179 4883 13751 209: 3493 16588 7031 5155
11150 17294 5241 10621 5786 5591 16509 2021 1324 12454 15061 1670
15143 16506 17002 8741 18091 4013 5959 19370 4239 12885 12174 3709
3431 9596 3378 1018 5963 13212 3150 2952 1619 19861 19114 15446
19582 12663 19603 210: 17777 843 11020 1337 18495 1493 6396 15336
14087 2568 4517 1157 10741 4553 14999 425 4258 17388 5327 748 11691
10174 17805 16628 10117 7980 8902 211: 9637 17261 13232 7387 10506
19067 12021 872 11620 13603 841 1450 2848 16911 212: 14398 1558
18339 13428 13431 4182 16260 10619 6127 12005 7704 5064 13072 4395
9032 19907 9588 478 12782 9917 13100 5202 10562 2044 4452 10775
3543 14840 14071 14020 10406 14218 1797 16955 3879 10312 12432
14824 7082 14554 15744 1508 15866 16232 6797 7077 5816 3597 3596
3311 15022 881 2638 906 12037 12039 3458 2270 8812 3217 9466 18102
610 9042 2431 18349 16650 14418 2251 4235 6826 1244 16651 13004
7852 14435 9553 9262 15603 4340 9235 1413 10538 8296 17223 16048
16071 2120 19847 10420 11239 18968 2995 7551 15854 12345 5685 18180
2381 4814 2159 16862 8677 13466 11435 6649 6015 8097 11230 16025
9440 1353 16151 16249 15930 11226 9398 2870 12373 15043 11765 10145
11071 285 9270 9537 9095 2350 13362 5496 7924 11756 2624 7967 6715
15074 17068 9275 10177 11007 14857 8951 12182 11643 13590 7684 1740
929 910 9093 10018 13344 16978 17544 6305 11378 1393 14108 15937
16631 11803 13126 11969 18524 9644 8425 8421 8420 10755 10757 16696
15994 1143 3549 5868 5126 7506 12547 12669 19032 4707 4710 4708
4732 4736 4733 8772 4730 4711 3385 1831 2528 2304 15098 13223 15197
11673 4386 11884 2336 14693 9118 9633 14548 12725 2399 18359 11011
14692 5652 5434 9755 9513 2681 4364 12903 19777 13525 4326 6859
6088 11611 3068 13940 3578 14440 8582 9826 19618 18012 17233 10057
18222 15021 9371 5985 14888 14001 1279 7826 14232 18172 7425 11502
15565 17431 8317 10294 17742 5564 5531 18144 7400 10835 14389 13461
13857 7113 16444 4722 2115 7276 18254 1237 5256 13951 2920 16719
1045 15371 19022 684 8004 2781 19811 19766 3279 18712 4940 14312
6426 10076 15772 7906 19836 11219 18985 2673 3933 14263 11860 1907
4672 15599 13737 4944 2252 15415 11543 3433 7746 17114 4115 4881
9445 14696 9525 14484 16112 5392 10428 1400 9349 7616 18930 2533
12585 10497 10236 18415 4874 19793 4633 12544 1995 17255 6130 10439
15261 6768 15985 17752 8504 12863 7487 14875 12020 2036 18127 9264
3628 5019 7498 15865 1448 9636 2840 3161 6207 2644 1059 2065 10031
2171 16927 4747 18701 19271 4174 1389 9893 12527 9018 10035 5039
10698 13943 7894 12671 5651 19586 12810 1254 3519 16114 4286 13948
14493 8294 14606 2586 14069 7712 16988 737 16797 16826 18966 5145
17610 6304 8895 7744 9280 9284 10647 16558 213: 12662 5295 19093
10971 15359 1163 18513 889 2151 3536 7105 3292 8466 18205 3579 1504
19486 13768 18047 6871 7174 11029 11505 17782 5697 18822 4892 12656
1284 3062 4697 5993 18343 18790 3057 7014 12265 10981 15204 11631
17704 17941 10633 19776 933 10363 8446 17800 9822 2953 13693 19862
14551 15692 17673 6081 5049 12581 6910 214: 15345 19484 4871 3871
6695 3993 11459 9199 15899 13821 14206 19489 13765 712 19072 11649
215: 1607 15669 13168 19044 790 9515 4511 13776 11573 13000 216:
4929 17143 10058 17506 15064 3332 5268 6968 16252 15949 6180 3000
14937 18454 13071 217: 6807 19716 480 14021 4179 12236 6337 12237
6338 6336 12234 4815 2261 18555 4706 7836 9176 18851 3443 9382 9439
8870 16691 16646 11516 11500 11515 11498 16670 16660 16683 11513
11496 16668 16664 11519 16686 11479 11481 7146 16643 16659 4850
17951 7144 4824 7163 4851 11234 4860 17956 7160 17977 4827 4826
4859 7147 11475 7165 17928 17958 4853 11473 7143 11477 5129 18893
18914 17526 17529 17548 17547 17549 18917 13686 19089 13683 4938
16999 13666 4494 12074 17097 12093 16874 12095 15878 17159 17154
17156 17153 5681 15126 10052 19712 4288 15399 742 11607 15654 7006
8041 715 2713 11522 7695 3581 7865 3776 15738 19821 17259 3948
13388 17427 15523 14216 8711 15423 19060 16814 14298 18720 7885
3967 9710 3568 3573 3575 8503 15486 12932 7870 989 3595 17756 9202
520 11205 14771 3574 2443 8036 19537 14641 6739 15375 2400 6753
3336 218: 13099 6217 4454 4699 11292 4702 1554 6467 15339 15722
11035 12964 16317 13914 17486 18809 14651 4189 9581 14957 2871 1561
17618 3835 14955 3868 9158 13663 10042 7575 19824 6200 10522 3600
4064 14590 12731 7201 19572 9060 4035 6255 3457 11320 7986 8818
219: 19325 10450 4487 12312 2564 10096 5641 1947 4367 6964 18031
1012 11041 15078 12255 5761 5061 9832 16834 18348 220: 8238 19207
18657 1358 2734 9320 13526 12631 17660 16548 15528 18501 18049
15274 19470 221: 2724 17089 9639 9208 17722 6817 11848 18904 13673
2347 11585 3054 7486 6056 1290 14932 14930 12636 14230 1572 15708
18912 18177 1699 10460 2090 5583 18802 606 5929 5041 19148 14224
9144 2643 17144 14142 14674 16275 222: 14820 18626 11591 649 3005
3037 5453 5430 14248 2371 4626 11661 9784 10560 16219 11699 223:
17739 13288 4139 15466 19773 753 1277 14333 4211 14510 662 7930
18703 3268 19400 17533 2302 9078 224: 13859 17816 12692 10417 7051
5444 12975 1611 7050 17105 5023 3397 13149 14751 10564 7331 7329
11868 9210 9211 18860 18879 14568 17920 14497 12723 19352 10587
225: 11265 17061 11261 17058 11267 17064 8014 8033 8018 508 8035
8038 8013 8017 3554 13411 12545 12528 11997 12566 11922 12520 11958
12525 12522 12553 11961 11979 5030 3464 3910 18901 14601 14600 840
15664 15668 15121 15142 15119 15686 15690 15657 15688 15146 15164
15716 15148 15656 11191 13623 12732 5050 5029 5011 5055 5035 5036
5032 5081 5058 5013 5078 5026 5059 5077 5008 5053 5076 11939 11976
12541 2898 17207 5179 5115 13674 13423 16078 9689 11174 5252 17279
17277 11177 1867 1865 1823 1844 1842 1793 1824 1820 10671 3139 1123
5805 12934 12755 7504 1091 17904 17909 17926 17902 17938 17907
17929 10598 17935 17933 5298 17980 10601 5383 14050 6585 11379
16583 17978 17814 4838 17812 2633 3257 3189 774 12008 9988 14268
18392 12567 14920 12980 11959 7682 7685 13235 4127 3740 3766 3770
3769 3795 14966 457 8521 19464 4847 13386 5024 13387 13401 5957
6571 7932 8369 10496 10492 10490 10495 10517 7943 4958 4956 4923
4986 4961 4951 5007 4937 4934 4984 4933 4924 4921 4936 4979 4932
4981 4988 18200 18196 7176 4739 5479 8351 7177 12886 9521 6464
17681 14504 907 16793 1869 14509 8882 19727 8798 1197 3419 14168
12846 15739 1115 14165 19715 492 8873 1119 497 1194 3251 1116 8876
13982 12860 14430 451 14969 6102 4358 2650 2649 11790 12925 5882
19696 9551 3867 19796 18933 18929 833 4908 14217 4852 4858 16064
14208 10736 4886 14214 16062 14172 10735 14210 10720 1885 14187
14190 14184 14207 14703 14707 14709 14706 14390 7658 7659 7661 2582
13794 5204 17305 16541 8914 16959 17283 9043 3924 2696 8656 6648
2675 18504 17311 6052 9425 9406 9410 9411 6051 9452 6033 15964 6267
6031 8489 8463 8865 9427 6268 8490 8493 15602 15621 14616 15649
15652 15606 11723 17247 9423 15595 15624 16148 2240 7428 11722
11363 2225 2196 5480 15579 15626 16142 7413 5483 11741 16145 16162
11725 5481 18072 10973 12451 12340 15511 12185 4348 12754 8722
14544 6171 10732 9492 19795 12165 15082 6617 9613 739 19520 2497
14005 11128 18795 14331 14329 14334 16439 12070 4053 3942 2387 2391
2394 755 754 2950 752 2377 19819 17119 7118 1360 2273 11458 13758
11215 11274 11229 13178 5110 16239 12884 18765 16637 19693 16831
17824 10810 15395 15420 17470 10005 16397 16394 14711 14728 15503
4401 801 12244 12292 12308 12219 9033 16763 14392 16804 16785 9566
16806 14409 14391 16762 9027 9568 9029 16788 9009 16780 14410 16781
16782 16802 3330 3357 3326 9001 3358 5789 14387 14412 3360 3356
1643 1805 14061 15787 5632 226: 19514 17912 10095 10091 10252 15927
5360 18385 12313 19719 12823 5182 1491 8631 4666 2562 9358 10461
3952 19610 9255 7782 16516 5161 6343 4894 5577 16734 1381 16378
14552 12707 11907 7651 10404 17767 2417 11311 17082 17741 11253
11254 7473 13718 6269 3242 7085 5224 8787 16268 19692 10769 8867
1434 16082 19530 14789 13615 18997 2954 9175 8092 9082 1813 1815
1817 639 3883 15427 2035 4435 3601 414 7005 8000 1812 1131 1455
6221 11231 10817 15601 16270 6118 2193 17500 14186 9141 3449 3089
13886 15786 15238 2001 3945 14973 16180 15317 18083 18695 227:
17372 8093 5527 2439 13784 14768 10702 11535 7750 761 18735 5946
17426 17988 10652 2928 18398 2009 9559 5094 12948 8522 1305 17785
5852 228: 2790 5299 15433 5501 18128 16466 14960 6564 8782 4397
19662 16008 10829 19118 18086 3428 17943 10935 15806 8101 1184 4968
6059 10388 229: 7539 3499 12437 19471 14158 11575 6399 15966 17187
13512 7688 2131 10858 4169 19567 7834 7585 14073 3954 1991 12940
7756 8167 18994 9449 15859 4341 17025 14373 12191 12982 983 13764
19774 16247 12665 12664 2619 18875 7515 11785 2849 10913 10717 4653
11887 905 1641 3563 6183 16765 17773 4166 4694 14328 11986 12084
1625 12695 9681 14082 17341 15047 17498 3530 3531 7241 7972 4790
15643 14365 230: 12430 9914 10470 14976 6139 18895 8072 18544 7257
6437 15066 231: 7185 13859 12150 12692 5555 6319 14735 16754 3121
2208 14915 18531 2991 7950 2419 4378 18922 4377 18705 17105 5023
3397 2203 5350 895 18953 5090 9210 9211 14568 19723 12433 3877
17892 19476 11685 9653 9066 16392 7989 5056 6545 19418 232: 10845
14019 10546 14942 7302 8774 13992 10864 7129 12564 10723 2440 19878
3172 18252 12532 9942 2129 18826 18723 17324 19791 8580 3395 5915
9604 7572 16359 8047 3408 5482 12915 9969 16513 1344 16044 19210
18748 11940 14402 19311 5620 2364 19283 5249 3646 17251 17844 14459
5315 9268 16190 19143 15124 418 3076 16204 17414 18692 13164 8191
10636 13894 3423 2842 1173 7663 13250 18208 14090 19680 15815 4805
10584 1287 15495 9256 2229 10166 19534 11163 2292 2687 19082 9365
2465 5381 18660 3073 9545 17750 233: 19344 12031 1229 15123 14123
19501 7767 5944 11173 4596 11614 10841 14129 2393 7360 621 14382
11612 8136 14668 7577 1652 2491 3392 2160 4767 17696 6615 12377
3471 2985 17194 17177 10988 5084 9695 16519 11445 3213 10846 16453
19346 18274 17478 17798 15663 6823 5131 14272 3777 19911 19724 6132
19722 14803 17148 1605 11787 14359 15796 645 10159 13264 12931 6357
4621 16774 18913 5746 7161 234: 11849 7119 1272 13625 6244 7780
7229 640 15779 14967 5388 14480 1349 17525 12768 10327 8976 5113
5895 15006 4428 16619 4613 3393 18954 4451 3799 17712 12488 15863
18697 18482 7844 235: 8664 9584 8904 18497 19265 8237 2617 7729
17728 8659 13842 11016 9197 10827 5351 3224 7379 236: 12797 7459
12082 1618 7740 16856 7623 8994 7602 19549 12596 7644 18396 13883
7266 18758 3328 18998 1660 12434 2139 19899 12252 5883 3266 19587
11474 5870 5443 237: 14746 16460 7754 9978 16450 18053 18050 18466
8866 10337 8245 1604 8568 13606 19627 18283 15611 10648 13871 9910
10297 3025 18486 8908 14474 11654 2349 10867 14488 13901 7336 11511
14836 1906 13400 5568 19527 4351 18319 6689 10519 15977 15760 7605
16652 14204 14665 727 9225 12804 2776 11108 14258 1882 4357 1972
8324 6435 18239 6728 18983 9555 13551 10797 18458 4622 14064 2771
1818 18410 13553 15912 10854 9475 10873 15530 14816 6283 703 8105
16216 7038 957 11908 13853 10718 11237 5623 10262 12248 6541 7053
4480 14011 17142 6691 7795 17981 2822 1473 3561 4545 13347 1348
11256 6511 11356 13955 6699 6555 6559 3185 16166 17894 9307 6011
10635 1391 13177 16458 702 18335 18740 16787 4175 10950 3412 4193
951 13870 19183 18644 947 16280 6184 13111 3667 10338 4125 14782
18479 18004 19084 12471 16645 866 1895 10049 11122 12521 7132 2660
12759 14534 18429 15615 17611 9347 12704 1525 7492 3451 18373 17125
5972 9777 16039 12350 4073 8962 17221 1961 1958 11156 12899 3908
7429 10977 15040 16234
17004 17293 13355 4977 260 10645 14133 9251 12220 12633 7087 9535
8894 11452 2346 6125 2062 6710 13299 12947 12943 4422 5817 13502
14378 13504 14379 4770 1451 19815 12842 4405 13837 9184 10051 10999
9565 12305 11330 16546 17073 1402 5263 7189 6160 2372 6567 1248
3858 14455 1766 2516 1656 14550 16864 14989 14368 15573 9312 7786
13516 2969 1952 2541 11207 10625 9198 19674 10334 18065 3495 8519
12201 3921 13249 18592 857 17545 11757 15455 2164 789 4350 3117
8649 11708 11463 238: 9324 12427 663 6064 9836 19556 9348 1900
17219 13657 7086 7381 6006 7357 10469 6466 8472 10588 17047 609
1637 10699 6450 14299 15101 10536 18759 2378 18013 9269 3343 12489
11305 18178 2269 16890 10629 18665 15962 9375 9437 16312 6501 11737
6339 10847 5290 4667 5522 18388 1804 11566 2369 15872 12465 7687
6209 11354 15667 15938 18971 18258 3774 239: 13622 9907 10802 1635
19607 8218 11862 18989 19820 1430 15998 4855 18040 3455 2471 6506
7587 11932 17440 16155 13643 12011 13012 11289 14270 17959 10271
5763 19020 5904 7809 240: 19167 14809 6116 11171 10424 1445 13848
1387 18366 9887 3732 10953 2175 241: 939 3338 2461 7962 16158 15297
19138 7398 3035 1767 9983 16494 10256 11165 13953 18376 4113 4116
10457 11995 19241 2063 19340 15435 14028 14611 14366 18488 9171
5494 10438 18123 11822 13093 15526 9516 18346 4375 8716 242: 12077
11851 16904 8919 18979 6443 17768 3571 5105 243: 12862 6311 17138
19420 12939 8147 18538 16023 6580 8709 16080 4700 2940 9554 5556
503 12303 7072 8226 11973 19415 12551 9852 13704 7982 1030 14887
12346 1435 1437 6242 16606 2041 5356 12706 9139 2872 10869 9028
7957 7190 19892 16505 9383 6589 11472 8394 8396 3626 16568 19220
18648 14425 15305 13545 8443 8427 1247 17078 14914 5103 19922 14358
18892 8972 16582 12124 19870 9538 19845 19867 19195 19221 19191
19323 19242 19215 19307 19275 19273 19356 19192 19173 19350 19279
19216 19852 19223 19250 19326 19848 19330 19214 19869 19351 19332
19276 19247 19177 19163 19305 19328 19851 19353 19197 19850 19358
19175 11034 19871 6578 17445 3832 3831 6233 7090 9456 6970 13917
13920 4539 4537 4542 4028 244: 1245 4324 12202 4379 6967 9036 9880
10540 13263 13138 18122 19047 2008 19483 2468 7439 5787 10979 15035
10982 3699 11097 14482 17829 11866 4198 14130 17239 6111 14956
17492 11385 4004 10448 9464 9894 15398 1231 12878 12829 3833 2080
245: 11361 5661 8686 17264 8328 8330 7550 10232 18956 315 8720 1696
5922 16632 14340 11996 8012 4522 16791 10038 5066 4969 4634 17213
14401 7284 2099 6718 8030 14321 1903 15554 15028 12204 3060 19178
11653 9316 17858 18132 18589 9667 17297 5418 17508 15450 6147 5012
18588 18020 862 4920 15050 16525 3064 2884 5607 12297 11396 18052
2912 17060 1697 17604 18870 1732 5968 18761 4044 5088 10593 5242
4527 13682 8054 16503 1962 19380 1623 10664 4597 15319 3478 16945
18584 3108 12724 15546 15764 15754 18354 18416 4890 19678 19130
15532 5213 11374 9753 14702 15217 15921 5855 18752 1904 15721 18203
18156 18864 8700 7560 16924 636 6811 16379 19046 11063 14929 16207
15425 17368 11052 14975 6574 9560 10349 3830 8250 2015 18785 8196
7713 11586 14490 18301 18792 15378 7699 18803 19816 16104 14468
17612 11556 1765 5525 2247 5751 11815 17200 10136 6205 16567 10487
18141 6202 8537 8518 7037 8133 6764 827 4941 818 8356 19365 7588
3017 6400 5637 8416 12444 510 12026 11634 9859 1460 18194 11314
2685 4226 984 19513 8932 12905 4112 18873 246: 13549 2112 6794 8833
12086 2984 5421 7868 9325 19092 9699 19026 1631 4555 804 8588 19422
8685 6218 17573 17039 12147 4754 10543 6834 6733 864 9209 4190 9301
16571 2360 4996 8081 11140 13006 10637 5281 17394 4637 9663 1828
12569 1215 14407 13300 6644 11688 17465 631 16880 3874 16141 975
8011 16154 18389 13579 15094 8944 3735 8021 2288 15334 16083 15801
12760 10618 13208 19889 18999 4870 13710 12132 15259 9267 11470
13144 17588 628 17272 14844 14000 19900 15096 12603 13869 6532
16013 5431 1192 10395 247: 14941 2202 18124 13094 7945 3968 8551
2458 18176 15639 12789 5427 16829 9519 1674 19789 16819 4712 18517
13990 16185 6271 15089 5575 1227 1331 10634 4978 6366 14209 9402
12738 589 6685 16243 2070 14026 12494 248: 4050 14993 16855 19797
5528 9097 18611 19631 18510 8376 8956 10289 11370 13805 18078 19606
8202 15826 16478 19041 5700 6765 8134 14325 10101 16816 4765 16287
5377 18675 7230 7964 876 9597 19802 11403 4833 17530 18265 13417
12779 8885 11923 18564 3790 19147 1406 7066 10573 7108 3783 4727
19785 1458 16375 8415 9522 10729 6071 9047 9113 3248 6378 1799
14779 3630 4294 7608 13707 16321 15368 12743 11299 2413 1584 11214
4012 2710 4268 19546 17625 6940 1273 5755 413 5952 3098 15675 11098
3286 8068 2690 1222 13381 15257 4756 4725 9351 7586 5770 10868 3420
12012 775 7742 8422 6103 15004 16010 12445 13845 17033 16212 3842
19401 4100 8417 13255 1233 5307 7664 11820 4960 4282 2333 17315
12161 4217 10485 8151 16203 1002 11057 5823 9298 1567 411 5686 2787
6556 17045 5742 3090 12019 12930 14231 15548 11694 8266 16248 1579
4806 2747 11349 6186 8303 4499 14141 8094 16001 12109 10916 10761
2142 4592 5947 1880 5294 16111 2594 9869 6575 13083 17382 14364
11324 2341 14545 1596 15428 18963 1929 10976 3736 16709 5662 4501
19083 17865 14066 8045 18782 2866 19741 12401 690 3507 2118 8721
10292 3096 7017 5861 9569 15479 6800 14522 15996 3926 12508 9266
7508 3882 1770 18129 2452 16753 7303 5038 17638 14421 11636 19188
2092 5721 781 700 15887 8872 5320 5466 10259 7441 13041 10874 7249
4514 9725 18158 17609 18140 18293 17059 18509 4888 9306 5484 7091
1826 19360 3124 1056 2563 3177 16370 10946 6177 11856 17835 15766
9377 14858 9666 2881 4713 18553 10081 3260 6030 16206 11892 10319
18672 3859 2663 15443 5089 10992 2411 12439 17434 6053 18213 1552
16896 16303 2170 4062 9729 5506 4195 2496 9435 18250 3643 10322
8871 11520 8307 10814 4172 3616 467 2220 2765 10994 7181 9294 9019
14302 16474 5797 4407 12612 9186 18928 4415 11982 14037 3959 1335
507 14948 9483 17387 6806 8933 14886 13678 506 12462 6785 2667
19322 5005 14242 11045 12403 2390 19573 11726 9949 5099 9853 16159
15920 13256 615 3439 10834 8240 12330 19303 17872 12750 7494 2539
12602 5373 16910 3341 11873 10613 7321 18101 12158 14798 8096 5837
1986 13521 5384 7186 4164 12183 5907 11172 15553 726 4059 6816 6374
16761 7732 6913 1868 15780 18634 16779 6640 3707 4959 10722 8632
18898 249: 8837 9354 459 481 15638 15637 10951 1014 1011 10934 460
456 1750 19841 3504 7770 7490 2370 12273 8627 10651 13584 13749
13618 14339 12485 7233 18749 12207 7057 408 14300 5215 19033 14163
1226 1228 5775 5424 8505 18275 18314 10203 8674 11707 15070 19005
8386 2693 9051 4549 8477 13649 15215 13783 10149 13027 13294 15862
674 9926 18724 12578 8169 1419 6285 15077 2147 14574 6516 1177
13510 9181 2520 4618 7286 7290 7135 6455 7288 9787 11002 551 2679
8942 1701 8928 8930 1097 17269 12778 15439 17593 12347 1453 10948
15909 19108 8061 16885 8964 5642 15311 18841 8881 9067 2902 10703
8544 18226 18698 18197 7766 17357 6778 13002 10083 15189 7944 1067
13701 18247 5067 8858 2375 10780 12562 6618 1888 368 13569 16443
14088 17735 6201 1162 9317 18221 17786 9563 9562 2386 3903 5156
18001 547 12480 13049 2495 8339 18977 12208 19466 10488 15681 11974
4385 19012 8291 16077 16024 5878 19014 8302 17408 16026 16018 16022
4841 9829 9828 17831 4690 2646 9830 13874 14131 9842 9827 9849 8403
9844 9847 9865 9851 8406 8404 6995 7012 8405 4019 10234 7448 17662
1677 19490 2475 6216 13658 5135 4005 982 15959 17990 3508 17774
17778 15478 7855 9687 17808 10782 7592 268 18627 12803 17776 12927
250: 12939 8147 18538 6832 13653 16376 2940 8025 9554 5556 503
12303 11973 12551 9852 1030 10923 12346 1435 1437 2658 15612 16349
9028 13226 19568 6589 11472 4670 17175 1302 15214 13545 992 5103
19327 18189 7434 13340 9873 7090 16545 6970 15069 19166 16647 251:
6603 10030 13559 6740 11390 7765 3614 11615 15027 11616 14213 11329
19168 2567 13201 10961 3308 11492 2325 6423 11527 3741 11560 8630
7463 1786 14408 10585 15696 14167 252: 17378 17392 17375 19787 819
9037 18778 15271 2631 2615 2616 2606 15288 2607 15269 15283 15286
15282 15287 15285 2609 2629 2613 2604 1472 3730 6707 4595 253:
16186 14031 2169 19019 14212 16029 16170 16973 2492 10956 13426
254: 9072 16169 1130 12933 1592 17856 19381 14964 19031 3682 255:
15936 5121 14535 14860 1527 1041 997 4031 18021 12960 12659 6385
17513 15910 6705 15441 15200 3272 16975 18974 12341 5796 256: 15904
2266 1296 6085 10365 4818 7630 6936 18652 4715 9134 15714 2506 5391
14801 12410 18804 2831 19264 7856 15547 14814 17301 17179 18891
11410 1475 11170 7067 17441 12196 257: 1879 1595 12797 7459 8363
12502 12756 5854 465 1425 1893 12362 12596 7644 16328 15689 4599
17970 5443 17245 10708 17790 258: 3828 1761 11325 17218 18307 14003
12997 4456 6236 19270 8499 7402 5087 10067 3938 3953 3939 17913
19475 5664 3918 12870 8678 9700 259: 12266 4307 12661 6842 6841
6413 17238 16962 15778 516 7796 7046 7155 8841 7260 9891 13142 2868
8814 9110 9530 14169 17860 3971 6657 13557 14715 9995 14954 9514
14428 5521 11000 8008 6231 6796 6894 12969 15894 13220 469 19780
17022 2260 17210 1668 1624 17554 9991 2888 10752 8717 1792 7940
17358 7238 580 582 583 2828 4970 4995 18273 1838 4998 4967 8488
14536 1477 13690 10891 14229 7730 15048 9774 18163 9481 10644 2992
6652 4943 7469 6637 19611 6346 6577 5487 15216 8436 7531 19737 9498
7963 8585 17924 10815 10055 9630 14139 8171 9921 12928 12473 13070
793 5190 2500 9328 1480 13432 15272 14189 13728 260: 14746 16460
18053 7754 9978 16450 18050 18466 8866 10337 10391 8245 1604 8568
8843 13606 19627 18283 15825 15808 15611 13871 2627 2601 19077 1485
5898 5916 8835 2570 9910 10297 3025 18486 8908 14474 11654 2349
10867 14488 9940 13901 7336 11511 19746 14836 1906 13400 19527 5568
4351 18319 6689 10519 15977 15760 7605 16652 14204 14665 2776 9225
1972 12804 727 14258 18708 1882 6435 11108 8324 18239 3913 10328
4357 12240 6728 18983 18808 5969 9555 13551 18458 4622 13553 10797
1818 14064 18410 6283 10854 2771 9475 10873 15912 15530 8946 16216
7038 957 13243 13853 11908 12331 10718 11237 5623 10262 12248 6541
17142 7053 14011 4480 2822 17981 7795 6691 3561 1473 17141 4545
13347 1348 11256 6511 11356 8651 13955 6699 6555 16166 6559 3185
17894 9307 6011 10635 1391 13177 702 16458 18740 16787 4175 10950
3412 4193 951 13870 19183 18644 947 18835 6184 13111 3667 10338
4125 14782 18479 18004 19084 12471 16645 866 1895 10049 11122 12521
7132 14534 2660 12759 18429 17611 15615 12704 1525 9347 7492 3451
18373 17125 5972 9777 16039 12350 4073 8962 17221 1961 1958 11156
12899 18769 11606 3225 3930 12985 3908 7429 18285 10977 15040 16234
17004 17293 13355 15499 2062 11463 11416 11708 6710 4422 5817 13502
14378 14379 13504 1451 19815 12842 4405 10999 9565 11330 5263 7189
6160 2372 3858 1248 6567 14455 1766 14550 2516 1656 14989 16864
9312 19300 19408 13516 2556 2969 1952 2541 11207 10625 19674 9198
10334 18065 3495 8519 12201 3921 13249 18592 857 17545 11757 15455
2164 789 4350 3117 14133 12220 7087 11452 6125 4977 261: 6807 480
6337 12236 4678 12917 9439 9382 11513 11500 16670 16646 16691 16660
11516 11515 11496 11498 16668 16683 16686 16664 11519 16659 16643
14322 2737 1932 12955 12317 19334 12435 12976 5809 18730 11183 705
12278 16937 13026 12999 1928 15878 8321 12130 11053 9846 17306
15324 10531 11328 12167 19474 12203 2827 878 9720 4228 5762 4288
13228 10010 14908 1017 679 14784 677 1487 10919 18296 10669 11200
9055 18733 17838 16389 728 715 1375 1396 6901 3743 12599 262: 11852
1409 16572 9074 6600 4427 824 15705 6709 10791 14587 9823 18325 515
12450 18527 604 6043 12455 2071 10310 18162 3176 2577 15203 12952
14467 6645 6698 12807 6113 4709 8824 6274 8311 3411 18810 17940
19805 17162 665 12693 1309 15412 18084 5235 8314 13722 15858 15969
18135 2305 13611 4030 14864 5363 4470 8271 5178 12458 263: 8837 481
15637 10951 10934 19841 460 3504 15638 1014 1011 459 1750 456 12273
4264 8627 13584 13749 19375 12485 18749 7057 408 7233 12207 13068
5215 5515 14163 8781 1228 1226 5775 5424 8505 18275 18314 10203
8674 2693 19005 14053 9051 8386 8477 4549 6597 15215 10149 13783
1372 1370 13027 13294 4287 12578 1419 15077 2147 2905 14574 17504
6516 6455 9181 2520 7135 7288 7290 2679 4618 7286 551 9787 11002
1701 8942 8928 8930 1097 17269 12778 13750 18947 15439 17593 1453
19108 8061 17556 5642 16885 8964 10019 2902 1548 5257 1728 7766
17357 5647 10631 15019 1888 18777 18221 17786 9562 9563 5156 2386
3903 18001 547 12480 13049 8197 2495 8339 19466 12208 18977 11974
4385 19014 8302 19012 17408 8291 16077 5878 16024 16026 16016 16018
16022 4841 9828 9829 9830 9827 14131 13874 9849 9847 4690 2646 9842
17831 8403 9851 9865 8405 8404 6995 9844 7012 4019 15785 10234 7448
17896 1513 13878 7287 264: 10139 8837 1318 481 15637 10951 10934
19841 460 3504 15638 1014 1011 459 456 1750 17473 5931 11424 7490
1243 2370 12273 4264 8627 13584 13749 14339 12485 18749 7233 12207
408 7057 19033 6702 13068 5215 5515 14163 8781 5775 1228 5424 1226
8505 8674 18275 18314 10203 11707 15070 2693 15215 13884 10149
13783 13027 13294 15807 4287 9926 12578 2905 17504 6516 7290 9181
6455 7135 2679 2520 7286 4618 551 7288 9787 11002 1701 8928 8930
8942 1097 17269 15267 13750 18947 15439 17593 9816 11871 13684 1453
19108 8061 5642 10443 12994 4925 6825 16676 7766 13011 12993 13010
17357 9582 4071 15183 1888 11394 1145 18221 17786 9563 9562 18001
3903 2495 17171 19146 18977 12208 8339 19466 10488 15681 4385 17408
19012 8291 5878 16077 19014 8302 16018 16024 16026 16016 16022 4841
9842 9830 9828 8403 4690 2646 9827 9829 13874 14131 17831 9847 9849
9844 9851 8405 9865 8404 6995 7012 4019 7448 538 4587 4576 14863
2709 10770 265: 10339 11051 4273 6693 11042 16475 7274 1052 18146
6194 6195 18166 10865 4396 638 10021 15834 6518 266: 12371 1790
10658 8747 10474 4583 267: 19068 13736 19730 2398 4156 8189 14176
3642 16457 13680 12542 268: 16601 2370 12273 10651 18749 7233 7057
12207 408 14163 1226 1228 5775 5424 8505 18314 10203 18275 8674
11707 14053 19005 8477 8386 7370 6597 2693 9051 4549 13649 15215
10149 13027 9926 8169 1419 6285 2147 15077 2905 17504 13510 7290
7286 7135 7288 2520 6455 9181 9787 2679 4618 551 11002 1701 8930
8942 8928 1097 12778 13750 15439 18947 17593 13684 19108 8061 16885
5642 8964 8881 18841 15311 10703 8544 18226 18698 7766 13011 12993
17357 6778 15189 13002 10083 1067 7944 13701 18247 8858 2375 10780
12562 6618 1888 13569 368 16443 17735 14088 6201 18221 17786 9563
9562 5156 3903 2386 547 12480 18001 13049 8197 2495 8339 12208
19466 18977 10488 11974 4385 8302 19014 8291 16077 19012 16024 5878
16026 17408 16018 16022 16016 4841 4690 2646 17831 9830 9842 9828
9829 13874 14131 9849 7012 9847 9827 8403 9851 9865 8405 9844 8406
4019 10234 7448 538 12803 17808 10782 7592 17774 7855 9687 15478
3508 2475 17776 12927 17662 982 4005 17990 6216 13658 269: 14096
14101 3422 9935 9907 11404 3149 1688 10616 10023 13889 4400 13959
6604 8205 13382 8564 13019 14460 15364 2532 8729 14099 19644 19650
19653 11186 19315 2228 13473 8252 19754 7420 12978 18550 13931
11080 8842 17395 1271 18960 9745 4617 1713 12914 5411 11902 15306
3958 14094 14091 14074 7042 19574 7859 17118 9165 9458 1459 14832
13165 14520 10512 17335 15352 14170 13097 5335 9461 13601 12995
8362 2662 13567 14785 3435 11646 11805 18390 4017 8969 6711 4962
8794 9291 7926 18526 14047 14393 7407 3866 5766 11775 19021 15525
1840 13661 14072 18260 270: 16324 10027 16399 19626 19600 18167
7908 9950 15476 16909 13757 17032 10125 16306 5372 19301 1764 9641
11812 14642 18106 18647 13221 9050 13225 2608 858 9561 5437 14594
5439 6002 12210 12290 5485 6003 13981 16840 17398 15782 13385 4464
894 19838 9505 11810 18088 18145 16187 1329 14353 14351 14349 10819
8846 9429 16618 17714 16634 13050 10112 19589 3140 14486 3444 16943
12766 19496 9070 271: 2051 2869 19263 12625 10041 11778 3745 15945
19372 16735 5498 12285 7206 9395 16559 5318 10659 1955 8048 19028
5197 13490 4306 15568
14829 8639 272: 16396 2024 2104 12134 16012 12790 10384 16093 7594
16424 3265 5193 1401 2076 7270 19843 9331 4976 4079 18320 7860 6861
14631 5218 2559 6330 4039 736 7978 11712 7058 13306 9913 17623 5261
3627 1734 12108 18532 10878 3240 10925 8343 13484 10007 17652 6295
15677 4058 5272 273: 12662 15327 19093 953 5512 10971 2513 18064
17932 1221 19584 4462 3536 19638 12776 5617 18924 7810 19428 13350
11809 13768 18047 11036 10750 3825 17782 18804 2831 18060 18535
9083 17800 9822 14551 11830 6598 8161 6081 5049 6923 6951 12249
274: 4873 8617 18355 6309 6356 19579 11946 18342 7376 17347 13047
4927 14649 12482 15068 14383 10603 3075 5557 16054 5542 5206 17331
19720 11637 5366 16537 14444 11823 2345 18756 7228 11287 3136 3972
2054 13176 4279 12726 19609 19002 3887 18393 12190 2873 4093 15745
19571 15950 5128 275: 3391 5673 17748 12970 2049 2756 10716 780
5570 15625 17600 2057 17079 14041 6494 10663 7787 10440 10550 12319
2852 13899 6288 6170 14404 7408 2857 4218 2808 18604 6362 17551
11818 511 5580 16146 1803 12576 13505 7130 9614 2287 6383 500 13389
19443 13855 8834 1779 5989 19035 7368 11731 19702 17630 783 15741
15774 15442 17674 2205 5474 2153 18915 13290 9728 17044 8267 18171
1334 15222 15195 7733 5047 8922 18281 12929 8992 12922 5696 12619
2026 6208 13543 17275 13703 15244 18936 16496 17942 12986 13118
16224 18419 10277 4864 9332 2640 16067 11844 6736 15403 4550 3500
4202 276: 9040 9153 13748 18710 9288 19447 1096 7103 8455 3624
18607 16311 18304 14306 9854 17310 16393 9187 17043 17574 17689
8273 13937 14386 4911 19063 18212 18817 18819 3310 3329 18815 3312
18959 8476 1386 2263 8742 15253 7396 3159 5367 18632 11510 4953
18618 495 19426 10261 849 13607 1099 15505 16314 14154 11065 14523
19079 3856 277: 6298 11547 9455 3560 3298 1224 6573 17886 3688
17535 6514 11001 16832 278: 9342 1148 8880 13995 9598 18620 16354
596 3713 12096 7035 13429 19465 13583 8623 4778 4775 4758 6813
14722 2970 16245 18993 16658 3106 6966 18030 8468 3622 3111 5706
1317 7832 18473 17421 13587 13609 3484 2614 11857 11213 7994 14581
14105 8667 8513 779 14862 6228 14830 2526 13752 2961 4199 676 14238
15588 17472 5776 1150 15882 3232 11072 7812 12851 3715 13664 13123
9048 17974 449 2155 3081 9548 12652 8280 15483 16721 8031 13645
18580 7580 3619 3479 6971 18732 15594 9508 10074 14377 14469 1702
11710 13599 19866 8968 12053 12047 4245 4259 279: 19107 3731 10544
1365 18558 3521 315 15072 9222 995 13810 10579 12184 1169 10820
8682 17063 17115 11426 7610 8335 6680 19590 669 12808 2095 3210
7981 8975 13797 19673 10108 12140 14891 4849 9589 10013 11489 5848
8508 16961 14635 3335 5341 9626 18112 14644 12785 9168 9484 8109
9837 3294 15636 3494 2017 19052 19769 16972 7811 19903 14579 7377
4121 7557 13806 6626 9547 10641 12326 19729 7235 1411 3644 10157
10154 2648 7794 10100 12628 15633 12168 7589 15853 19745 14933
10255 1291 17736 2414 7188 16138 9089 9415 11728 12818 6609 8114
10065 12609 4638 16498 2517 12709 17410 15136 17048 16713 18469
19832 12689 1479 15584 12811 17562 13267 13892 10949 9327 472 5713
11273 9716 15698 5082 17282 1612 7565 10794 11434 8216 10240 6588
15316 15700 6461 7231 10826 9945 1966 767 9017 8428 9216 1791 12429
5223 4910 19009 13274 12648 16308 4798 9611 5747 13303 2839 10405
4049 4046 969 5871 13542 17764 7597 9272 18722 14315 13433 12396
17102 19120 3267 2911 2934 2480 15593 1566 15118 17099 13495 18798
16391 13630 12936 4072 17390 11981 14556 12247 14559 12268 14560
14577 14558 12267 14576 12226 9387 332 2109 7841 18828 9615 11137
19936 14986 3553 9388 17887 11929 4554 18866 5440 3007 845 18184
4097 2805 16099 12066 16033 3934 3003 1594 10929 12343 16214 17420
5606 4643 10415 14917 6601 15702 10449 19711 13552 1507 19835 1698
9321 11696 2863 14802 17698 1498 17572 14405 13598 12043 2262 567
1583 2618 3734 18793 19614 14449 16036 8175 4878 19636 13279 17286
5486 17542 9087 15980 2290 11904 428 10747 9550 13537 3804 8971
5843 3250 1514 18175 14056 11021 12535 9436 8113 10697 15175 13365
7725 5679 15598 19442 16718 6306 9803 17134 10762 2355 13282 9344
18414 962 10707 13732 15247 16140 14944 8665 3901 12556 13238 3850
16213 15720 5407 7133 17222 15571 4342 952 16118 708 14879 19403
2997 14894 498 14617 11730 18476 3794 6451 3609 16585 3917 3066
16371 13698 6380 5627 5614 3881 18493 6416 12573 15178 11348 7043
8200 17880 11947 15915 7621 7041 8209 18926 10681 14294 3702 8567
18263 9236 1978 3932 19800 9976 13414 8046 11624 1429 3768 17319
17779 7609 16512 3551 630 3171 7227 9996 16499 5172 8855 13654 9117
3812 16292 19434 10320 1982 3126 14582 9645 1807 19478 7519 12941
17861 14481 4909 1899 3459 1922 19333 4083 1541 12260 11304 16432
3209 9163 2838 17263 2745 9173 17072 5826 7351 9534 19739 19536
7374 17185 14542 17759 5670 4857 6297 13448 7546 11655 18214 11988
3497 12119 18421 2238 9138 8088 14015 6428 10563 6419 14637 4955
9470 1521 11999 10694 19034 314 18961 1819 15545 10660 10122 1038
15537 16448 8891 10556 10553 13996 16085 16717 16720 16629 3416
6712 18028 18027 18046 18045 18018 2046 2280 11438 7044 432 10580
10575 17329 2438 2435 12466 13227 13331 3373 4354 1137 14182 14180
17288 17285 17287 17309 18279 17281 5134 13133 13134 7591 7579 7578
7590 7622 7593 7619 8710 11257 10155 17393 5497 7611 7613 3368 3371
3345 3347 3346 11146 18562 15875 3327 11270 617 12175 16942 6398
14919 6019 6427 16524 15844 4568 13742 8545 8646 17237 10441 15117
4903 16684 7548 4237 4891 11339 18131 14205 916 15666 812 8359 2239
6482 16408 10426 1946 16250 7313 7296 10169 13861 16153 7466 6375
11598 1049 14326 7059 9757 6237 2331 4295 3739 1600 4339 9125 15701
18725 10147 17364 5245 694 5869 7830 14712 3680 8784 12459 4338
15013 3889 4760 2323 2136 16586 3470 11223 13993 8676 4320 6154 958
7689 4997 6877 12357 17840 823 1007 2433 5629 7080 8534 2047 12554
18572 15080 13812 14010 15955 19762 16570 11714 12363 13476 19136
3340 8548 16230 13481 15749 6845 2819 2803 2874 2798 2851 2774 2772
2847 2769 11161 2801 14362 3590 1712 8501 2235 2242 2245 2243 18869
17430 17096 16063 16061 4419 4399 7500 7499 7479 7507 7501 7478
2530 9228 852 5988 15329 19925 15542 16445 15520 16918 16920 16451
14285 16922 15560 3473 2729 2751 2748 2730 1449 313 6008 10997
19320 14355 13633 280: 15402 17754 15436 17629 9518 8079 14324
10849 9618 16771 13444 11169 12000 16733 11507 10938 18192 18292
19813 13096 7912 8166 14940 10205 3198 12339 17093 6121 18940 9368
16294 13760 16750 10258 19864 717 17453 996 3004 19619 15600 2042
7099 14437 10711 13380 19640 16532 2201 1802 2309 6647 9806 6879
9457 8208 12113 10109 11945 9605 12639 15237 13105 11307 15891 3950
15781 412 17950 8118 5602 1129 18116 15989 10069 5174 1902 2069
8549 11084 3460 2119 3544 5463 19760 6025 2277 7245 14680 12142
11315 10103 11686 7410 5674 18381 3114 6700 7140 6390 1801 5927
9743 14343 16776 14417 6038 14869 8795 12626 14704 15315 19906
16747 15411 2945 6780 4297 8158 3758 5621 3569 4034 12467 19872
13169 1081 8095 1736 18688 9902 11293 1423 19566 17541 1444 2075
19482 17202 17020 15746 15846 16117 2321 9835 4123 7173 5998 4409
19765 10646 9885 10056 18669 12506 11744 14243 2630 5877 7111 5671
1529 1675 7444 6146 3023 3898 18187 1165 11993 15839 10330 6061
5491 4227 3781 9058 17595 11079 11202 7362 10144 11840 8333 13318
3170 6878 6916 16201 15777 19884 5750 12644 18885 13200 8731 18368
8181 14701 7566 13576 7966 16096 3440 4942 4573 2833 10605 7925
14032 3404 4390 11831 17429 15424 19414 16495 16622 11298 10131
6694 18316 8214 4497 14461 11010 15850 4991 15394 15401 5554 7553
7642 6491 5072 16712 18970 17407 1013 5799 15429 13670 4021 17706
15034 10501 15109 5127 9817 12262 12837 1717 18387 17051 1748 3020
8327 5847 16045 1172 5230 13057 10892 8743 3975 4171 5329 579 17409
16177 5306 5472 11154 9931 4136 5374 2437 15963 2773 17124 17810
18806 13785 2757 10243 16360 5612 16322 12186 2514 18051 19245
15150 17624 1481 7727 15029 17521 3890 2359 5655 15000 19595 10596
9889 9655 9987 11358 19111 11956 4272 6625 9408 15892 4615 17792
13508 980 2199 5653 15516 15373 15610 13373 9133 4362 10774 17361
10744 477 554 11553 14415 17110 12358 14292 13332 11804 11811 9941
13978 11248 18909 19512 16233 3315 13090 18834 11850 7862 19388
5804 19237 19460 4704 13932 6772 6661 6659 2180 12650 18850 12384
12309 430 13903 13110 18218 9954 6627 8550 4388 18332 16577 10907
816 14925 18100 1185 19304 652 9511 10006 6151 12106 6150 12493
7145 15562 2025 18478 7025 7026 4485 14496 7121 15494 13310 16051
8638 5236 11905 7716 16724 17428 13977 12065 14350 8192 14225 12919
19150 3456 17883 4875 7007 6429 17497 12548 9527 12164 398 13983
6190 3241 2352 5757 2425 19500 3188 13614 16480 1042 2584 18280
17452 1855 15112 8640 9646 8269 17863 2113 19671 4472 8213 10111
7758 11259 15393 17215 10615 14115 18114 19469 2426 8337 9601 19412
1557 13838 5949 16547 12016 6009 17996 16020 13608 18118 4335 2510
2358 6156 10954 3031 10072 16042 9333 971 1426 6023 16744 18147
18700 17680 17679 4200 2825 14313 17189 9300 3978 3376 2704 12344
17083 7635 8941 10128 19694 13357 12141 8860 8119 8265 15908 3690
8287 5054 16229 19707 8187 12767 8492 1917 743 3160 17877 8353
12737 8570 6610 16241 17568 17643 18099 7570 6986 13046 14790 11141
18103 19929 4945 4863 7823 1957 13295 8615 7666 19503 18182 17400
18683 13167 474 4854 8015 15919 17049 6539 8967 18743 18229 1022
7970 16839 19367 17290 18980 13370 3941 6507 16184 8973 2098 2078
3254 17794 10311 17483 2883 18643 13517 3729 1887 1368 14447 2804
10407 12834 17444 18805 15242 15905 18413 11332 15147 5790 12206
12866 11142 2298 19828 19319 18179 10150 5563 17462 11283 10674
17994 7653 11921 8670 18362 11531 10803 14348 18130 4425 7792 3742
4069 13020 8929 12909 5891 3491 11742 10960 1707 7447 11965 14102
16993 19751 4688 6075 5409 928 14103 17862 5849 11486 3424 13098
6417 1263 14274 3937 4584 19652 3860 4922 7959 16775 281: 8694
12679 18529 10066 2678 7179 6485 2766 9063 12300 13395 10238 7333
12286 11528 7686 10627 19423 16297 3477 14307 14332 4683 6386 14764
18946 11842 6708 4206 7511 14161 4948 18236 16555 19706 16935 9276
1705 8251 17729 14148 10222 1114 14562 7443 7171 12646 525 3814
3651 11471 13348 17253 3355 5958 18560 9986 5060 4869 4769 4656
9642 648 1410 2931 18886 8791 11423 16482 11075 17009 10151 4552
4158 14670 5141 19526 19457 6070 18446 10680 4669 10087 10957 4753
6048 16338 807 6752 7620 2892 903 10046 10520 10397 14613 1203
19013 13180 13847 9021 8726 11936 9469 19630 17914 1539 16852 2684
10230 15144 7414 15169 16341 18719 4705 14987 1054 1301 8183 10260
12916 14291 9101 5800 9224 19194 2786 14896 17070 13453 10805 8590
1236 3657 17563 5588 19064 6364 15198 2518 14895 12534 11548 11147
5143 4729 18858 19784 5715 6997 19753 10859 13393 5535 514 6792
15326 7576 4957 14042 1374 8856 9698 10313 12942 14998 18662 4919
12571 16319 920 17169 6335 5044 6370 18927 5610 2268 12436 6087
3030 2039 16323 8447 18750 16277 2412 18682 18358 12549 18406 1390
8657 15832 8609 6974 6281 8591 1357 5973 16208 12678 10423 18094
17711 4111 10235 10848 1743 3109 9476 6992 16202 18853 17948 2213
12570 4347 4954 1510 15130 3018 12888 12264 16727 16884 19498 10768
14695 16944 12594 14452 18055 12881 19189 10784 2720 13143 5352
7662 15194 18003 5874 16708 14822 14749 15941 2031 15715 10075
12830 6407 9819 6114 8177 6890 5432 15138 19857 18139 1174 8978
8120 9900 6781 18900 282: 9488 13616 15590 2297 2284 4876 8155 4503
12483 1657 3791 899 884 18026 18515 18521 18025 17955 559 18537 557
16157 10429 13191 7467 11321 7432 14150 2723 12764 12504 16859
19196 19198 19204 18436 19288 19227 19280 19200 19248 19885 18438
19282 18463 19259 19224 19904 19285 18461 19231 19909 19908 19292
19887 19310 19924 19920 19912 19255 19201 19905 18443 19860 19222
18441 18425 19252 19253 19233 16129 15836 3701 13372 6579 3072
10251 10921 11241 1852 14936 13471 2795 13570 13592 5773 13546 3179
4658 5425 19564 6136 5441 19623 527 19056 16702 16685 2755 4581
1965 4319 3694 3892 6668 6060 18290 1483 12475 6925 2154 2749 11421
12831 15432 10442 12991 12491 11934 17139 10324 10227 2579 15389
7706 18794 1036 10124 11030 10073 15797 12617 3046 10666 11189 6165
6563 8600 2237 11718 1494 17677 1968 11225 3759 19617 1963 1230
9162 14979 2576 10989 16101 1141 17522 11388 19518 14314 9391 12490
9259 9858 9861 9878 3027 8297 6634 2580 564 536 513 13257 12751
2800 4947 6596 6727 12672 13807 4898 6067 7002 9776 9305 5205 17859
10706 9919 5631 3026 14137 17947 13952 10969 17270 11134 11135 1464
1994 7337 4302 19858 14835 3132 18541 18685 15115 13477 7242 5034
4232 4229 7849 3936 9400 7074 1711 13593 1267 7020 6203 5752 11017
19211 16500 5464 16481 16479 11262 9474 2657 9912 17012 17455 16989
2421 11405 4134 4135 13791 3760 4746 7250 11301 16165 19238 6369
8863 6260 10640 806 1373 1942 9798 10528 1672 6827 6035 18378 2844
12503 12481 18439 9335 6502 16929 6137 3738 14085 11558 7893 670
2543 714 7694 12393 8619 7916 5416 4052 6540 18074 16465 12699
15044 7738 6074 5428 10870 18932 19505 13905 15650 18814 14236
16809 16695 16811 6358 6377 9099 13233 15830 15855 16971 9952 13720
19312 2882 17849 13346 11894 13927 10068 15728 15724 1669 10842
5211 5573 11524 15993 10964 11561 8701 8076 2340 9030 12177 8173
15874 13078 13080 15578 16552 11954 17694 13066 11930 9502 8839
10015 14690 15514 14413 543 5118 19602 14943 17995 1533 15898 17708
13695 8703 7375 12243 12246 10408 1134 7047 7069 1384 664 14725
13989 14727 12311 550 588 2405 3105 1325 3085 4328 12896 10416 5716
5734 5080 10206 14083 5736 12511 7935 9350 11129 2367 15487 18863
6310 16136 16616 17976 3605 11762 6296 7735 3664 15502 16857 13747
16858 1704 8970 8242 3966 18776 2037 13001 18364 8423 12792 14596
1694 2481 2503 17868 531 18007 7674 7678 2470 5590 15264 15917
13152 11243 11441 18206 19145 19302 5018 2605 15951 15519 11689
3205 8293 4752 4648 2589 3319 3377 6240 2569 2542 4651 4796 4774
4647 4644 4101 4693 4777 4022 4018 4020 4714 3995 4133 4060 4668
4197 1988 16865 1441 16612 758 17827 17825 6420 6905 12143 5753
17242 18540 553 5233 15097 3540 2308 14280 13379 15914 9672 9677
653 5107 10689 462 16974 6046 7495 7028 14188 17597 18006 10434
5334 13292 9733 15419 1170 1187 16770 17137 16828 16680 5597 11937
16404 17267 5157 13849 8607 18671 4234 14051 1528 11389 17864 17893
17962 561 12269 12414 14893 13196 10758 18547 563 558 18543 532
5357 13969 17925 3015 560 3362 5820 7763 960 17879 10047 537 3873
8224 535 555 556 585 17895 562 2019 11745 11746 8692 18545 533
19830 13796 13716 3761 15630 15770 10070 7827 6148 2018 7825 7828
8464 8157 7805 3409 3407 3366 3430 3387 3429 7806 7768 7804 7797
7772 7799 7771 7776 7822 7820 5838 18949 16551 2875 14511 15973
10383 9041 11443 17876 17871 1216 3604 18867 11364 7984 7564 5619
16508 9249 19817 11408 4489 7775 16600 11204 11343 6760 16109 12509
11456 1471 12963 1159 14149 17898 4525 8069 5516 3522 17718 17147
2073 10352 9958 12355 4590 8758 17922 17979 15581 2550 4547 11843
1492 9863 9732 12824 4458 4446 2432 8850 10795 2573 12282 1601 2523
1313 4569 6686 7903 18104 7660 11455 7569 10308 14385 17379 16758
283: 15531 6256 3677 12101 13037 10190 10248 11043 822 16817 7460
8374 602 15713 1872 6858 11835 10862 12518 10351 1380 17170 18399
10269 9599 10039 5643 1398 5309 2941 13830 626 17116 19140 11467
18598 13371 8055 11493 2929 12146 10801 14962 19804 16176 17439
2811 496 2695 19371 12254 9322 16426 17763 3303 7474 8440 6587 7356
10022 7182 7542 19708 4269 14632 12375 12229 13513 5894 17174 11372
14162 15843 13669 2318 4478 4374 2133 1853 5420 9504 9838 8996 1878
5702 13717 1634 18754 3402 15053 14759 16550 3082 4918 14755 14416
9281 8312 16172 11469 9564 18095 19075 13665 14192 9419 14729 2368
10551 14620 6612 19450 454 8874 19239 7854 8878 17842 15819 18002
13934 12440 12425 7219 13659 3489 18080 14572 6789 15128 8759 17772
11836 13231 12637 8764 17762 17882 18242 8340 4916 1839 12027 518
6044 6520 14817 9982 15655 16991 6478 8575 17011 7819 19454 11008
3852 16238 12816 18889 19856 3049 6004 4420 14200 19451 3399 3287
757 4411 3141 3803 3354 2485 600 11184 11333 15248 4728 19407 13987
16072 13120 493 7888 811 14946 2583 8687 9939 14742 4000 19160
16329 17352 7798 9075 11087 16289 13321 3576 5304 6605 16610 12700
5370 15030 7544 2014 8691 16557 17396 12352 3485 16266 13480 19873
8847 11387 13745 2392 8223 5726 12517 14311 13416 14639 10763 14203
18402 3129 12616 9840 18774 18715 15765 15812 8125 18204 4445
799 8531 4265 17783 16560 14953 11093 3886 5539 6863 15521 13084
19629 18230 15314 6456 5130 7907 9874 4068 9185 2982 8263 8178
11555 12456 2286 17432 6568 1183 1923 13056 400 18905 11046 17586
15182 10135 12752 9213 14279 7205 650 6593 6212 18450 15704 9964
7123 5422 14997 16892 14156 16838 19853 13287 1951 6717 14456 16168
7220 6525 16069 12673 11280 9572 7263 16613 1000 3100 1497 17231
9541 2750 6439 3980 10290 15824 17100 4572 9670 8215 6984 12801
10455 3696 17104 11037 10557 7817 7141 11642 18768 18492 12742 7415
8653 14608 13015 13405 13121 8857 17133 16228 14222 5302 7237 7488
5999 3641 566 2958 11175 2130 2764 16189 18380 6548 9673 14038
14035 7139 15190 5426 19562 18437 17501 644 2919 7071 7261 14062
13482 15191 11743 19472 19437 6920 13727 15618 15631 19891 11927
10932 8010 16223 14499 6723 19485 6581 14854 14856 7405 12499 14495
885 6550 15800 14507 17436 16225 11331 3216 8486 4438 7918 5382 661
9656 5184 1208 3516 15538 15408 11613 11439 5451 15131 18608 4901
4791 17602 8576 6930 9997 16304 16968 284: 14546 4993 1029 7514
2917 10152 11335 9594 14726 17937 6935 14151 10273 5997 7000 10002
6010 16908 7529 2137 5389 15290 8071 6027 4413 15840 17540 6072
10477 13780 16667 4735 851 17780 8426 4344 3194 6508 4023 12288
16472 15236 18423 2712 6253 7180 8745 16575 12645 11798 15025 14872
12821 12055 5069 8763 5400 5829 7669 2817 5344 1590 8606 18818
16682 18975 16411 12120 1632 2145 10107 3618 17320 7159 19374 8892
12001 19521 2499 7464 11752 2395 19468 2094 17945 13685 3703 7073
8473 15278 11750 6376 722 1788 12276 2177 3659 2253 13161 10545 826
3249 6401 10373 5991 4410 9993 9994 4785 9814 10962 18365 854 5075
4178 13328 18181 19525 14890 1603 6161 1186 310 587 2986 3592 12649
3080 15617 11601 13501 13181 6206 12788 7897 2732 11732 7914 4586
18225 17758 4002 14369 2043 9570 3991 19823 19209 11521 18784 14970
6131 8658 15987 11465 8315 19681 17975 1559 5862 3880 11271 2108
14500 11285 16316 19436 10432 7652 11110 19523 2843 17746 1118 7774
19578 18424 4835 14580 15727 5582 2332 2821 17034 16853 10210 12320
9478 7636 16940 5379 13944 8103 651 3131 7232 11772 19236 15809
4006 529 19806 7702 4689 17509 5782 10158 6488 7040 13260 10093 686
10592 15289 19347 9038 13009 13925 7052 14143 8739 14688 12107
15485 11400 6474 3104 3107 14423 1690 5872 1941 9668 3685 11900
10682 15943 14081 8352 10943 8232 18499 14024 18737 9701 5604 13245
3263 5326 14254 14253 14255 14251 19565 4990 2198 4887 16593 4192
623 16711 18391 10980 7532 10316 16098 911 19278 8228 17323 16757
2097 18771 1562 8768 11770 13930 13210 9302 4394 18666 1679 19230
14127 8256 3565 3486 8305 17338 12200 14095 16106 16554 5101 9627
11323 13709 19928 15448 7178 6851 9856 17693 5450 14758 6440 4840
16948 10481 18098 12809 9820 8612 5560 2328 16487 4316 9793 9587
6024 4610 3518 14472 8379 17788 16283 940 18073 12073 12239 12641
1517 4520 4253 19655 19157 7457 14257 13738 9744 6631 11117 7100
10958 1663 19658 830 850 981 19109 1871 7127 18552 12232 3002 16895
13330 7142 5045 8449 15114 15111 19687 19859 2450 5262 5468 1622
15404 10115 3091 1432 7857 9421 10367 16178 16767 18032 14517 10922
4845 5596 17422 17656 14974 16264 17181 461 10535 13155 18603 4629
7200 12897 12379 19551 3158 16835 2030 2535 10521 8602 5328 6340
5196 12908 4510 6303 5493 710 15944 18673 17709 16365 17701 3274
14621 2173 3229 12063 8399 9226 19709 3984 17274 5355 11541 16698
15860 19429 17598 1573 5354 3074 10753 6512 4856 14767 3042 14876
3811 856 14273 14271 2338 13640 5456 5459 8719 10281 6810 7883
15726 15508 17683 3047 16285 12882 13574 3723 18442 11447 14287
18277 8323 8320 19125 8734 18846 8429 8735 6351 18016 7745 18581
16456 13515 4296 16800 3915 7965 11716 12382 5285 552 18741 19308
12228 713 1275 3193 601 4292 18067 7027 15113 285: 14398 1558 18339
13431 13428 4182 16260 10619 6127 12005 7704 5064 13072 4395 9032
19907 9588 478 12782 9917 13100 5202 10562 2044 4452 10775 3543
14840 14071 14020 10406 14218 1797 16955 3879 10312 12432 14824
7082 14554 15744 1508 15866 16232 6797 7077 5816 3596 3597 3311
15022 881 2638 906 212 12037 12039 3458 9042 8812 3217 9466 2270
18102 610 2431 16650 14418 2251 1244 4235 6826 16651 14435 13004
7852 18349 9553 9262 4340 9235 15603 1413 17223 8296 10538 16071
16048 2120 19847 11239 10420 18968 12345 2995 2159 2549 3987 7551
19585 5685 4861 16862 8677 13466 11435 11230 6015 6649 8097 16025
9440 1353 16249 16151 9398 15930 11226 2870 12373 15043 11765 10145
11071 9537 9095 5496 2350 11756 7967 13362 2624 7924 6715 15074
17068 9275 10177 11007 14857 8951 12182 11643 13590 1740 7684 910
929 9093 10018 13344 16978 6305 11378 17544 11969 1393 15937 11803
16631 13126 14108 18524 9644 8425 8421 8420 10755 16696 15994 10757
1143 3549 5868 5126 7506 19032 12669 12547 4707 4710 4708 4732 4736
4733 8772 4730 4711 3385 1831 2528 2304 13223 15197 15098 11673
4386 11884 2336 9633 9118 14693 2399 12725 14548 11011 18359 14692
5652 5434 9755 9513 2681 4364 19777 13525 12903 6859 4326 6088
11611 3068 3578 13940 8582 14440 19618 9826 13275 18012 9371 10057
5985 18222 15021 11502 14888 14001 1279 17431 18172 7826 14232 8317
10294 7425 15565 5531 17742 5564 18144 14389 13857 7113 16444 7400
10835 13461 4722 2115 13951 19022 5256 18254 7276 16719 1045 2920
3279 19766 684 19811 1237 15371 10076 8004 18712 2781 4940 11219
2673 19836 18985 14312 15772 3933 6426 7906 15599 11860 1907 18897
14263 16522 4672 13737 574 16112 15415 11543 3433 7746 4115 9525
5392 9445 2252 14696 17114 1400 9349 4881 14484 18930 2533 10428
7616 18415 10497 12585 10236 4874 19793 4633 12544 1995 17255 6130
10439 8504 7487 15261 6768 12863 3628 15985 17752 5019 2036 2840
18127 12020 9264 14875 7498 1448 1059 9636 2644 15865 6207 9893
3161 10031 16927 2065 7894 2171 4174 1254 18701 10035 19271 1389
12810 4747 12671 19586 10698 5651 9018 1931 4286 7712 3519 12527
13943 6181 5039 13948 16797 16826 18966 5145 17610 6304 8895 7744
9280 9284 10647 16558 286: 9313 1653 17351 14049 14657 1383 1015
18068 4392 10779 16383 9920 487 9934 2830 3247 2351 18430 10228
17404 17658 5336 5337 17655 1350 17686 17699 17685 5353 17632 5339
17682 1645 1642 1640 1620 1616 8916 8212 9895 2832 2783 4293 6486
10048 4251 5819 5822 5393 7543 8453 7971 5457 12283 11526 4620 4594
4593 12447 12446 12443 12421 12418 12419 4579 6639 4625 4624 4598
8851 4628 4580 4631 14489 13819 14308 18593 5807 15337 2081 12213
5581 14765 7934 18948 16079 6446 10336 6213 8673 10129 12010 19004
19003 13184 10392 11721 7649 9054 15693 14867 14868 7482 1784 9674
1270 19176 1199 8500 19667 4212 4448 3986 12515 15430 7679 18854
16043 4242 10959 13366 2878 11272 5100 3297 7169 19051 3475 13999
17136 17946 7102 9260 9177 5942 18576 8622 9353 18500 14477 16377
4784 16796 16425 17653 13145 16135 7307 4655 9544 12992 17516 2232
1408 2230 1599 11103 9661 8918 17531 7259 10941 16332 19728 2216
11076 2670 18069 18160 18744 1206 2891 5478 5142 4382 2683 19169
7606 11398 12629 18745 3786 17936 5887 3425 5214 8401 14931 18934
15251 6533 4671 6531 17015 1412 5092 8805 5545 6049 12372 578 17463
10263 1161 2814 5758 10686 7449 1330 10508 15984 8027 1288 5176
19668 1710 19355 3454 13918 14445 2162 14905 2727 17196 7122 9749
10816 13053 15837 11705 1700 2315 17710 12014 19134 14514 14744
2761 1547 12385 7818 16211 18823 9872 2759 2806 12795 9882 6415
1151 49287 8821 10229 14451 7419 9477 11295 15793 15660 11783 701
2955 17659 2504 17348 16342 15186 6406 8247 5380 13891 1315 1256
19915 1724 2012 14065 10299 8282 616 13375 9102 16056 11991 9014
9223 19193 5659 12275 17960 6092 13451 8592 8527 7016 15734 1394
3654 2224 9086 6674 15201 5282 3178 18367 19531 17843 8903 4152
2519 4236 14485 8563 18395 2904 12192 17438 7558 8708 17132 14463
2926 3606 14880 4126 13192 11148 18831 19343 3854 3197 1662 15209
1164 6993 19768 5846 5825 13392 3964 18837 3676 6815 1563 14197
4983 16851 1080 10309 13336 6679 6681 19226 1139 18217 6677 11476
10458 11376 11534 15281 15284 4323 17519 4170 18686 3737 12407 9314
18763 18235 2522 424 12163 6909 13949 13705 2221 17613 6620 10223
18017 9192 4434 7858 4011 19099 15849 2703 7837 16579 10683 5630
13463 4008 5853 15102 13390 15974 3534 9370 7120 13281 10677 6277
7442 19758 3296 12002 5857 6669 12717 15152 973 15151 5971 2689
2692 14737 15184 19235 11966 9246 1051 16723 7949 2967 13928 1691
17376 8438 6021 5801 5705 16980 13383 18063 14100 12568 5238 12085
17418 17386 7929 19078 16133 15135 4577 8414 19149 3621 10044 6007
16987 3208 3567 14540 9431 6837 9742 14981 18407 10889 5572 15933
2966 14048 10199 19043 8146 11747 2107 1220 19179 13356 3167 14446
7973 13997 10221 14201 10975 11488 8663 6381 2578 2575 14911 6552
16339 768 1468 18066 12089 17874 13394 9319 919 12747 7881 17584
17587 6226 19910 7627 18249 9111 10610 1083 6509 17564 5851 15935
14750 1415 7919 8561 13034 1757 7298 10482 17985 2674 18011 18159
6510 8905 4745 13968 5850 6365 18717 6735 7063 6097 7741 6692 16850
9791 1250 1976 287: 14033 3069 10887 17423 15804 10725 1039 8823
13656 1864 18512 3164 11788 9149 8808 5438 15880 3992 800 12872
18261 8766 14438 3187 10085 4070 901 13535 2677 8285 10764 19705
18563 18309 4608 19842 15814 13706 2652 1108 2600 11436 8221 18056
9465 14769 5688 5397 3900 1598 6393 5004 17760 15162 12125 12647
7151 11629 14327 14070 16315 15318 15939 1024 17173 7378 11794
17214 14034 2307 3146 11136 745 14629 1219 8349 9131 14106 2574
6164 13786 12212 288 9930 5003 15379 17113 11587 10350 16113 1863
10534 16794 10418 3427 14528 9660 19113 14120 18931 693 10649 15876
620 11764 4473 14614 15160 2527 7562 15931 15942 9412 10452 1278
9417 14663 6750 3059 18739 5729 11588 8690 1571 1240 12230 10197
3943 9396 19704 5587 3849 6395 2512 1364 4248 9916 17140 19660 9460
2383 2545 10421 10451 7528 17188 5982 10710 4743 17869 13867 15895
18341 13127 9124 11340 15482 4846 15991 3001 8402 17663 13283 11123
13596 1151 10214 18536 11869 2959 17628 2207 1422 5187 12698 16068
18921 3969 18923 15856 13136 15313 11878 7997 15604 14266 18654
16893 14982 19298 8644 8268 18840 18022 3726 3772 13530 12761 10851
9602 19087 12685 5286 4627 16074 10376 1178 17450 2560 825 10966
6542 5902 17622 13520 16539 494 18321 9756 4078 13338 11983 15481
17256 1850 10700 6895 19331 5603 16542 8629 14464 8541 11955 15582
1615 9393 11799 9705 10217 3894 5124 17122 10037 19916 3503 17417
13729 5423 16076 1843 12859 17649 1593 15088 11536 19105 6404 17208
9567 4208 3180 3864 9156 12512 15134 8390 16883 10879 8150 9719
12593 14926 14282 17273 6293 6538 13207 13438 1179 13854 1534 12605
12235 11858 6290 13936 15363 13652 15361 19700 3899 1614 9390 19686
12223 19528 1763 6129 2975 18334 4649 2415 18042 5063 4001 2807
5550 868 2976 14624 3321 10604 2149 11085 14039 5615 8502 14424
9369 14136 4541 3019 11951 18036 14808 13308 6822 19066 15323 12540
9754 11217 12299 6562 19219 2211 1980 409 1970 8961 14118 1424
14183 13802 3223 12957 11050 12155 17192 14643 15307 6911 12324
7373 11544 13247 17437 12217 11827 14360 17578 12753 6662 19268
1665 7212 10672 7843 1289 5216 4585 19139 7937 16633 5644 16320
11433 9472 12965 12360 3202 11429 11427 19689 1587 773 8864 14046
5266 9201 17227 10510 7291 10904 10655 4965 17040 2183 16534 15799
9967 13632 6850 19691 8788 16409 4673 17193 14098 11916 2451 4834
18460 18183 14691 19318 1489 12987 16441 17707 16821 18525 6452
5795 10306 808 5051 9434 4528 13359 1168 13904 3700 15002 12152
6475 2944 15440 18551 1414 4231 14124 8358 6492 16565 17743 5954
16843 954 5541 5859 18096 12827 15357 11338 14884 7974 10246 5834
3697 450 16197 8091 11058 8780 10822 12036 3632 1407 632 5735 2700
8621 4284 18605 5544 3482 7892 18630 6701 13975 18530 11623 10209
10341 12178 19162 4832 13434 19639 7936 1306 11386 4376 19409 7887
13961 19229 11753 10931 1751 18762 698 7891 10920 8887 5093 14119
434 10475 8949 9491 15868 17642 9386 8713 17515 11664 12640 4630
9414 7285 9296 14419 9418 14870 5136 2943 8767 9080 9232 9233 9981
8666 11032 6927 1544 17476 16917 19548 12188 2244 15586 11795 14909
4751 13962 9106 429 18945 4616 5199 10178 18224 7863 288: 14033
3069 10887 17423 15804 10725 1039 8823 287 9557 13656 1864 18512
3164 11788 9149 8808 5438 15880 3992 800 12872 18261 8766 14438
3187 10085 4070 901 13535 2677 8285 10764 19705 18563 18309 4608
19842 15814 13706 2652 1108 2600 11436 8221 18056 9465 14769 5688
5397 3900 1598 6393 5004 17760 15162 12125 12647 7151 11629 14327
14070 16315 15318 15939 1024 17173 7378 11794 17214 14034 2307 3146
11136 745 14629 1219 8349 9131 14106 2574 6164 13786 12212 5003
15379 17113 11587 10350 16113 1863 10534 16794 10418 3427 14528
9660 19113 14120 18931 693 10649 15876 620 11764 4473 14614 15160
2527 7562 9412 15931 15942 10452 1278 9417 14663 6750 3059 18739
5729 11588 8690 1571 1240 12230 10197 3943 9396 19704 5587 3849
6395 2512 1364 4248 9916 17140 19660 5811 9460 2383 2545 10421
10451 7528 17188 5982 10710 4743 17869 13867 15895 18341 13127 9124
11340 15482 4846 15991 3001 8402 17663 13283 11123 13596 1151 10214
18536 11869 2959 17628 2207 1422 5187 12698 16068 18923 3969 18921
15856 13136 15313 11878 7997 15604 14266 18654 16893 14982 19298
8644 8268 18840 18022 3726 3772 13530 12761 10851 9602 19087 12685
5286 4627 16074 10376 1178 17450 2560 825 10966 6542 5902 17622
13520 16539 494 18321 9756 4078 13338 11983 15481 17256 1850 10700
6895 19331 5603 16542 8629 14464 8541 11955 15582 1615 9393 11799
9705 10217 3894 5124 17122 10037 19916 3503 17417 13729 5423 16076
1843 12859 17649 1593 15088 11536 19105 6404 17208 9567 4208 3180
3864 9156 12512 15134 8390 16883 10879 8150 9719 12593 14926 14282
17273 6293 6538 13207 13438 1179 13854 1534 12605 12235 11858 6290
13936 15363 15361 13652 19700 3899 1614 9390 19686 12223 19528 1763
2975 6129 14424 9369 868 4001 2807 5550 18042 4649 2415 5063 14136
18334 4541 2976 3321 2149 14624 10604 14865 4260 2050 11085 14039
5615 11951 13308 18036 12540 14808 6822 19066 15323 9754 11217 6562
19219 2211 1980 409 1970 1424 14183 3223 14118 17192 13802 12957
12155 11050 12753 8961 6911 12324 7373 14643 15307 11544 6662 13247
17578 14360 17437 1289 5216 7937 4585 19139 16633 5644 16320 11433
9472 12965 12360 3202 11429 11427 19689 1587 773 8864 14046 5266
9201 10510 17227 16534 7291 9967 10904 10655 19691 8788 16409 4673
14098 2451 17193 11916 14691 4834 18460 12987 18183 2944 1489 19318
17707 10306 5795 6452 13359 16821 18525 16441 808 5051 4528 9434
1168 13904 954 5541 5859 17743 14884 5954 16843 18096 12827 15357
6492 16565 11338 19639 5834 10246 3697 7974 4376 19409 11058 16197
8091 1751 18762 450 1407 3632 8780 2700 8621 12036 18605 5735 632
10822 5544 6701 7892 10341 13975 4284 3482 5093 14119 434 10475
8949 9491 15868 17642 9386 8713 17515 11664 12640 4630 9414 7285
9296 14419 9418 14870 5136 2943 8767 9080 9232 9233 9981 8666 11032
6927 1544 16917 19548 12188 2244 15586 11795 14909 4751 13962 9106
429 18945 4616 5199 10178 18224 7863 289: 5523 4313 17448 17841
12777 10355 19605 19897 13150 13788 8075 5624 13087 8533 15156
16282 4418 5362 17314 16470 9691 6830 3539 8947 11303 15847 12513
12958 12763 11550 1760 7062 5192 9953 3474 3481 8116 13424 12304
5690 13692 18678 7503 12740 12452 4939 13248 3994 5264 3961 4726
4657 19545 17062 16253 1040 7214 5231 11985 5953 3077 10399 11073
3556 17461 15325 9938 4761 10626 12944 12889 11220 6105 15005 17775
7340 16006 1428 19624 11919 12601 642 3843 3912 4096 12974 13254
2664 16161 8861 13261 13575 9768 14949 19126 4880 5824 9299 14874
5687 2782 17505 17726 6275 3175 13677 4398 4025 5376 4677 18843
7388 15255 14128 2596 1938 14569 13062 4742 13091 16870 9122 11112
12762 3052 11166 2182 15922 13893 6314 10549 17230 10781 6932 11582
16011 6946 9000 6251 9407 11924 9877 6838 15556 15794 11808 19431
2295 1090 12514 7454 18403 2529 18862 19419 12173 3773 11501 1975
14174 16124 8277 11572 2186 4994 16607 12215 7089 1851 16812 1689
3238 11697 1993 14319 8424 1113 3997 2880 18417 15224 9191 8757
16205 13252 3135 18125 1294 2016 407 744 18246 19479 15883 18721
4036 14388 15680 19029 5232 12705 8217 16267 8222 11264 961 12449
16916 13761 3615 7172 14831 14301 16476 14882 7390 14607 14441 5660
11599 11359 4092 17343 13568 581 5071 859 5934 10571 12380 8016 725
9697 6714 8705 17074 1644 11944 12826 11957 4502 5499 5549 14898
1200 7101 18165 6291 6133 13712 11143 13419 3259 14157 11554 16748
6359 2357 941 11901 18451 1984 931 4926 6943 18727 6551 17793 4278
4905 3893 11593 10509 15596 4274 3302 3510 8231 18704 13258 4695
1347 8641 4776 16869 9696 16749 12938
4466 5831 2176 13023 7184 5489 8886 12920 6529 871 15574 8579 17493
7527 18633 2515 3708 13536 7991 3379 16897 290: 13013 2572 15390
1939 14078 15522 9968 9379 18412 18308 9944 6870 12684 3058 2845
13741 4261 19592 4701 17626 13030 8728 11077 4091 4089 17112 14261
13039 2448 19588 19632 10171 3875 16215 6248 6904 17027 14757 14672
18336 6783 16361 13415 7175 19494 2544 3982 12302 12865 16091 6469
16191 15776 6721 2879 4779 2699 14780 16535 10778 10809 10295 3281
8912 10017 11633 16868 2259 19421 16353 2746 14045 11766 14772 5191
8574 17749 1617 17592 15384 291: 17667 17054 19440 15213 7334 2141
3727 9845 11081 9526 12337 15918 15597 3255 10276 970 16639 19309
2222 15291 19583 14945 7813 9966 1286 1910 10491 19293 16030 19104
463 11758 9810 14676 9957 4241 14669 292: 9221 9220 6193 3253 14842
13721 4160 4741 7322 10303 16057 13497 13494 13514 3142 3155 3203
3201 3204 3231 3227 3230 3228 5987 3206 17127 17830 17527 19410
19413 12404 13866 13360 13864 13860 1092 18317 16638 3344 11672
1048 5311 11768 18957 9486 5520 4119 3271 14470 11906 13464 5650
8589 1858 2994 2964 2947 2923 14662 12783 5730 10701 3718 10132
8593 12855 6873 15376 15374 19023 14646 14645 9650 10687 844 17322
17326 6887 4652 3798 14561 15275 12003 17738 3837 3822 3819 3846
15647 8778 10880 17001 8637 3841 8776 18718 14843 3863 3817 13443
13421 13442 13498 3923 13787 3280 3276 18190 4216 12478 8586 7030
8614 293: 13730 7076 16121 2661 2089 15753 13834 1549 18755 5880
14792 946 10952 15392 19376 15572 11083 1945 12301 294: 19341 4724
19896 3258 5111 599 6431 13589 6942 6192 6252 13509 19882 4075 1265
10657 10589 5296 15003 18223 18248 2327 1261 9965 4027 15223 16926
14122 18287 13182 19642 13929 3304 730 11185 4607 2628 9008 18151
2691 11457 19048 6876 6857 15174 15158 17254 6991 11670 4110 4109
12772 10184 4872 12819 12694 6264 7048 9607 9648 12614 8990 7650
19393 5863 3904 10368 7157 16438 13128 17560 7535 1575 10105 10134
568 13578 4304 9818 7538 9257 18048 15120 18444 17647 18149 3239
2052 591 16792 10643 9825 9839 18609 3056 3720 12370 9361 1519
17206 2733 2404 2274 10638 1810 1681 794 5509 8149 6109 16956 4055
7526 9906 3806 4482 3406 8669 11941 8434 17353 1718 16055 16094
11109 13291 14317 16938 8688 12395 11729 19065 14012 4384 7554
19069 18471 16995 10788 16414 3527 4972 4041 7910 6178 2598 12998
9686 12367 8259 9712 9092 15589 17494 13102 3555 4223 2428 5186
13868 17026 19357 3670 16415 4465 5164 14396 8679 11667 4509 11014
4526 10211 3983 11652 13984 11918 3989 1532 19637 1516 8361 9956
2740 19492 19752 7125 504 18859 1252 13539 6656 17606 8695 18363
828 4343 4885 16387 12180 16021 6095 15293 7951 15342 16946 16808
5255 15684 11760 19481 5992 13140 3529 12315 13875 7700 1035 5477
5476 17832 8331 8176 7166 12559 4721 7461 1735 1862 4162 19901
18566 16648 17875 1044 17901 16027 3656 5177 4493 8389 7751 16700
19251 7033 8539 8515 6547 17753 6527 7218 4305 18907 2441 16894
8368 15641 7365 8148 6445 5873 7736 9634 9685 9688 5086 9147 19697
6980 13974 14084 14057 14234 16981 7596 2200 9879 6560 7032 4346
9104 12902 9061 16803 18628 5254 6524 19495 6553 1235 13979 4589
5122 8755 5028 18168 8279 14683 17291 3546 19846 17369 19927 12627
14146 16105 17234 15988 10866 12145 8926 6958 8927 4271 16119 10894
10871 14052 4521 4546 19411 17524 542 18878 6595 2294 11153 9855
9127 6844 8065 9659 15206 10237 10286 5228 7708 16193 16173 1816
2487 7234 18474 15871 18498 18386 9860 16437 13117 11881 10754
18738 9129 5234 17737 539 13246 11876 5966 6301 18615 2022 1249
15906 6134 1486 1737 17536 16641 16126 2117 18070 1727 19256 772
771 16084 13449 415 1588 4731 11912 13036 12904 10167 10170 10181
11796 7931 4436 19552 15340 9069 6065 9576 14476 4033 6144 6355
9580 11373 3845 10555 6026 1538 14252 11149 12524 19249 7977 8112
14058 7344 7310 12622 12584 4642 6874 16745 440 1628 829 15558
18297 1682 2486 4188 5990 15852 3101 17511 647 6316 8897 18910 1311
5918 17076 9692 4085 13054 13063 16969 6300 295: 10020 1439 9915
16163 18062 10972 18887 18356 4431 1884 10727 13800 12798 2858 7343
14738 2791 14434 9539 17180 8662 5767 8275 2125 6101 2121 7866 9540
2780 5578 19289 18868 12356 8526 2380 13296 3693 16875 2379 13074
19810 949 14344 1531 16649 14889 14044 4090 3535 14795 14076 11583
15037 3570 4564 3572 16352 16416 1362 5033 6120 979 439 15995 19886
17585 14283 1830 6945 9194 11025 1647 6731 8953 4154 14591 490
17151 13499 11392 12621 8859 12715 16405 19405 18614 9282 12579
6899 1876 7114 15543 19677 5323 11508 12990 19006 18475 3862 7010
19748 1860 15260 4534 296: 4540 6354 1755 1754 17371 3891 9752 4896
3390 13183 12597 8675 19057 6229 4548 17590 5708 4363 6198 17103
9094 2389 11801 14837 6762 297: 4370 7533 2597 5919 4081 1338 1069
10498 19778 13926 7098 7281 7968 12746 5461 13420 11565 11748 7477
1523 7224 17897 18082 14625 17717 1367 15055 3466 18152 3922 5925
14810 16421 9929 17561 14752 18760 19598 12786 11882 9247 5551 4562
3594 11415 17495 11692 12861 16921 838 16714 10602 15187 4256 6989
12555 10731 13076 7618 12876 10208 869 12690 7839 12981 17982 15557
6454 5181 4327 12171 8233 18590 17657 298: 16642 4906 6546 8807
15527 1974 3476 14666 13472 17605 12833 9706 9679 4373 19761 15703
11680 18191 19790 19596 6939 14155 3044 7880 10088 3490 299: 13175
3648 1095 19091 6471 18453 11391 11863 12390 14399 8543 2686 18964
17580 17565 300: 15769 8388 13307 16886 9617 9239 11198 2854 892
2335 11948 5159 11294 8727 8073 11276 15356 19272 11054 301: 10572
5247 19764 5097 11353 18884 8479 9341 11247 19532 7752 2918 6108
14397 6315 302: 17017 11635 5436 17789 15563 8900 12793 3944 11383
6001 6054 7021 17088 19664 6262 2642 18990 4151 14818 7272 8345
5258 16301 1266 4095 18986 14878 10156 303: 9378 2246 16919 2665
10325 6537 6515 8525 17336 2540 304: 3039 5198 11864 16540 18772
6379 16878 17787 1436 12983 13229 16103 16127 8943 12794 16102
16100 4759 19321 8190 2913 15179 3181 5096 12048 6045 692 3816 5920
473 489 14828 10454 1781 15219 3647 12472 10767 4582 2698 3361
17036 3796 8573 19690 16677 8338 13554 16002 17464 6225 3244 7491
16139 13938 11138 14770 18781 12979 6776 12028 7778 6586 305: 6411
7134 8906 16488 13440 2289 8244 2283 7468 6684 10347 4285 18467
13646 6223 12937 4443 18455 1856 14107 6321 2537 3970 3246 12156
16596 16256 12890 15409 19151 4718 5175 11879 18126 18711 9204
13563 9579 16836 306: 12951 10398 11346 19186 15452 7802 7096 13491
13681 11482 14807 3733 17000 671 17496 8110 12773 17405 2034 7911
17108 17121 17123 10527 13104 9923 15163 15161 15672 1133 7107
15159 16049 2789 5414 19524 9684 307: 17075 12342 704 19539 6820
19480 17050 17008 19402 15902 18440 5435 2820 13268 13272 6889 3099
441 18043 8140 7516 17639 18575 12426 10811 14604 7842 4222 5559
17265 12910 16743 603 5928 7036 308: 9770 17349 9595 3084 14563
12042 12040 1361 7692 7690 7693 11111 13156 9807 11464 16693 16672
16674 16694 16675 11027 16798 4507 7680 7462 15210 11446 7522 12111
10983 13675 15116 10650 17747 18142 6554 18344 14846 3610 1916 1918
1920 10514 10515 5814 16298 16963 8419 5633 10896 11971 11972 11360
9381 15416 8935 9451 16130 7252 12038 12592 11897 11899 9157 12294
15591 3634 3640 6918 1028 1025 1010 7954 6924 6937 3533 5048 13534
13474 10893 902 10939 9585 2197 2948 10393 11720 14453 7240 11026
11092 11088 13756 13185 13798 13762 13187 13799 16822 9450 14147
11126 10984 11917 14454 6394 8064 8050 1033 4682 5085 13556 3724
3725 3721 3722 3710 18855 16128 4243 13813 13676 13159 17397 9345
13367 5940 18157 464 11157 3264 3704 3705 16760 646 11952 7153 3598
8442 9161 5867 7339 1404 15165 12587 12670 19102 11078 3824 18746
9973 5144 1144 7739 4130 12607 1009 1004 1006 11992 1285 16777 7909
4484 8987 406 3215 13397 1047 5188 16783 5519 18583 11625 11004
11056 11059 6050 3963 13604 8117 14971 18716 17730 12013 7877 17204
5195 19070 4915 13843 19554 4417 15788 11190 1693 2846 16330 11950
7243 10375 3691 3684 3687 17915 16005 1671 19540 10499 10225 15761
1933 15767 2505 8246 16343 19493 18295 12056 10278 16556 16134
13206 13209 16090 16088 16110 9103 10581 9834 3635 13203 4829 5828
1395 2655 597 19923 5896 16913 5772 1466 3658 3683 821 19840 795
820 15202 10661 15913 19756 19772 405 19504 5314 3672 6313 9762
9767 14249 2904 2899 2900 8635 18813 7724 8042 16951 14555 9160
2926 14241 2897 2907 6915 6922 6897 9718 18623 16845 656 654 659
16108 18227 12270 12018 10063 10061 13077 8431 11119 11118 16150
16966 13427 3374 18426 7386 11478 3612 3613 3636 6900 6903 16373
19217 6757 19030 10360 13103 6818 14125 6759 13921 13923 1196 13924
4819 16656 1658 8471 1659 11962 14022 10872 13157 16640 7948 18681
18699 9529 8931 12442 1512 3143 16501 19154 6032 6908 18549 9542
12945 3916 18656 12708 15592 14240 9740 9737 14661 1110 13204 886
14017 9925 2939 15031 19131 1242 483 2701 9085 19779 2445 3183 8143
14002 9189 19747 2742 4298 18369 5914 14761 2103 10374 8783 8779
8777 17066 2087 8596 4717 10877 14774 8813 8334 16562 10853 8024
16960 13916 13919 15016 15751 12115 10855 2038 746 14851 19743
15226 16549 2267 6875 7927 1005 9649 9959 7485 19025 14411 14374
9783 13960 2711 9164 3716 2547 17424 18941 14075 13323 5543 14815
2165 1756 13839 16367 15644 11187 13216 6388 12024 17755 13911 9905
17809 19499 10196 18637 7216 7392 11656 13835 19161 16251 11503
6720 2060 15709 5305 7634 17852 18207 16818 3165 3167 12327 9373
14446 11406 9769 2446 11776 15103 15104 9805 5938 19634 3749 1241
11031 13445 16440 11033 3821 17474 17796 16654 11605 11600 17919
4230 11602 17229 15737 7785 3844 3847 519 688 4144 11023 1132 10390
15833 11665 4077 12918 16563 15661 7525 9509 17053 887 778 9741
9759 13398 9612 8193 17328 1726 10300 10279 9459 4992 8844 9571
11468 16657 10900 10059 4404 16431 11209 1678 3452 10909 10062 4426
1897 4429 8284 16799 18573 16149 16964 6742 6744 5361 16614 5359
12651 15347 15877 15350 15344 4440 3577 3488 4442 10910 10911 14004
10912 10807 15901 13213 4453 4455 4477 4149 10914 4145 4506 4504
4500 4481 577 17702 16492 16427 16446 11291 16449 16422 11428 16623
11430 19757 14384 11120 19257 11484 6191 11608 846 12045 12044
14753 16772 3629 13129 8555 3650 3653 10024 12068 12069 1079 9792
9323 9336 9318 12730 11003 10356 10985 6919 18233 18757 9875 9790
9802 11466 5912 9363 883 11049 11994 14457 3872 11895 9736 10974
19097 11659 19098 11658 9788 309: 16801 13132 15648 16447 8652
17833 5712 8074 10690 3442 8077 16017 4336 4334 18570 13315 10785
16462 16900 17538 1921 19152 17383 12486 10936 16527 2668 17086
13286 16325 15106 13376 6034 10789 18519 10507 14473 19441 13022
10831 1501 10537 763 17615 18865 8762 11975 17249 1355 1352 1731
3928 897 5821 9053 17271 7633 3483 11371 720 6786 10425 4104 19770
14043 16580 16413 15086 13278 2632 19416 11567 16915 397 7115 6155
10323 4935 10287 7095 9624 14788 15196 8456 15783 6496 1418 747
8160 15292 7226 422 8460 777 12388 7677 4483 18613 12189 5109 11791
10558 6465 10804 9227 17539 7309 3145 8845 16327 2894 1639 629 7456
18787 8517 4205 13792 7154 8540 19386 12121 8153 18037 14847 2324
11604 5469 9801 18468 17366 14140 12739 8760 7404 15266 14677 8556
8459 8628 6999 13739 10675 6389 19049 14756 908 978 13391 15622
2116 3418 6687 7203 11089 7349 10890 12729 12757 12691 8258 15154
5394 2810 6594 13059 13162 13166 13276 13782 13550 9631 11740 13230
18487 12114 13130 765 19329 16985 16984 5165 16244 3083 4150 10606
8313 3214 11734 4026 14277 12261 15383 15691 14747 10250 7726 5530
9870 13909 4204 5398 7793 3405 7816 1214 655 5288 9771 18267 18824
18445 5386 6029 17183 14760 17340 14748 14228 707 15468 7547 7657
4329 6352 16661 19814 2314 3041 8145 7582 19453 4067 11236 9815
11632 10505 5399 5401 5701 3262 7403 12127 7921 3166 6436 1351 6654
13970 6833 12197 8822 8554 1854 17475 5955 3220 10389 18522 18779
11151 13163 16644 6673 19122 8310 11845 11773 14281 8380 9076 16982
13329 19679 12412 10783 16471 12667 4679 11260 17650 666 16258
15997 18585 8921 10213 7197 5699 9843 6432 2595 1776 740 11551
15041 10033 14055 10738 16517 16515 13985 6821 4687 3337 14627 7093
14433 13024 16428 7275 3380 4043 18350 608 14968 2172 16860 13317
8029 18848 11693 11576 15045 11780 19931 1062 11038 10639 11224
11221 2227 3034 6350 5148 7749 12030 8913 7568 3467 5635 3053 4469
12468 7698 10930 8896 10567 5095 10895 11854 16996 7668 15453 7431
7583 8611 5945 3403 2901 7239 8207 19926 4267 5634 2493 15967 18640
17304 14193 13803 13885 12365 19133 15544 17454 12090 5312 11677
14113 8439 6438 1093 1935 8879 6523 2410 4492 1778 17725 1981 17098
9203 11167 16434 12877 12668 8547 1722 11460 9512 9552 5897 11717
12745 12744 4809 4793 17479 11627 8724 14508 4543 10264 4971 8893
12051 5043 4355 1461 19129 13714 13778 4337 1055 12988 17873 9876
17972 770 14159 4321 9397 9625 4982 12453 12399 15388 13950 19243
9355 1752 1060 16605 8940 17507 5292 4698 6678 1898 8775 4290 2447
16769 3911 11807 5120 6985 4114 3219 17056 14361 17019 4749 8281
1577 17419 16965 17327 5163 19684 6013 18109 15816 18702 17013
15234 16355 14223 9490 1545 18506 18978 18616 9311 10823 8457 8366
18318 6280 1223 3810 19685 10902 7638 4578 7075 3196 14114 9172
3199 14132 12017 11925 1300 1299 18838 3719 12015 11644 5046 3195
19721 18624 15377 19934 6278 1999 1378 12351 5552 13478 7204 16346
1190 12660 10133 10832 11577 11662 11896 13158 19477 7317 4352 1153
5239 13031 8108 17846 9200 7760 2474 1105 8336 10576 16823 13973
16952 16876 12397 479 1881 1602 6077 2802 12398 4605 2551 8786 2140
2855 2834 9841 17023 18871 17373 17344 16728 10808 18676 16746 8129
2385 8180 3562 5933 4787 2996 10676 15651 12169 19544 7801 7900
2278 855 7521 18383 2546 1234 12032 19919 3652 721 8963 1814 10032
8605 10275 18902 12424 12526 310: 4993 14546 1029 7514 2917 10152
11335 9594 14726 1405 17937 6935 14151 10273 685 5997 7222 14658
11749 10121 7000 10002 6010 16908 7529 2137 5389 15290 8071 6027
4413 15840 17540 6072 5406 10477 13780 15060 14826 18210 10116
10120 10142 16667 851 4735 17780 8426 10161 10163 18631 4975 4344
3194 6508 3557 11735 16259 4023 12288 16472 15236 18423 2712 6253
7180 8745 16575 12645 11798 15025 14872 12821 12055 5069 8763 5400
5829 7705 7669 2817 5344 1590 8606 18818 5065 13075 16682 18975
16411 12120 1632 2145 10107 6360 3618 17320 18641 7159 19374 8892
1683 10110 12001 19521 2499 7464 11752 11792 2395 3322 19468 1246
2094 17945 13685 3703 7073 8473 15278 11750 6376 722 8952 1788
12276 2177 8655 3659 2253 15358 9315 2534 8070 15012 13161 10545
826 3249 6401 10373 13341 5991 4410 9993 9994 4785 3349 11306 9814
13902 10962 18365 854 5075 13311 4178 13328 18181 19894 19525 14890
1603 6161 14478 9243 587 2986 3592 12649 3080 15617 11601 13501
13181 6206 12788 7897 11732 2732 7914 3063 8862 19937 4586 2634
18225 17758 1058 4002 4561 14369 2043 9570 3991 19823 19209 11521
18784 14970 6473 6131 8658 7280 15987 11465 8315 15256 19681 16402
17975 1559 5862 3880 11271 16235 13872 2108 14500 11285 16316 19436
10432 7652 11110 16345 19523 2843 17746 1118 7774 19578 18424 4835
14580 15727 5582 2332 15462 2821 17034 16853 10210 605 14264 12320
9478 13425 7636 16940 15239 5379 13944 8103 651 15792 12912 15640
3131 7232 11772 19236 15809 4006 529 19806 7702 4689 17509 3421
19306 5782 5183 10158 6488 7040 13260 10093 13627 8732 686 10592
15289 19347 9038 13009 13925 7052 14143 12780 12107 8739 14688
11400 7874 6474 3104 3107 14423 1690 5872 1941 9668 3685 11900
10682 15943 14081 19665 8352 10943 8232 4088 18499 14024 14211
18737 9701 5604 13245 3263 5326 14254 14253 14255 14251 19565 4990
2198 4887 7549 16593 10140 4192 623 1967 10165 10137 16711 3289
18400 18391 18244 10980 7532 10316 16098 911 19278 11727 8228 17323
16757 8819 2097 7207 18771 1562 7815 9992 16477 8768 11770 13930
10114 10138 10097 10099 13210 9302 4394 5658 18666 1679 11104 1773
19230 14524 6799 14127 8979 14095 16554 5101 8256 3565 16106 3486
17338 8305 12200 9627 7178 11323 13709 19928 6851 15448 5450 14758
6440 4840 16948 9856 17693 10481 18098 12809 9820 8612 5560 2328
16487 4312 4316 9793 17788 16283 4610 8379 6024 3518 14472 940
18073 4253 1517 12073 19157 19655 12239 12641
4520 7457 14257 13738 6631 9744 7100 10958 1663 19658 19657 850 830
981 19109 1871 7127 18552 12232 3002 16895 13330 7142 5045 8449
15114 15111 19687 19859 2450 5262 5468 1622 15404 3091 1432 7857
10115 9421 16767 16178 10367 18032 14517 10922 5596 4845 17422
17656 15742 14974 16264 17181 461 10535 7200 13155 18603 12379 4629
12897 19551 3158 16835 2030 2535 10521 5328 8602 6303 12908 6340
16365 17709 15944 18673 710 17701 14621 2173 12063 3274 3229 8399
3984 9226 19429 19709 17274 5355 16698 11541 15860 3074 17598 1573
5354 6512 10753 4856 3042 14767 3811 14876 856 14273 14271 2338
13640 5456 5459 8719 10281 6810 7883 15726 15508 17683 3047 16285
12882 13574 3723 18442 11447 18277 8323 8320 19125 8734 18846 8429
8735 6351 18016 7745 18581 16456 13515 4296 16800 3915 7965 11716
9558 12382 5285 893 552 10143 18741 19308 284 3200 12228 1654 713
1275 3193 1053 601 4292 7027 15113 11082 1186 311: 13531 15014
19184 4117 8168 10792 10850 19284 5526 10160 888 19833 19024 13115
9575 3212 14092 11196 16286 14686 5558 17090 17081 16907 15798 5743
12604 593 8040 15926 19225 11909 5613 8553 2850 19417 15940 5913
17199 13547 15258 14739 402 14718 10577 2249 2275 7788 11681 6460
1636 8604 12864 2293 8498 7265 8520 7952 12354 16688 12128 1747
6881 10130 17339 3222 18097 865 18477 16994 5237 19366 395 5648
7676 8241 14849 16511 1950 3133 15141 12703 6805 4165 17466 13619
14951 10472 5901 7341 11090 7199 6418 19254 13897 13967 6098 8683
6978 11297 10739 18490 11417 13326 15170 8985 4283 10620 10050 2430
5975 15405 7764 12075 11938 5271 18351 2785 12328 14687 15954 10173
10759 17131 7683 6392 9694 19508 11227 14431 10991 10955 5333 17596
14716 12198 17690 13772 11828 9888 1787 3935 15629 403 11568 12658
9592 16152 16381 18209 19448 16752 14848 2937 5447 7889 8469 9447
13876 12634 18731 15627 12984 10207 3116 8826 6318 8934 3762 3827
1708 7049 9274 12720 9444 9420 7354 14664 6570 4045 1225 17512 4782
7308 1264 7406 3352 15122 18219 1822 2876 14134 2291 3261 16739
2348 11942 11875 9500 6270 9871 6173 5722 16368 13322 16507 13154
9751 11960 1438 3048 4276 19604 2680 11597 11557 8211 1536 11015
10386 6448 17614 15609 11101 10314 7156 6322 4619 11816 14775 1023
3639 18377 18621 18433 10468 17302 1949 17435 9669 4544 1990 7409
11278 9214 16595 11711 18807 4491 14566 15498 15400 17691 19615
14984 2401 18622 13403 8647 3919 733 12287 9690 2714 4040 7221
18345 19682 16469 10317 16625 3496 2388 12510 10301 19547 15032
8347 1998 2048 14532 10402 4795 13067 6630 12135 4047 7346 15387
1896 11660 3389 19879 2061 6988 19738 13900 4474 2621 17457 11124
8595 8920 19461 11829 16912 9638 10219 14338 4225 12172 9374 10078
11281 3024 15023 11963 14871 13655 10963 11216 16949 17195 6722
17870 4862 12097 5511 1927 6433 11365 12620 12118 8817 4187 18833
16333 4734 2143 19881 4183 19538 447 7211 18329 5917 9943 14372
8806 16953 312: 9795 6261 5149 5170 5168 12820 7573 12233 9643 9693
2068 11819 18352 16729 2793 17847 7225 12946 8557 1832 444 10898
1956 4691 7777 13647 19902 13284 13061 12536 5153 5151 16950 2990
2989 2988 3487 2993 9433 3848 1198 1195 12880 1340 18680 17799 7617
13361 9528 3585 6230 9821 3940 6243 18903 7878 9794 11915 6414 8130
3463 15900 17558 2602 313: 19107 3731 10544 1365 315 15072 9222 995
13810 10579 12184 1169 10820 8682 17063 17115 11426 7610 8335 6680
19590 669 12808 2095 3210 7981 8975 13797 19673 10108 12140 14891
4849 9589 10013 11489 5848 8508 16961 14635 3335 5341 9626 18112
14644 12785 9168 9484 8109 9837 3294 15636 3494 2017 19052 19769
16972 7811 19903 14579 7377 4121 7557 13806 6626 9547 10641 12326
19729 7235 1411 3644 10154 10157 2648 7794 10100 12628 15633 12168
7589 15853 4368 19745 14933 10255 1291 17736 2414 7188 16138 9089
9415 11728 12818 6609 8114 10065 12609 4638 16498 2517 12709 17410
17048 15136 16713 18469 19832 12689 1479 15584 12811 17562 13267
13892 10949 9327 472 5713 11273 9716 15698 5082 17282 1612 7565
10794 11434 8216 10240 6588 15316 15700 6461 7231 10826 9945 1966
767 9017 8428 9216 1791 12429 5223 4910 19009 13274 12648 16308
10486 4798 9611 5747 13303 1346 2839 10642 10405 4049 4046 969 5871
13542 17764 7597 9272 18722 14315 13433 12396 17102 19120 3267 2911
2934 2480 15593 1566 15118 17099 13495 18798 16391 13630 12936 4072
17390 11981 12247 14556 14559 12268 14558 14560 14577 12267 14576
12226 9387 332 2109 7841 18828 9615 9388 17887 3007 11137 19936
14986 3553 4554 11929 16099 5440 845 18184 2805 4097 18866 12066
14917 16033 3934 3003 1594 10929 12343 16214 17420 5606 4643 10415
6601 15702 10449 19711 13552 1507 19835 1698 9321 11696 2863 14802
17698 1498 17572 14405 13598 12043 2262 567 1583 2618 3734 18793
19614 14449 16036 8175 19636 13279 17286 5486 17542 9087 15980 2290
4878 11904 428 10747 9550 13537 3804 8971 5843 3250 1514 18175
14056 11021 12535 9436 8113 10697 15175 7725 13365 5679 15598 19442
16718 6306 9803 17134 10762 2355 13282 9344 18414 962 10707 13732
15247 16140 14944 8665 3901 12556 13238 3850 16213 15720 5407 7133
17222 15571 4342 952 16118 708 14879 2997 19403 14894 498 14617
11730 18476 3794 6451 3609 16585 3917 3066 11348 16371 13698 6380
5627 5614 3881 18493 6416 12573 15178 7043 8200 17880 11947 15915
7621 7041 8209 18926 10681 14294 3702 8567 18263 9236 1978 3932
19800 9976 13414 8046 11624 1429 3768 17319 17779 7609 16512 630
3171 7227 3551 9996 16499 8855 13654 9117 3812 16292 19434 10320
5172 1982 3126 14582 9645 1807 19478 7519 12941 17861 14481 4909
1899 3459 1922 19333 4083 1541 12260 11304 16432 3209 9163 2838
17263 2745 9173 17072 5826 7351 9534 19739 19536 7374 17185 14542
17759 5670 4857 6297 13448 7546 11655 18214 11988 3497 12119 18421
2238 9138 8088 14015 6428 10563 6419 14637 4955 9470 1521 11999
10694 314 18961 1819 15545 10660 10122 4646 13358 18428 15805 12921
13439 13112 10688 18791 3218 10153 5875 15881 14320 2938 13237
12193 16805 2072 13687 15276 12110 9603 12516 3748 11719 18595 2719
7437 2981 5840 16220 2127 19699 11867 10298 3040 4738 17635 17582
9714 2444 8326 10594 16463 11355 16573 18034 10940 2168 14219 13085
7255 8220 15653 19511 18649 7840 8684 2272 9676 5395 14310 13304
17528 4645 2741 19834 12291 3607 4917 15188 1038 15537 16448 8891
10556 10553 13996 16085 16717 16720 16629 3416 6712 18027 18028
18046 18045 18018 2046 2280 11438 7044 432 17329 10580 2438 10575
2435 12466 13227 13331 3373 4354 1137 14182 14180 17288 17285 17287
17309 18279 17281 5134 13133 13134 7591 7579 7578 7590 7622 7593
7619 8710 11257 10155 17393 5497 7611 7613 3368 3371 3345 3347
11146 3346 15875 3327 11270 617 12175 15844 16942 6398 16524 14919
6019 6427 4568 13742 8545 8646 17237 10441 15117 18131 812 16684
7548 4903 4891 11339 14205 916 13861 4237 5869 14326 7059 9757
15666 8359 2239 6482 16408 10426 1600 4339 9125 15701 10147 6375
17364 694 11598 14712 1946 16250 7313 3680 8784 12459 4338 15013
3889 4760 2323 2136 16586 3470 11223 13993 8676 4320 6154 958 7689
4997 6877 12357 17840 14977 10169 1152 12474 11366 1049 7296 3453
823 1007 2433 5629 7080 8534 2047 12554 18572 15080 13812 14010
15955 19762 16570 11714 12363 13476 11640 13481 6845 19136 3340
15749 8548 16230 2819 2803 2874 2851 2798 2774 2772 2847 2769 2801
11161 14362 3590 279 19320 14355 13633 12071 4122 10856 1712 8501
2235 2242 2243 2245 18869 17430 17096 16063 16061 4419 4399 7499
7500 7479 7507 7501 7478 2530 9228 852 5988 15329 19925 15542 16445
15520 16918 16920 16451 14285 15560 16922 3473 2729 2751 2748 2730
1449 6008 10997 314: 19107 3731 10544 1365 3521 18558 315 15072
9222 995 13810 10579 12184 1169 10820 8682 17063 17115 11426 7610
8335 6680 19590 669 12808 2095 3210 7981 8975 13797 19673 10108
12140 14891 4849 9589 10013 11489 5848 8508 16961 14635 3335 5341
9626 18112 14644 12785 9168 9484 8109 9837 3294 15636 3494 2017
19052 19769 16972 7811 19903 14579 7377 4121 7557 13806 6626 9547
7168 10641 12326 19729 7235 1411 3644 10157 10154 2648 7794 10100
12628 15633 12168 7589 15853 4368 19745 14933 10255 1291 17736 2414
7188 16138 9089 9415 11728 12818 6609 8114 10065 12609 4638 16498
2517 12709 17410 17048 15136 16713 18469 19832 12689 1479 15584
12811 17562 13267 13892 10949 9327 472 5713 11273 9716 11114 15698
5082 17282 1612 7565 10794 11434 8216 10240 6588 15316 15700 6461
7231 10826 17284 9945 1966 767 9017 8428 9216 1791 12429 5223 4910
19009 13274 12648 16308 4798 9611 5747 13303 2839 10405 4049 4046
969 5871 13542 17764 7597 9272 18722 14315 13433 12396 17102 19120
3267 2911 2934 2480 15593 16755 1566 15118 17099 13495 18798 16391
13630 12936 4072 17390 11981 14556 12247 14559 12268 14560 14577
14558 12267 14576 12226 9387 332 2109 7841 18828 9615 17887 9388
3007 16099 12066 845 18184 4097 18866 11137 19936 14986 3553 4554
11929 5440 2805 16033 3934 3003 1594 10929 12343 16214 17420 5606
4643 10415 14917 6601 15702 10449 19711 13552 1507 19835 1698 9321
11696 2863 14802 17698 1498 17572 14405 13598 12043 2262 567 1583
2618 3734 18793 19614 14449 16036 8175 19636 13279 17286 5486 17542
9087 15980 2290 4878 11904 428 10747 9550 13537 3804 8971 5843 3250
1514 18175 14056 11021 12535 9436 8113 10697 15175 7725 13365 5679
15598 19442 16718 6306 9803 17134 10762 2355 13282 9344 18414 962
10707 3609 13732 15247 16140 14944 8665 3901 12556 13238 3850 16213
15720 5407 7133 17222 15571 4342 952 16118 708 14879 19403 2997
14894 498 14617 11730 18476 3794 6451 16585 3917 3066 16371 13698
6380 5627 5614 3881 18493 6416 12573 15178 11348 7043 8200 17880
11947 15915 7621 7041 8209 18926 10681 14294 3702 8567 18263 9236
1978 3932 19800 9976 13414 8046 11624 1429 3768 17319 17779 7609
16512 630 3171 7227 3551 9996 16499 8855 13654 9117 3812 16292
19434 10320 5172 1982 3126 14582 9645 1807 19478 7519 12941 17861
14481 4909 1899 3459 1922 19333 4083 1541 12260 11304 16432 3209
9163 2838 17263 2745 9173 17072 5826 7351 9534 19739 19536 7374
17185 14542 17759 5670 4857 6297 13448 7546 11655 18214 11988 3497
12119 18421 2238 9138 8088 14015 6428 10563 6419 14637 4955 9470
6734 1521 11999 10694 1819 15545 10660 10122 4646 13358 12921 18428
15805 10688 13439 13112 10153 5875 3218 18791 2938 12193 15881
16805 14320 13237 2072 13687 15276 12110 9603 12516 3748 11719
18595 2719 7437 2981 5840 16220 19699 10298 2127 3040 11867 4738
17635 17582 9714 2444 10594 8326 16463 11355 16573 10940 2168 14219
13085 7255 18034 15653 19511 18649 7840 8220 2272 9676 5395 14310
8684 4645 2741 19834 12291 4917 15188 3607 13304 1038 15537 16448
8891 10556 10553 13996 16085 16717 16720 16629 3416 6712 18028
18027 18046 18045 18018 2046 2280 11438 7044 432 17329 10580 2438
10575 2435 12466 13227 13331 3373 4354 1137 14182 14180 17288 17285
17287 17309 18279 17281 5134 13133 13134 7591 7579 7578 7590 7622
7593 7619 8710 11257 10155 17393 5497 7611 7613 3368 3371 3345 3347
11146 3346 3327 11270 15875 617 12175 15844 16942 6398 16524 14919
6019 6427 4568 13742 8545 17237 8646 812 10441 15117 16684 7548
11339 18131 4903 4891 14205 916 13861 14326 4760 4997 7059 9757
15666 8359 2239 6482 16408 10426 1600 4339 9125 15701 10147 6375
17364 694 5869 11598 14712 1946 16250 7313 4800 3680 8784 12459
4338 15013 3889 2323 2136 16586 3470 11223 13993 8676 4320 6154 958
7689 6877 12357 17840 14977 10169 1152 12474 11366 1049 7296 3453
16153 10586 1007 2433 5629 7080 8534 2047 12554 18572 15080 13812
14010 15955 823 19762 16570 11714 12363 13476 13481 6845 19136 3340
15749 8548 16230 2803 2874 2819 2798 2851 2774 2772 2847 2769 2801
11161 14362 3590 279 19320 14355 13633 12071 1712 8501 2235 2242
2245 2243 18869 17430 17096 16063 16061 4419 4399 7500 7499 7479
7507 7501 7478 2530 9228 852 5988 15329 19925 15542 15520 16445
16918 14285 16922 15560 3473 2729 2751 2748 2730 1449 313 6008
10997 18961 315: 2185 11361 5661 8686 17264 8328 8330 7550 10232
18956 1696 5922 16632 14340 10579 4523 1169 11996 8012 4522 16791
8335 10038 5066 4969 4634 17213 14401 7284 2099 15619 17243 6718
8030 14321 1903 15554 15028 12204 3060 19178 8109 11653 9316 17858
18132 18589 9667 17297 5418 17508 15450 6147 5012 18588 18020 862
4920 15050 16525 3064 2884 5607 12297 11396 18052 2912 17060 1697
17604 18870 8264 17736 1732 5968 5593 15864 18761 4044 5088 17149
10593 5242 4527 13682 8054 16503 1962 19380 1623 10664 4597 15319
3478 16945 18584 3108 12724 15546 15764 15754 18354 18416 4890
19678 19130 15532 5213 11374 9753 14702 15217 17235 15921 5855
18752 1904 15721 18203 18156 18864 8700 7560 16924 636 6811 16379
19046 11063 10405 4049 4046 969 14929 16207 7597 9272 15425 17368
11052 14975 6574 9560 10349 3830 8250 2911 2934 2015 18785 8645
8196 7713 11586 14490 18301 18792 15378 7699 18803 19816 16104
14468 17390 11981 14556 12247 14559 12268 14560 14577 14558 12267
14576 15277 12222 16710 12224 12226 9387 332 17612 2109 7841 18828
11556 1969 9578 7424 5369 245 18873 16602 11137 19936 14986 3553
4554 11929 5440 2805 12674 16033 3934 3003 1594 10929 12343 16214
17420 5606 4643 10415 14917 6601 15702 10449 19711 13552 2863 1507
19835 1698 9321 11696 14802 17698 1498 17572 14405 13598 12043 2262
567 1583 2618 3734 18793 19614 14449 16036 8175 19636 13279 17286
5486 17542 9087 15980 2290 4878 11904 428 10747 9550 13537 3804
8971 5843 3250 1514 18175 14056 11021 12535 9436 8113 10697 15175
7725 13365 5679 15598 19442 16718 6306 9803 17134 10762 2355 13282
9344 18414 962 10707 13732 15247 16140 14944 8665 3901 12556 13238
3850 16213 15720 5407 7133 17222 15571 4342 952 16118 708 14879
19403 2997 14894 13269 498 14617 11730 18476 3794 6451 4601 7256
19396 2011 16585 3917 3066 16371 13698 6380 5627 5614 3881 18493
6416 12573 15178 8209 11348 7043 8200 17880 11947 15915 7621 7041
18926 10681 14294 3702 8567 18263 9236 1978 3932 19800 9976 13414
8046 11624 1429 3768 17319 17779 7609 16512 630 3171 7227 3551 9996
16499 8855 13654 9117 3812 16292 19434 10320 5172 1982 3126 14582
9645 1807 19478 7519 12941 17861 14481 4909 1899 3459 1922 19333
4083 1541 11695 12260 11304 16432 6090 18117 19599 18305 3209 9163
2838 17263 2745 9173 17072 5826 7351 9534 19739 19536 7374 17185
14542 17759 5670 4857 6297 13448 7546 11655 18214 11988 3497 12119
18421 2238 15982 9138 8088 14015 6428 10563 6419 14637 4955 15467
9470 1521 11999 10694 314 1765 1819 4359 18984 14947 13368 6080
8594 12041 17552 5525 3665 12817 1783 2890 18428 15805 9454 10688
13439 13112 10153 5875 3218 18791 2938 12193 15881 16805 18770
14320 13237 2072 10668 13687 15276 12110 9603 12516 3748 11719
18595 2719 7437 2981 5840 16220 19699 10298 2127 3040 11867 4738
17635 17582 9714 2444 10594 8326 16463 16175 11355 16573 10940 2168
14219 13085 7255 18034 15653 19511 18649 7840 8220 2272 9676 5395
14310 8684 4645 2741 19834 12291 1038 15537 16448 8891 10556 10553
13996 16085 8752 6235 10378 16031 9773 9748 8634 1983 5010 16717
16720 16629 3416 6712 18028 18027 18046 18045 2247 17307 5751 13958
2046 2280 11815 11438 7044 432 17329 10580 2438 10575 2435 12466
13227 13331 3373 4354 1137 14182 14180 1685 6522 17288 17285 17287
17309 18279 17281 5134 13133 13134 7591 7579 7578 7590 7622 7593
7619 8710 11257 10136 10155 17393 5497 7611 7613 3368 3371 3345
3347 11146 3346 3342 14996 9789 7217 2775 6179 6205 617 5167 7673
18690 12874 2313 15844 16942 6398 16524 14919 6019 6427 13742 5524
12408 15818 13998 17952 15058 2676 443 16567 8545 9337 11440 6373
12996 16653 18303 8646 8910 18008 14978 10441 2409 16827 2374 16684
7548 18131 4903 4891 11339 14205 916 13861 14326 7296 2136 7059
9757 15666 8359 2239 6482 1101 8053 8470 8156 10487 1094 11152 5629
7080 6211 8534 2047 12554 18572 15080 13812 14010 15955 9662 19762
16570 11714 2862 11347 12363 2885 13476 19136 3340 13481 6845 8548
16230 15749 18141 2803 2874 2819 2798 2851 2774 2772 2847 2769 2801
11161 14362 6202 8537 8518 7037 8133 6764 827 279 4941 1712 818
19365 8356 7588 3017 2235 2242 2245 2243 18869 6400 5637 915 8416
12444 510 12026 11634 16063 16061 4419 4399 7500 7499 7479 7507
7501 7478 2530 9859 1460 18194 11314 2685 4226 15542 19925 15520
16922 15560 5221 8058 14961 984 19513 2729 2751 2748 2730 8932 313
12905 6008 4112 8720 316: 5462 8702 15896 14144 15464 19725 9380
4042 545 16095 1526 8816 16014 8322 3022 1553 2635 18516 10285 6421
5546 6489 8482 8478 5585 16227 2190 10760 1465 2255 11395 7990
14793 10172 16192 7992 12560 3462 14906 12272 16970 2134 2867 2758
7489 6672 10233 12166 15802 1638 918 18996 2473 4032 8620 3162 3756
12149 13374 1308 1307 11769 13333 10034 11874 12574 4659 9026 13327
2753 14426 2429 11931 6246 16533 2250 3513 4318 4974 3869 15232
3820 4665 16608
13406 16766 3671 4310 8986 18992 8995 19933 19932 8988 7545 3888
1293 17163 11580 4168 4167 16335 4007 13342 13297 2864 18382 15220
6521 15457 12531 12431 4213 7363 7483 7328 2936 8542 8532 1997 2463
1996 7728 9652 15509 7851 12529 2192 8974 9761 13146 8836 9376 9401
2717 11802 11806 2558 2553 18119 3929 423 19081 8210 9326 5275
14503 3110 14226 5291 5571 4513 5138 6994 5137 17365 1785 5345
11683 6635 7064 18861 13492 12133 805 10146 9632 15755 16143 8100
13038 16967 19575 19594 3996 16196 1721 16725 14719 12580 11240
5778 618 8966 8955 19713 9948 9946 3468 7884 16898 1191 18670 7152
18629 2566 12157 3148 14714 9023 16483 14008 17781 13594 7412 4148
15789 2191 19158 8063 16731 853 6697 3043 4675 3548 5951 5342 3186
6258 12561 3976 16231 7192 3154 16854 9100 11130 3448 9146 12098
14067 15841 18357 879 3661 7236 1239 14934 1964 8301 13913 18880
2792 2960 8392 12600 19792 13251 1556 8938 11739 11738 17186 14126
8704 6167 15076 7923 10995 8699 4137 19459 2768 11490 2285 468 3256
3962 3526 6749 785 4270 18693 14414 12394 15009 15406 7292 12653
1253 9468 10332 19444 7625 16015 5343 16491 5692 14537 13107 10267
5745 11145 6063 13844 12413 7513 1555 19155 8411 14705 6809 12611
16041 16179 16590 16403 14059 5845 15694 17067 9812 2968 9811 11761
5358 7512 10371 10369 6536 5488 6872 5893 5937 18324 3446 15225
17313 18360 16566 17334 12104 16784 4519 11690 1160 2536 14367
18839 10104 11824 2933 10654 19865 12293 12274 12271 7670 10692
7530 2086 7632 7453 5948 6608 10756 17250 3010 4463 3520 2896 3517
10296 1847 11132 11288 6317 16569 19515 4801 16530 3438 12214 12211
3078 7475 6795 4591 15447 317: 2231 3383 14866 16544 7418 9107 4315
9446 19269 318: 4830 2716 9062 17385 658 13025 18942 1913 7312
19788 1716 3538 4143 17321 815 18938 9848 2344 5906 7890 446 6885
11380 759 13452 4140 13032 458 17467 1500 8433 6737 15173 681 2424
16493 320: 1136 7051 5444 7050 15227 18825 10186 7331 7329 11868
7003 6249 4750 4038 15916 19050 15491 14585 17723 15235 17636 18713
18024 13564 3655 7523 2416 321: 15725 14852 5781 6187 9364 6675
12873 12050 2178 18054 18447 870 12686 19055 13293 6047 3502 15480
6688 6643 16132 12103 18489 19742 17678 2836 12364 8309 14776 7846
17350 17117 11028 7278 1841 322: 19529 18972 10678 6073 19135 18539
10466 12487 17080 14595 7367 8165 4913 16318 11781 323: 9079 9955
19786 2490 2106 6931 4177 11411 14494 401 16768 10346 324: 11512
8262 6323 11449 12376 15280 6345 6342 6758 18459 5739 3469 6326
6372 4249 12966 5720 5704 5725 5724 5756 5810 5785 5764 5783 5788
5723 5727 11451 8412 19543 15504 16837 1442 3587 7395 11317 13364
750 15231 7715 3807 7915 8643 10574 16564 2736 16086 7850 13459
10318 9779 731 10396 10077 6470 798 7137 6756 6754 14009 17356
13319 2236 18981 17018 6664 803 19647 1274 15145 18856 9265 1809
1806 1811 9896 17998 8581 15642 5684 17295 7254 7960 5636 8730 9734
9831 19625 7753 8179 7998 5300 6623 12543 16137 18696 5777 1167
4524 1077 11970 3765 4567 14316 9593 16226 5513 12102 3788 19164
15971 3631 12022 13285 9219 5808 2023 12867 12139 14800 9148 9151
5703 19445 3505 1417 9937 12900 5835 8793 17627 9980 10119 15928
3285 12749 534 19027 7116 10086 3784 18420 9372 3809 16983 19749
4147 2561 14685 19010 5508 15932 1043 8298 15444 13412 8608 15947
13976 8067 3584 19918 10353 11595 11284 14370 8853 4973 8385 5589
1798 1207 509 7947 11095 15540 3437 7615 7612 10821 10244 18075
7438 19651 8598 9205 7814 14515 14553 7875 9013 4551 17654 6535
4686 15510 9928 14939 9130 13820 10857 11701 13734 9606 633 7757
10685 964 7436 502 18371 10090 18370 5553 15365 16452 3436 13450
1356 18559 15301 2361 18638 10471 17333 9747 13715 12322 6368 6349
6775 6779 6773 6777 7269 6774 7262 6328 17232 17226 17209 17212
17211 5741 6344 6348 6476 12159 4157 7574 8332 6766 5759 5760 12701
2721 10839 17534 8451 14104 12537 13141 14902 14903 4314 14916 4309
13007 10518 3401 7876 1104 8383 14730 14720 12059 1106 6982 1031
15090 15093 6907 17634 8009 484 7723 1281 5926 776 6614 7009 15697
12348 13028 2330 12655 15081 6495 13560 18571 14791 19199 6442 7955
9241 7078 6798 10503 15218 8382 19172 938 11115 6504 5016 5836
18241 7022 17216 3591 6384 2085 8286 9708 14861 18111 4952 13581
14185 2853 14506 14516 6690 10730 1934 2612 10612 11767 14612 3998
3009 16097 19296 15380 18005 12479 8182 4663 14202 1064 1476 5622
11833 13437 12832 3282 17447 4447 5740 1103 6324 5794 13746 19613
13540 3119 325: 16615 14754 4057 8715 5965 14877 1866 8104 17176
4366 14518 9167 9065 10806 584 14195 17198 8201 4762 17268 2697
6174 2254 8981 13193 12420 6622 8325 5839 16464 6347 11709 18411
6294 10583 326: 11367 14736 13964 6308 3036 11437 10175 5974 9622
2625 2363 10917 11431 9207 5827 1582 5507 327: 10691 6846 18734
10944 7446 14465 1433 5628 16454 18586 10494 17172 7904 9524 7196
17084 9159 445 5749 328: 1385 15829 19449 11943 5936 18464 6928
7520 1074 12457 797 5586 11159 6704 13511 5517 4980 15756 19404
11162 11144 11682 8750 6782 14571 796 7484 18881 2581 19645 16081
10523 17997 6738 18661 512 13506 959 14965 17458 6459 1020 16188
3545 7761 17240 19190 3663 4263 17715 14220 329: 8714 3717 5566
10565 1859 668 9746 12025 1427 7417 530 14121 7988 11832 3990 15583
10329 7800 2906 9120 14177 14111 16467 2762 4786 19733 3949 3152
8034 16589 3235 4766 6719 8060 10844 12009 12714 15999 13202 19675
9263 15341 11412 11538 1175 13971 9989 3509 7314 9081 5349 6882
4703 8235 2796 5908 10247 12378 16419 16844 12799 14375 5098 6430
9862 12923 12774 18038 9506 4345 14983 1075 17160 6513 3472 1454
13829 6880 6497 17891 11910 6204 10379 14721 14990 10513 5022 5978
10881 5325 4900 11308 9150 18664 5881 12552 6583 6062 13051 8792
15886 882 6896 2502 9724 4391 16217 12349 5780 2148 13289 1827 6149
7304 5083 12091 2946 7643 17480 13408 14394 7079 11009 5514 9020
19826 17411 8276 9974 16420 12687 6975 522 5006 10709 7584 18456
15757 4215 10986 2166 5442 6286 9293 9482 944 7759 18574 18169 6499
4685 7873 8584 17543 9884 7382 7352 7061 17128 14603 3906 11674
10746 1503 13826 14262 12721 17380 11192 13270 15052 9833 12894
19206 19502 19182 12719 6650 17024 11885 12688 5510 1496 14710
18472 12216 4163 6189 13422 1597 8924 12843 10721 10028 1759 14239
14549 4604 10040 7920 1403 10667 14918 7430 13189 11618 14583 3524
4966 17459 839 13522 3834 6998 2999 6865 7167 13043 1901 11888 1209
6472 14813 788 3885 3956 7158 18767 16576 15007 18092 16591 6954
6172 3857 2555 2459 2669 18256 10830 6666 8480 11442 12577 9271
14060 9245 14655 15710 17030 7711 7709 3351 6487 4533 2082 17184
8698 14994 13896 7835 8142 814 14855 3617 935 10433 15296 18619
17848 3434 4185 18019 521 13339 14025 5754 12949 10467 9496 5158
19096 2135 576 993 12906 11309 11504 5691 9623 12589 3192 8950 6480
10293 11074 16003 19115 966 1849 14063 14904 657 1848 16007 14717
4142 16489 12381 2754 8869 14112 8121 19313 10861 19088 3712 6558
7559 10653 9717 16715 18087 10084 17845 19383 16584 14634 8560
18766 7631 18659 1540 9731 6883 17687 15181 15549 6519 11663 486
13767 15635 13224 15907 5070 10188 9295 9297 19656 13831 9250 18896
6462 10456 4816 6498 11564 14166 10726 13462 2420 6005 5935 11870
8991 1892 3542 12006 14673 1339 15348 11771 7607 19801 18944 7773
6641 18882 7567 18085 12565 330: 904 12835 3861 1482 5979 9709
13954 6123 6143 16986 17855 18668 1909 5240 12004 9616 16459 11407
4107 15822 4812 9206 13190 986 738 8516 7209 9218 9155 15166 1046
9972 9234 14160 10926 9007 12402 13404 14565 1314 331: 14505 19187
14237 2004 13667 15330 8712 2983 10833 6141 8832 10908 14924 5518
1621 10016 549 17499 11318 10215 16348 19205 15747 5139 1782 19628
11628 15831 15263 14491 13634 7647 10268 16730 6410 1484 1366 16382
14778 1874 332: 625 19038 18542 16881 3521 18558 315 11296 10579
14173 1169 11180 14471 696 8335 11062 1469 7394 18110 7821 19076
5860 4906 11277 2138 9025 2195 14684 8109 11176 9010 14713 5710
3301 16679 1176 8811 8290 4792 18465 8807 17003 8983 9258 15527
15529 14912 17091 17736 13621 7248 8229 8007 10799 19406 11113
13933 9338 9443 15873 16790 5467 4303 11067 15230 15245 17641 9012
19616 6000 14666 2725 14734 17332 12078 13496 13912 13472 10405
4049 4046 969 16147 12289 7597 9272 835 17757 15353 2123 17605 2911
2934 13460 10176 18009 1003 13239 12374 3372 17390 11981 14556
12247 14559 12268 14560 14577 14558 12267 14576 12226 9387 2109
7841 18828 11137 19936 14986 3553 4554 11929 5440 2805 16033 3934
3003 1594 10929 12343 16214 17420 5606 4643 10415 14917 6601 15702
10449 19711 13552 1507 19835 1698 9321 11696 2863 14802 17698 1498
17572 14405 13598 12043 2262 567 1583 2618 3734 18793 19614 14449
16036 8175 19636 13279 17286 5486 17542 9087 15980 2290 4878 11904
428 10747 9550 13537 3804 8971 5843 3250 1514 10411 13008 5960
18175 14056 11021 12535 9436 8113 10697 15175 7725 16718 13365 5679
15598 19442 6306 9803 17134 10762 2355 13282 9344 18414 962 10707
13732 15247 16140 14944 8665 3901 12556 13238 3850 16213 15720 5407
7133 17222 15571 4342 952 16118 708 14879 298 7886 19403 2997 14894
498 14617 11730 18476 3794 6451 16585 3917 3066 16371 13698 6380
5627 5614 3881 18493 6416 12573 15178 11348 7043 8200 17880 11947
15915 7621 7041 8209 18926 10681 10164 14294 3702 8567 18263 9236
1978 3932 19800 9976 13414 8046 11624 1429 3768 17319 17779 7609
16512 630 3171 7227 3551 9996 16499 19516 8855 13654 9117 3812
16292 19434 10320 5172 1982 3126 14582 9645 1807 19478 2739 7519
5561 12941 2436 17861 14481 4909 1899 3459 15199 19324 1922 19333
4083 1541 12260 11304 16432 8512 4387 10025 6653 9022 6289 7641
9339 2859 11610 9145 8381 8830 8028 17745 3541 482 2045 8753 3209
9163 2838 17263 2745 9173 17072 5826 7351 9534 19739 19536 7374
17185 14542 17759 5670 4857 6297 13448 7546 11655 18214 11988 3497
12119 18421 2238 9138 8088 14015 6428 10563 6419 14637 4955 9470
6734 18494 1521 11999 10694 19034 6803 18967 314 1819 14284 18428
15805 9706 10688 13439 13112 10153 5875 3218 18791 2938 12193 15881
16805 14320 13237 2072 12833 13687 15276 12110 9603 12516 3748
11719 10927 18595 13468 2719 7437 2981 5840 16220 19699 10298 2127
3040 11867 4738 17635 17582 9714 2444 10594 8326 16463 2150 4184
2007 11355 16573 10940 9501 2168 14219 13085 7255 18034 15653 19511
18649 7840 8220 2272 4799 9676 5395 14310 16236 8684 4645 2741
19834 12291 4917 15188 3607 13304 10053 19863 17528 10883 622 943
12590 14403 18836 9723 10918 19559 18677 1038 15537 16448 8891
10556 10553 922 3334 13996 16085 16717 16720 16629 13354 3416 6712
18027 18028 18046 18045 15020 9679 4373 2046 2280 16254 15252 11438
7044 432 17329 10580 2438 10575 2435 14080 12466 13227 13331 3373
4354 1137 9112 9115 8626 9114 14182 14180 17288 17285 17287 17309
18279 17281 19761 5134 13133 13134 7591 7579 7578 7590 7622 7593
7619 8710 11257 10155 17393 5497 7611 7613 3368 3371 3345 3347
11146 3346 19018 18562 4131 617 15703 18059 11680 15149 6854 10204
14371 15844 16942 6398 16524 14919 6019 6427 4568 2096 17412 15137
6371 442 13742 19059 13740 18548 8545 8646 10441 19790 9654 2971
11164 12791 16684 7548 18131 4903 4891 11339 14205 916 13861 14326
7296 7059 9757 15666 8359 2239 6482 16408 10426 1600 4339 9125
15701 10147 6375 17364 694 5869 11598 14712 1946 16250 7313 3680
8784 12459 4338 15013 3889 4760 2323 2136 16586 3470 11223 13993
8676 4320 6154 958 7689 4997 6877 12357 17840 14977 10169 1152
12474 11366 1049 3453 16153 10586 7466 18725 19446 12713 6237 2331
1068 5956 14116 4295 5532 19458 9119 2957 3739 1926 5245 13988 1520
18033 18302 19042 17337 4538 4879 14267 1733 12961 6254 3681 7830
2074 16366 1543 11786 9016 14318 19058 3564 8810 13044 12256 15026
4210 6152 6276 7015 19314 14293 8809 10942 9006 4237 5540 4066
15205 2922 7718 9533 18546 10937 14422 11800 15473 1522 19100 8937
2303 11886 7722 16621 18323 896 9132 14097 11222 15658 13479 11450
7853 10321 18832 7922 19744 6867 15240 11684 18174 1205 4611 5375
16992 2152 19112 4946 19596 16620 19036 18565 19016 18568 18507
18505 18503 18502 5629 7080 13753 1126 8534 2047 12554 18939 18572
15080 13812 14010 15955 6939 14155 19762 16570 11714 12363 13544
7253 10903 17939 19714 11964 15759 1212 3044 7880 13476 18635 9531
13481 6845 19136 3340 1470 15749 8548 16230 16655 5529 5322 6804
2189 2803 2874 2819 2798 2851 2774 2772 2847 2769 2801 11161 14362
279 18610 1712 3375 2235 2242 2245 2243 18869 9808 16261 14527 6784
8618 13941 16063 16061 4419 4399 7500 7499 7479 7507 7501 7478 5967
5140 7332 2530 9726 5117 1829 3095 15542 19925 15520 16445 16918
16920 16451 15541 16209 16922 15560 15559 11352 19040 2729 2751
2748 2730 6517 313 6884 6008 333: 417 6257 8790 13789 16313 14699
10733 16574 8828 5654 11626 2210 11562 3826 12856 1109 334: 8128
4221 17278 17046 18251 12696 6743 3779 8868 11244 10704 12642 6965
16337 9523 335: 18958 12883 14235 19663 17014 1182 2397 1912 8514
1875 540 4252 8725 6215 16237 4063 729 2593 724 8219 17866 6912
6447 18691 2763 5446 2066 15270 17205 19591 336: 12057 12728 5689
13469 14260 4138 1692 19601 15628 9152 17930 16195 2028 8172 17751
16934 16401 337: 3127 12366 13856 16497 1925 19818 4950 15674 5793
15496 15493 2188 2206 5492 1835 6671 11106 1524 9890 1111 338: 5950
18742 15397 6802 19876 12654 8206 17804 1971 16998 18602 18337 4016
5119 2979 4844 10291 19710 18136 5748 10284 7029 5412 5278 11920
1908 6210 762 9715 12314 6273 6084 19740 8804 15410 16699 8756
13731 3128 3144 10582 15564 339: 11402 410 12775 2861 5260 5387
19121 1001 11194 18533 19260 466 4275 18650 7675 1919 4821 7323
16863 9487 12895 16977 15299 2339 8707 2334 9658 18289 19688 13814
2715 19839 17620 11898 1578 12967 4603 13211 930 19517 19938 15569
15063 8098 19557 8485 18333 16609 14853 9045 18679 9143 17460 7537
13585 18153 18578 1495 6307 4430 14054 6332 15889 17603 11064 948
2013 18684 6751 1586 17167 17443 8350 2105 8487 9467 13324 1745
1746 5173 4505 11300 7665 13801 6730 2418 12477 12495 19560 8467
6040 6166 5180 14885 7681 18736 6363 10978 340: 6122 1518 18485
14501 10446 5892 19266 13779 16587 8559 9244 8689 11841 16626 17021
341: 10333 3897 11487 9936 11989 10885 8445 11048 9005 17316 16689
13965 11249 5452 8186 2457 10098 342: 3006 14845 848 12847 17916
1499 5698 15349 8481 2524 12033 2479 19698 8954 6422 13377 17520
343: 6892 17589 9778 16830 14586 3829 7946 7552 7372 2949 6234 6869
12368 9735 13309 2554 7769 4764 8126 18134 2656 2408 9463 16486
19750 2406 15386 7481 14227 7803 18842 18844 4369 15153 18161 19455
3782 7008 2599 4449 1837 2204 6481 10484 18534 15820 3300 18577
14013 8365 13671 11914 3706 344: 4518 2816 7393 8749 3714 18587
12336 13565 834 14466 2212 18658 3417 2925 345: 471 17836 1008 8569
18625 14950 11789 8572 1102 10998 17791 346: 16406 9039 13935 10414
14191 11070 347: 6068 14653 2218 9394 348: 8023 17381 14654 13733
18394 10569 4912 9979 1032 937 13915 10361 1651 5040 10191 12530
349: 1648 10364 2132 11847 18514 11591 2053 350: 13558 19061 6182
985 13642 11733 7195 10366 10180 4120 16484 13042 18195 12100 8507
353: 4575 1730 16597 3415 11245 13804 9868 8538 15670 7953 11687
14656 1891 354: 19676 7455 15869 13573 16262 19202 4246 19397 2728
12796 9583 7861 5152 12632 18294 12083 15810 355: 2962 6855 10045
10561 12389 12391 2422 18108 12563 12416 4416 11255 8450 16833
11444 14745 12519 14732 12470 14479 1758 356: 8236 1667 6732 4010
6333 999 12332 2454 12092 2743 7397 3753 12781 16704 6391 18035
3785 1321 8174 19017 8597 16697 12677 9178 12405 17672 12838 19432
2856 7305 5899 5645 1905 926 4508 6831 11594 13483 19577 13571
17403 19874 357: 18266 7571 19462 4882 3947 18198 19775 19771 19267
9864 15678 7599
19000 3750 3851 12623 17165 17571 14922 2910 16737 8164 5433 14442
13244 5939 1262 17766 9254 2064 358: 4219 1193 4372 8802 10387 9975
16842 12280 12281 12279 17094 11872 2301 709 13862 19439 1073 17661
15857 5981 5996 10915 2469 18211 13345 7869 8911 18950 8909 18952
8225 2963 8049 6934 2376 6638 15632 8089 9727 17744 4332 3625 2818
3137 17921 3318 16305 764 13749 12825 10113 546 11893 19759 13294
15366 11250 13771 8642 15051 6891 15845 12578 4987 6516 18800 4333
18374 4360 14269 17370 9277 18408 4808 4029 12225 19338 787 15279
9722 10304 1332 9721 3088 11139 6037 16019 8571 5449 4424 5365 2467
3432 7502 1283 2362 17765 12194 5682 9922 14289 9064 13109 2442
5695 13898 1251 6239 13774 860 2056 6839 6840 612 11105 11286 2407
12769 13122 9044 13743 3836 3447 8243 6953 359: 12802 5201 10302
17468 16751 7450 9413 19290 5280 19612 15469 15474 13637 6949 3348
2194 4865 3207 16722 3441 14427 7198 3775 3802 12181 360: 19570
9059 8115 3359 3370 16218 4471 2588 3190 1342 1327 7790 7330 15451
1506 12550 3293 13170 13832 8062 11326 2156 5293 5104 3243 672
10504 12238 7614 14171 3051 10249 4843 15762 12428 361: 3065 950
8348 12335 19427 14109 13455 16581 18561 19522 5471 7987 13435
14619 9480 6424 5284 10400 10362 7024 1443 7277 15946 6856 5638 955
2157 1720 9881 18969 15813 7701 12321 3920 8603 9215 3907 11529
14014 6824 2329 8668 10607 9238 2565 4098 9193 16372 18237 17803
19666 2639 7110 12353 660 13242 17052 10200 12815 11310 17724 3805
1392 19435 12595 5074 10118 5073 8413 6434 8087 3649 6611 4839 1944
2006 14636 15167 19849 3029 5269 18965 9850 11641 4433 12460 3711
13980 19654 16418 4220 14462 14907 12618 7928 11377 1861 16288 3333
15079 11178 5930 14221 15099 7106 13591 11342 6453 9428 3138 4529
18919 10578 11967 3123 15132 7246 1399 19641 1877 15059 10489 18120
12869 12088 11647 7054 2316 4238 4224 17713 13334 18328 6713 10427
5017 1537 13171 3450 18694 3746 11425 11266 11206 14036 8884 13754
3840 5694 12680 14821 15322 12298 15719 13691 13441 2511 17203 3588
3365 11160 4803 13723 19123 12875 17986 9764 4606 4403 4531 10348
11668 16400 16295 8960 3752 5598 15851 14513 7149 15712 16423 6990
14475 12448 4402 11453 9183 3764 10933 3278 7656 10372 17668 18799
2893 12854 5169 17911 17413 11409 13265 438 10127 17867 12591 2654
6083 11279 17487 15623 19297 15212 13033 4207 10444 16705 11619
8797 17292 14740 6849 19452 2484 14138 14897 9108 16156 4383 5020
6543 6549 11168 3290 6961 16871 3270 15512 2895 14733 15488 2322
14667 15731 18753 18312 12557 13197 3823 7435 13672 2033 5162 8163
7655 6409 8511 14850 8444 8899 15067 362: 15752 17695 9990 5932
15211 8418 6238 13628 14659 485 972 7628 19124 17248 10742 14839
7306 6162 2258 9285 363: 15587 7536 11706 19935 8746 17583 13021
11251 695 6660 14178 15828 10282 7088 4536 3957 2909 10712 8289
1371 15458 19277 18642 4003 15273 4301 13644 7311 11813 1680 735
15095 1457 1128 3122 9711 3793 1155 18202 5222 16291 5429 8099 7324
11246 909 2980 6952 4744 9471 15817 19001 6196 6665 2449 16000
14068 9442 4512 15890 11197 18150 14278 19695 6747 10772 7524 8625
11005 5911 9704 1140 3532 6565 12151 16037 13454 9169 3324 9517
19274 3692 4037 3896 1082 8078 12333 17688 6926 9760 17178 8565
15303 18874 5274 3608 11418 15790 13582 9809 13016 12263 17135
18105 2163 12533 9985 14786 6012 15171 3012 7976 18090 4450 16120
10148 9586 1280 9405 9430 10773 8152 17676 19007 13766 7476 16279
18231 12787 12805 1089 16681 3955 2488 673 12058 8888 7696 12741
2767 7629 364: 18935 11480 10102 2128 611 16123 4822 16369 3396
5943 18617 12251 9404 19062 5185 6458 17236 4412 6457 4432 11891
4444 17731 5068 13351 18269 1188 18262 18243 18264 18245 15524
18268 7045 10216 15254 15585 9962 6016 6018 5983 10695 4247 11334
2233 11399 14429 17303 18326 5114 4325 13602 3559 12081 18081 5579
15294 19095 8364 12907 14773 15870 10476 3925 8957 18029 8308 2927
19174 3169 6014 4048 6676 17157 11935 4361 4103 7348 3589 16742
2320 8945 12971 1753 2476 9137 14089 16990 18646 2973 10662 10632
13827 11754 1568 5396 13363 13991 6017 6232 13824 9366 1808 7556
18911 16773 9136 4823 14910 396 19794 17197 7423 9283 517 4437
17158 6128 6636 12989 7092 2462 14623 6769 11837 19463 3305 13186
5079 2059 2342 5833 5569 1515 5189 675 4488 17733 8803 17720 1127
7371 13624 3909 4748 1217 8255 9253 16038 5961 6590 16131 19015
11099 19890 2161 12546 10713 925 11622 813 16009 13081 15471 17703
8306 13620 1120 7320 18557 7452 7470 13172 15551 3325 1780 2126
16004 10743 14145 11018 15477 18982 10765 13029 15065 4084 8672
12316 10611 3273 11834 6279 6327 17101 19914 19661 12771 7385 9866
8494 1768 15903 6534 13088 18689 5562 9170 7829 3895 17666 16326
15100 13815 9286 8993 6944 16914 7289 8901 17669 12277 14660 16867
11040 1316 3645 2585 2770 4895 1589 399 6220 18600 18397 917 5160
14743 19767 11532 1354 13972 3182 2310 15434 2040 5147 8430 14935
13335 16362 10812 11006 12064 11533 16872 18830 15354 1960 11327
15506 19294 14823 1796 7366 15566 5646 12469 10813 15208 13430
17355 4773 1940 14330 14354 12329 4797 2889 595 9772 14913 17811
14573 7131 12710 6929 13194 15743 17161 14900 19555 4173 17031 5984
7342 16617 8400 3525 3512 3611 3679 3669 3674 10419 9782 9140 17719
6888 2353 8032 12136 15475 934 10480 19362 17217 16756 19071 18554
12338 17276 2282 13612 1421 5210 19246 14938 4414 10326 861 19438
16936 5565 10516 7011 365: 13631 2299 17761 900 706 18107 11275
3061 17510 586 1894 15867 13092 19868 17581 6902 13125 3191 17566
7355 11590 2877 15662 10745 10009 17671 18076 2217 2055 11552 10714
19648 16707 9924 9503 19883 12972 8648 6287 14196 10126 7645 11125
1084 2706 1420 1739 11228 2641 9918 13470 15676 15827 15328 968 809
8295 15038 16125 2326 13641 2592 15180 7187 19519 8254 7001 15948
419 12496 7126 5605 18193 15842 6741 17608 8770 10875 17317 16846
9813 6726 15155 11019 16116 16028 16531 1857 17795 11282 12582 8230
14029 9438 10288 9360 9453 5792 14648 3353 782 13500 19798 3960
2396 13841 6828 19659 4186 5277 570 7389 8319 1775 5548 697 6933
4532 9927 7345 5347 17289 12608 7808 2718 9932 14265 15975 13060
14443 6331 18432 13873 12306 4371 15241 12122 15732 5458 13523 8923
17770 1063 17401 17362 14436 7068 13174 12734 11814 1336 10705 3094
7480 832 4811 15561 12242 19576 792 9707 13586 7094 13475 8199 5714
15555 18801 1992 5212 10193 18728 19345 15911 1076 1019 2978 11483
2478 14400 8170 1943 17567 15417 5317 11195 14991 18347 7672 19335
19822 11797 13910 8435 16903 15659 6408 13305 6028 491 12784 19379
8454 3118 1686 10876 13040 17491 11393 2077 3299 874 13487 11838
9664 10552 2610 8066 13005 12361 6853 3757 2887 15577 10529 14247
16087 18789 15976 16374 10771 4277 14521 12901 18215 11495 3853
4255 1629 12007 2466 9883 11432 13572 8080 5273 3638 18449 18786
16514 12076 1511 6812 17300 5015 8838 9304 10185 7905 11345 15952
766 13089 2956 613 8059 1979 12501 8535 16930 3363 10502 9217 16331
12258 3097 9416 10257 11596 2590 11883 5567 18847 4461 15605 10990
10192 17106 6082 15250 19258 19683 6624 17570 5102 9195 8650 11158
366: 7895 6135 5404 5150 6602 19424 2002 11648 2102 11953 8198
18899 10410 16171 8925 5672 16257 7833 4196 7416 8915 14363 1706
15695 9308 7164 13079 367: 17228 9424 6076 17145 3914 10656 6091
14671 7138 1890 732 16240 11984 11978 17029 18186 17451 1323 5667
18155 19103 8458 10719 1530 1664 4884 11793 11316 1027 569 6663
18298 6906 1989 11679 8546 17917 7183 4074 5267 8898 17813 368:
10139 8837 7747 7598 13605 1318 9532 7490 2370 12273 4264 14618
8627 10651 13584 18749 7057 7233 408 12207 5515 14163 1226 1228
5775 5424 18314 18275 10203 8674 1800 2693 19005 8477 14053 9051
10777 4549 6597 9738 7370 13649 5856 10149 13783 12613 17815 11337
9362 13027 15862 4287 18724 8169 1419 6285 15077 2147 2905 14574
1177 13510 9181 7135 7290 2520 6455 4618 7286 7288 2679 551 9787
11002 1701 8930 8928 8942 1097 13702 15267 12778 15439 18947 17593
12250 12347 19108 15478 7855 9687 17776 17774 12927 17808 3508 7592
10782 2475 17662 17990 268 18627 12803 4005 6216 13658 8061 16885
5642 8881 8964 10019 2902 8544 10703 1548 4925 12994 10443 7766
13011 12993 13010 17357 15189 1888 18221 17786 9563 9562 2386 5156
3903 547 18001 12480 13049 8197 2495 17171 18977 12208 19466 8339
10488 4385 19012 16077 8291 17408 5878 19014 8302 16024 16026 16018
16022 4841 9827 9828 9829 8403 9847 9830 9842 4690 2646 13874 9849
14131 17831 9865 8405 9851 9844 7012 8406 4019 499 9951 11218 13124
538 13569 369: 6354 13600 13597 13402 8495 11462 4678 17651 2271
18508 9403 8227 19337 18039 11414 13507 8796 16388 13135 10409
13151 13116 13153 14811 13648 16385 17968 17992 18014 17987 17989
17964 17984 17963 18015 17961 17934 17931 18010 17966 17884 17850
17851 17885 17905 17839 17878 17900 17881 17854 17903 17360 12858
19170 13662 7781 19569 19153 13650 13595 5154 17820 2688 17837
17834 17821 2707 2705 17822 17363 3754 3771 3775 3751 3778 3747
17028 370: 13195 17260 15550 15298 16070 13528 18906 6667 3884
19731 16485 15332 11024 4720 7335 10724 9903 16073 9800 2005 2226
17918 14439 11382 1930 15821 6790 3307 2184 3678 6020 2483 16932
11759 18061 8407 17553 9683 10479 3498 7938 11193 10141 10064 5340
13160 1065 1050 1066 8697 5057 1948 19456 10401 4381 5626 3147 4807
15513 12411 1085 17692 15803 9678 13378 594 3808 6353 1729 12195
14567 9675 19783 10272 1789 7097 4737 17591 9334 11855 9329 14859
6302 14086 8083 15671 12954 13947 17819 5460 14531 15105 16035
18528 15990 19316 10591 3086 18057 7458 4476 12898 14681 5385 6706
12606 19781 4024 18079 12117 14093 15233 18591 7496 19829 4740 7646
6599 7671 19185 12054 10751 9600 19620 8234 8875 8090 5475 9292
16255 13529 6245 17823 2300 18651 19369 988 5505 16473 15879 14117
9448 8299 7128 1260 12844 6801 11913 12153 3878 16199 3426 6628
5890 3275 9071 19553 19622 5677 2987 5813 8587 5410 7917 10123
11678 19390 5338 10026 16740 6793 1954 9499 1886 18780 8529 17665
18483 19399 13823 10599 10014 11713 13942 16536 6829 13457 12144
4468 15992 10473 13518 2219 10524 420 421 14605 14570 6724 1269
15608 11671 5125 6124 12849 7013 12911 427 17957 7104 18943 14199
10798 14689 13399 9608 5287 17769 9135 11977 16390 8020 11559 16849
475 16412 9947 18271 8288 16395 11055 10283 8052 3695 4102 6848
1576 5301 7717 994 2477 9188 4330 6788 6219 12712 8475 8239 14804
13446 8249 3014 11497 719 15957 17377 11889 19701 16334 9730 14899
17716 13795 14007 15861 9886 16290 1777 16807 13147 5390 5656 16417
11861 16848 8738 10094 18845 16925 17514 13485 17384 15049 13629
16050 15795 15733 10818 17446 13946 5886 1298 18331 2672 1478 4124
15983 15934 3583 13711 1676 19213 15367 19827 17298 12179 5594
17065 6157 13890 4408 9574 11208 1762 15748 10092 2000 6115 8274
11506 15771 13770 17670 13724 4250 18579 18707 18706 18338 11926
18667 19212 9804 19854 14597 7979 5533 5584 7353 4291 17318 16669
687 880 11774 18653 11461 14110 6979 18340 8989 12588 16340 12839
4132 12893 15176 12464 5250 13418 19208 6505 2317 5276 12735 5595
3237 2548 769 11313 4897 4054 13148 18282 8680 17055 9088 13069
7258 15620 4755 2281 4479 14873 14276 18888 1613 2084 2083 2525
15057 8026 1769 6771 18311 14286 18220 3288 3011 1673 18154 842
9128 10218 12711 4076 14589 13986 9857 976 14336 10790 16144 19128
6185 10202 19394 10344 5962 15092 13101 13349 1772 5842 17973 9011
7194 12615 6592 16662 6996 4557 2794 9462 13636 1591 641 17482 711
1379 4654 19782 16899 4214 17607 4609 14972 12409 6334 8398 2027
15897 1125 2319 5865 18872 5227 19597 6259 16933 6862 12848 17576
9766 5970 13699 9543 10511 17456 371: 8393 9015 13413 2587 10796
17631 11121 433 4963 9556 575 5319 15391 10530 9031 4635 9004 9073
3306 11645 3981 19101 19141 19812 10554 17490 13822 14492 17246
1376 15351 19391 12231 4365 5194 2111 5408 17546 18797 5903 6042
1218 4848 19643 4899 6748 1341 2965 13343 10436 3965 12507 16052
10080 14622 15172 1156 15431 19244 5027 7279 4985 13179 18612 9230
6746 8566 5265 5208 3815 11698 18164 13048 16879 4441 8300 11252
16442 505 453 12406 1723 6094 17550 19037 5884 14923 4421 18481
6530 12498 17991 3134 8890 3350 7985 7864 10001 10340 991 5289
19317 10465 7319 2659 5841 13312 1585 8483 2167 16410 18310 17967
11530 8907 847 867 8377 12438 12935 1630 8528 11657 16778 524 942
13396 13561 12813 15308 14883 14448 5321 8706 4558 4612 4061 16274
14724 2777 3112 607 6814 11242 4393 19491 1312 1238 437 18920 2620
5133 8260 9619 13014 8506 10965 2373 14342 18448 19165 17471 5405
6607 1741 14959 15075 16265 13994 1258 14304 8744 10343 19837 12926
7213 3174 10993 14602 10617 7779 10187 9485 15515 19398 18257 19349
1202 15472 7901 12094 13651 1646 14731 1180 12154 16047 18288 2248
8051 19670 7996 7995 3855 1551 14194 18409 5592 11704 13409 18199
12052 7391 8999 15302 4964 15580 16350 15735 14708 15312 6145 15699
6159 15221 18322 10493 501 6886 13963 12295 8982 13436 16066 11980
4664 9057 9034 16666 15687 2427 11094 13668 3320 14079 9090 11181
8003 4257 11638 6544 6957 3999 12296 10381 2653 4772 4810 8751
10568 10162 5600 5649 15893 14376 16307 17697 9785 10884 16276 6169
6176 1037 9196 8367 7783 14533 9056 15139 19917 12676 3038 10673
5503 7023 15682 10198 898 1297 5858 14777 6955 17092 945 3032 10231
16741 4106 11060 10054 18270 12505 13314 1821 7784 11537 6572 3603
13635 5021 9052 7601 6284 8815 10431 18883 18773 17616 11724 3501
16356 11013 14805 9024 14557 7384 17109 7703 18849 14347 5364 6468
8936 3979 19281 10693 13957 3977 13486 7380 17077 372: 4087 6477
5737 5639 1608 6099 373: 11821 15570 15362 17684 12417 16687 6089
15073 1359 9786 19736 8877 10254 5707 15924 3173 5784 19735 9392
2552 15018 11302 10241 18422 14812 5768 9824 19506 6981 10715 19649
3163 9998 2058 6405 6584 7848 14615 15185 2020 16636 15445 11375
18820 10331 1794 6619 8395 6787 15426 6658 7293 10195 2815 19672
5744 14420 15534 15370 14303 15177 12323 14958 14797 11491 452 5665
6655 9310 16947 3284 15338 17037 6312 3537 1320 3839 1070 13922
11570 9105 5536 15484 14763 14487 9068 12682 9682 10905 17502 4201
4406 8085 7056 8194 19718 4289 19086 2788 10609 1276 11826 13369
15331 5251 8761 16059 6055 8829 15454 5769 404 6808 15129 1725
10525 13488 7737 8203 2464 17537 3566 8056 4191 10305 16861 7136
8740 1328 6582 7383 9441 7191 15884 17130 5403 5900 19080 12583
10011 3547 16174 11235 17910 9121 5888 18291 17740 7117 12383 15552
8043 8998 8613 9367 10541 17801 8616 10220 1388 8599 4600 15321
13524 873 3602 1369 11422 7039 17579 16732 1166 9497 14498 11817
691 12683 11133 10036 10168 9240 7540 16302 2752 10828 18058 9911
15729 17807 1719 19117 1924 3905 6227 4868 10358 11763 18113 5209
8329 10740 6938 9635 14512 17399 18121 12733 13966 5279 1953 1959
15567 18234 3818 5220 2744 18925 13352 15418 7193 635 9142 8342
1452 16526 8583 14626 10595 15517 3675 15449 18995 1550 12913 1574
10748 15207 689 10793 16997 12087 3593 9495 12369 16764 13313 16678
1034 9702 12049 15046 15685 1505 4817 6591 10004 17280 1057 2209
16242 6770 11621 11066 19348 13114 5818 18606 11096 2735 11091
16599 13119 10928 11061 18170 18353 3156 18093 8497 15157 14638
11736 1201 11263 18000 17999 11949 914 4681 8852 14381 624 7004
5368 718 17732 11589 12259 1569 3028 5910 8948 4159 8737 11933
15646 4723 2738 4789 376 8578 16888 18857 6948 802 19507 5226 6977
6646 17557 2557 374: 16075 17346 1488 14198 4254 1650 2972 17330
12199 963 19510 17391 5693 10970 15087 18216 17575 18987 17965
11155 16523 17488 9549 11549 10008 17518 8820 12423 1502 16789 5728
716 10447 10464 16976 5052 6412 5108 3314 6292 8733 16269 15372
10825 12586 10413 10380 12879 10280 19593 3339 9629 3902 1154 10253
8577 7055 2366 3283 13877 9422 4904 10226 1456 18827 6632 11525
14700 16759 2841 3514 3755 17006 17415 7347 3067 13384 3558 2974
1846 3323 6629 3184 19561 19703 15961 8827 2835 9303 1071 10179
14834 435 12500 4768 8437 7517 2760 14250 12828 10786 1268 13055
13052 12497 16738 9520 19359 10335 2636 2886 9647 6341 6642 7315
12387 19717 11540 13887 5657 16407 18284 16813 16160 19921 18470
14030 12523 13879 8162 18330 4380 12845 8696 15369 16716 14346
16690 4719 3277 5678 5680 10403 3623 5774 2508 1744 19808 4467
13035 11579 7401 2215 4902 11210 11968 15518 8984 3055 3245 18372
7421 8387 11179 7162 12630 5371 6163 5316 14827 17771 590 15838
18775 7975 11865 15784 2067 15507 17191
10843 14530 15228 9977 13811 16873 2501 8524 6493 9867 12072 8122
14458 2666 15333 9399 4828 15607 12871 15613 1771 16671 7034 11542
15229 10987 6960 14341 14525 7247 4763 19509 5283 18405 8785 16222
14297 18434 14175 18636 10459 13065 17257 16561 1889 14928 6483
19913 2456 1117 3838 6107 6860 7721 6402 1845 12187 10183 3985 7714
6914 17425 9651 13259 791 15634 15360 10630 8039 9174 19497 1774
12575 11290 11928 14723 17308 16624 18115 9049 7707 4914 15036 6484
13266 7824 18137 7109 4623 13337 19831 7899 18876 14992 15343 4660
14819 3465 1564 18556 19844 4349 19085 9971 13851 16931 2174 8283
19354 11651 4757 15968 6959 10967 11188 17645 8006 6247 8959 10307
13566 11039 13744 17853 863 11617 7364 10342 548 17035 13825 1142
13781 9657 5941 1581 17069 12968 15470 16299 4281 15614 1937 4559
14610 4696 13538 7697 3637 7603 11413 2179 7933 19467 8355 1987
19608 967 15718 3480 8769 12950 16296 15497 18747 16455 2726 19646
1232 9750 12105 12718 12852 5921 12836 3388 7411 16468 8997 11368
1122 7595 14296 9278 19558 12461 14633 16726 17802 7427 18821 8484
8671 7208 18639 13316 13956 4535 18240 4560 14766 13755 10837 5042
14741 10899 6606 836 5977 3221 7913 9252 12441 5332 6266 4788 11839
7268 17954 2356 3394 7563 11666 4588 13840 956 15465 19389 17489
4423 4266 13131 10548 375: 16510 13467 14806 18916 13489 19287 6963
11609 12123 6168 14647 17406 19726 10559 16928 16901 7555 12727
15736 13018 2908 998 11571 14927 11630 8370 8372 13218 10996 17016
7958 9384 13045 13447 8144 1147 15001 2276 2823 913 14592 3364
18232 14988 977 13106 11518 14539 12112 17129 2915 10863 1580 4515
5798 8253 8082 416 19385 19171 1100 16820 6199 4571 18783 1742
10182 13173 16635 17155 13139 6972 5995 17971 7440 13865 9190 7070
17784 2010 723 15385 11201 10394 4498 13493 13320 14352 678 13273
448 5709 11312 18597 11116 7510 15024 12386 4804 16278 7734 1363
7445 19533 12959 1292 16198 16824 12162 2158 10445 8558 12176 1883
5802 17888 7202 9357 9212 9289 15706 15848 18962 11998 16594 7471
9904 6557 5534 7297 14395 12716 14630 1709 8057 5106 13882 13240
3414 8391 13833 18908 11344 5879 12334 16882 8131 13298 15056 11047
2998 9898 12476 14335 16430 17095 2214 1606 8257 16665 13613 6921
7748 14323 15979 5733 12116 6947 8474 13708 15492 18185 6299 13610
18148 14215 18714 2930 12129 15295 15268 14650 18384 18143 3511
6197 6361 17944 17442 5229 2611 565 16358 16357 15084 16089 1181
17594 9098 2241 19299 12023 7283 10377 6819 4949 8736 13205 2694
19763 13769 19621 13407 9290 16521 1825 10029 12822 2903 15953
15986 11911 13793 11546 2234 14881 9908 11523 15533 17577 11068
17517 17721 17374 10696 12227 19181 13058 8185 15758 4153 877 8465
19011 4495 6613 16284 8138 18523 4389 15763 3113 17826 2647 1098
10483 10463 19364 11397 11401 11381 6868 15127 1474 16902 5599
19363 17325 9309 19361 5037 4928 3787 16923 4146 16939 19535 15773
19339 15539 15125 18484 4203 18687 9493 10628 16954 18751 5417
19261 19262 19234 1714 19898 19336 19855 19291 19286 19880 19877
19295 19895 19875 1295 10357 6086 10734 11846 6683 12722 18816
18796 7791 4566 18951 15711 11715 3398 18259 16246 1255 3813 1431
15683 7433 3767 5490 5324 10201 9680 13775 10924 11448 5618 3801
6703 2837 11877 2626 19090 4080 8371 9573 3492 13188 3045 16273
5297 2403 2365 6847 7359 12126 16429 13888 376: 11821 15570 15362
17684 12417 16687 6089 1359 9786 19736 8877 10254 5707 15924 3173
5784 19735 9392 2552 11302 15018 18422 14812 5768 9824 19506 10715
19649 3163 9998 2058 6584 7848 14615 15185 2020 15445 16636 11375
18820 10331 1794 6619 8395 15426 6787 6658 7293 10195 2815 19672
5744 14420 15534 15370 14303 15177 12323 14958 14797 11491 452 5665
6655 9310 16947 3284 15338 17037 6312 3537 1320 3839 8693 1070
13922 11570 9105 5536 5454 15484 14763 14487 9068 12682 9682 10905
17502 4201 4406 8085 8086 7056 8194 4289 2788 19086 10609 17621
1276 11826 13369 15331 5251 8761 8829 15454 5769 6808 15129 1725
10525 13488 7737 8203 2464 17537 3566 8056 4191 16861 7136 8740
1328 6582 7383 9441 7191 15884 17130 5403 5900 19080 10011 3547
16174 11235 17910 5888 18291 17740 7117 12383 15552 8043 8613 9367
10541 17801 13465 8616 10220 1388 8599 4600 15321 13524 873 3602
1369 11422 7039 16732 1166 9497 14498 11817 691 12683 11133 10036
10168 9240 7540 16302 2752 10828 18058 9911 15729 17807 1719 19117
1924 3905 6227 4868 10358 11763 18113 5209 8329 10740 6938 14512
17399 18121 12733 13966 5279 1953 15567 3818 2744 18925 13352 15418
7193 635 9142 8342 1452 16526 8583 14626 10595 15517 3675 15449
18995 1550 12913 1574 10748 15207 689 10793 12087 9495 12369 16764
13313 16678 1034 9702 12049 15046 9046 15685 1505 4817 6591 10004
17280 18300 1057 2209 13541 16242 6770 17146 11621 11066 2735 11091
11096 19348 10928 13114 5818 18353 3156 18093 15157 11263 11949 914
4681 7472 8852 624 7004 5368 718 17732 12259 3028 1569 5910 8948
4159 5738 8737 15646 4723 4789 12067 16888 18857 373 6948 802 5226
6977 6646 17557 2557 377: 8393 9015 13413 2587 10796 17631 11121
634 433 4963 9556 575 5319 15391 10530 431 9031 4635 9004 9073 3306
11645 3981 19141 19101 19812 10554 16183 13822 14492 17246 1376
15351 19391 12231 4365 5194 2111 5408 17546 18797 3789 5903 1218
4848 19643 6748 4899 1341 2965 13343 10436 3965 12507 16052 10080
14622 1156 15431 5027 7279 4985 13179 3070 3071 18612 2494 9230
6746 8566 5265 5208 3815 11698 12148 18164 13048 16879 8300 4441
9892 11252 16442 505 453 12406 1723 6094 17550 19037 5884 16347
17111 14923 4421 18481 6530 12498 17991 3134 8890 3350 7985 7864
10001 10340 991 5289 19317 10465 7319 2659 5841 13312 1585 8483
2167 16410 18310 17967 11530 8907 847 867 8377 12935 12438 1630
8528 11657 18567 17817 16778 524 942 2110 13396 13561 12813 15308
14883 14448 5321 8706 4558 4612 4061 16274 14724 2777 607 3112 6814
11242 4393 19491 1312 1238 437 18920 2620 5133 9619 8260 13014 8506
10965 3168 2373 14342 18448 19165 17471 5405 6607 1741 14959 15075
16265 13994 1258 14304 8744 10343 19837 12926 7213 3174 10993 14602
10617 7779 10187 9485 15515 19398 18257 19349 1202 15472 7901 12094
13651 1646 14731 1180 12154 16047 18288 7639 2248 8051 7996 7995
3855 1551 14194 18409 5592 11704 8432 13409 18199 12052 7391 8999
15302 4964 15580 16350 15735 14708 15312 6145 15699 6159 15221
18322 10493 501 6886 13963 12295 8982 13436 16066 11980 4664 9057
9034 16666 15687 2427 11094 13668 3320 14079 9090 11181 8003 4257
11638 6544 6957 3999 12296 10381 2653 4772 4810 8751 10568 10162
5600 5649 15893 14376 16307 17697 9785 10884 16276 6169 6176 1037
9196 8367 7783 14533 9056 15139 19917 12676 3038 10673 5503 7023
15682 10198 898 1297 5858 14777 6955 17092 945 3032 16741 4106
11060 10054 18270 12505 13314 1821 7784 11537 6572 3603 13635 5021
9052 7601 6284 8815 10431 18883 18773 17616 11724 3501 16356 11013
14805 14557 7384 17109 7703 14347 18849 5364 8936 3979 19281 10693
13957 3977 7380 13486 3384 378: 13086 19930 5574 17164 15382 2809
1565 9389 817 831 5123 14538 9536 1834 1833 13082 18812 19542 12765
13660 1610 1609 15422 15421 1627 13773 5445 16272 10838 19127 19142
9628 10532 7251 8084 9179 10060 3233 3234 10382 15456 12062 12060
8373 598 11569 18041 16210 3876 5308 5310 4105 19053 17469 5866
18520 18518 17299 16592 13763 1649 2826 1088 5244 17633 8002 6325
8510 8509 17342 10265 8523 11182 2722 2708 14181 17828 10012 8552
6241 10590 5676 10274 8661 3295 6576 8917 12079 16538 11322 11319
15823 16815 16795 13353 14432 2003 1985 7807 7667 1870 12245 15011
15010 7081 7327 7326 3951 11419 11420 5668 5402 2091 2088 10840
16194 9002 9003 8127 8141 1440 6106 6104 17126 17005 4082 9591 9590
1915 6725 10684 7300 3317 3316 9330 19487 19488 14152 14153 2122
1914 1911 10665 14295 14309 2813 2489 17345 11755 18173 14628 16281
4570 426 12610 17201 17057 16060 16058 9609 987 9610 15673 921 1086
18306 11336 2498 17087 17085 12034 12035 14678 14675 1138 544 16271
12318 12812 4308 19378 14598 14599 7369 18594 7648 17481 19008
14345 5166 7493 13726 13725 8384 3400 16200 7902 8410 8409 13137
16958 16957 15723 12257 12253 15978 12973 4460 4459 13017 1570 1078
2531 15107 15091 8965 13199 13198 19203 19218 2645 523 5806 17244
17241 9229 13301 12697 16182 14543 17648 7956 541 7604 12160 14921
4194 19094 17637 18276 18272 14246 14245 6265 11350 6263 11351 1447
9984 5779 16703 16701 8272 8270 15033 637 11702 3382 13215 6987
17734 17485 17484 8448 11578 16065 14275 12748 19807 2279 9116 2296
2779 13325 13222 5611 2538 12956 15346 10824 1158 4300 18255 6983
15168 4680 19799 12284 6950 16092 3445 2799 10239 3660 12422 2797
18788 11987 9273 10359 2829 13626 19734 592 14588 3599 6112 6093
6110 572 5844 11779 4108 3093 11517 11539 455 10947 11127 13214
3927 11199 12891 15461 15460 7465 5719 5718 3367 13458 13456 1149
14244 6969 12138 9126 1661 15396 5031 16825 1467 3689 6716 6528
6526 8496 18877 379: 16324 10027 16399 19626 19600 476 10737 6503
2637 5207 7908 18645 13757 8204 17032 1213 10125 9070 5372 15085
10478 7338 18023 9641 11812 14642 18106 9561 2651 1666 5437 14594
5439 12210 6002 12290 5485 6003 14353 14349 14351 10819 8846 9429
2453 10112 16543 3140 1146 1124 1121 15438 2354 380: 15042 683
16630 18404 5663 2482 10670 11782 18811 18709 11700 19144 6755 9799
13818 6126 2455 8789 381: 17923 18890 5500 4771 6745 5378 3236 1210
11603 19433 19342 15300 17953 11454 9261 12841 11591 5415 5453 5430
8278 14564 6761 17927 18569 14781 10539 6214 3620 3528 17899 8748
12221 5504 5502 9489 6425 8958 382: 19132 10614 8139 10836 16040
11545 7879 2100 1703 14547 383: 1189 16611 3586 4641 19633 17705
17150 8849 3552 7294 2029 6153 18596 384: 741 1633 5923 8765 16673
14609 3698 6449 2951 2093 11563 614 14593 14762 6397 7731 14290
17038 4311 14640 385: 11357 13113 2101 9359 2343 5986 9703 12857
8854 17190 4889 13808 12572 3386 7845 17983 4794 386: 1016 16046
10106 6272 16847 10542 15062 5576 4893 5448 15407 7831 16578 18278
2702 7215 9961 6250 3252 18599 16433 3226 17007 387: 7223 8111 7867
1322 9479 3291 18674 5132 12538 3515 18852 16518 14682 5419 9781
388: 13217 19387 1319 13219 1333 2384 4322 7600 13280 5924 13688
2181 16877 16122 5091 927 6100 3686 389: 11269 4678 13073 1755 1754
8654 3662 17371 3891 3973 2187 15246 18462 4802 3390 619 643 13183
4014 1542 10000 19057 6229 1262 14305 14256 17590 5708 4877 3775
8462 390: 17182 3673 6158 5731 8195 15335 7264 7267 5346 10728
11675 2337 19110 7961 8491 12046 10224 6729 18918 18228 3153 8723
3309 3313 6222 15956 4632 12325 6835 6387 2622 4496 9897 10860 8044
10003 13262 13236 6403 5331 4640 1303 4602 1304 18435 16053 18480
17646 17644 17675 16363 4781 3269 8610 10852 3582 5717 9242 9340
9346 13271 1345 3780 11086 14006 2264 5062 5146 16786 891 1282
11639 2472 9621 19732 15665 10462 16384 18138 9510 19888 19119 4931
18991 13945 18937 13580 18401 13863 1936 4639 10245 760 13679 14995
17389 5259 10385 9763 15381 10901 10886 10888 12415 15437 8840 7871
4684 12209 17107 11203 16435 8624 13410 8530 10422 11650 15110
13639 12675 13689 19825 4662 13277 5625 9279 3369 5976 1377 2144
4086 18044 16889 6767 18418 11859 8188 6500 16529 16528 13234 14259
18077 11012 10071 11825 16181 6479 3800 9154 19377 924 923 7361
4574 14380 13895 17168 14901 17969 13735 17599 10270 4099 18973
3523 14337 8378 4490 18286 5203 2114 13850 9109 9096 7060 4161 6561
1211 9640 13790 19180 14796 5771 15355 14027 6282 12681 16502 4842
4262 4930 15500 16490 14450 18361 15929 1171 7624 13836 15083 19541
1490 8022 9426 19755 13700 10435 8346 13719 8681 19368 10906 19116
1509 3865 12853 3125 5889 8316 18315 9077 436 1977 8354 5002 5025
16553 5171 17152 9352 17908 9960 9963 4907 2079 14164 3744 3102
18375 4128 4129 14794 391: 5830 10547 4780 6670 1560 15309 12977
11581 15243 10500 7299 19550 4614 11584 18988 17503 13880 18201
7124 6036 17359 15414 19074 13696 4180 810 12806 13939 1259 2434
12029 5666 8718 751 18491 15717 1795 4475 1738 627 5732 18253 8660
11107 16309 12868 4299 14698 14679 14502 8248 11990 9758 16167 9385
10043 10430 16906 12099 5791 16810 13759 8562 2921 8889 10315 14077
2682 18457 14406 12241 10194 5832 13548 15304 4676 392: 18452 9432
5303 14233 9970 7637 14980 15265 7581 3016 10412 19809 393: 19384
19382 682 6463 1021 6039 6041 6057 7898 7896 13532 13533 8037 9356
16887 16866 7150 7148 11880 11903 7084 7083 3381 13816 13817 12463
4530 2311 2312 10624 10623 15054 2256 2257 14016 14018 8801 6140
6142 8800 6138 8357 8375 8360 3666 3668 11044 11022 9473 9494 5001
5000 4831 4836 18327 19232 5812 5815 9999 7941 7942 749 3079 7426
7451 8001 7999 8005 19635 4051 15885 12205 2860 786 784 15576 15575
12310 12307 2306 3461 7640 10242 394: 12840 13577 8132 1535 5905
3211 3797 12558 6843 17949 15017 9231 1310 17041 6367 18764 8461
15888 6866 6864 7983 8980 10079 15489 11102 9933 7325 1749 8441
7720 17727
Example 3
Consensus Sequence Build
[0198] ClustalW program is selected for multiple sequence
alignments of an amino acid sequence of SEQ ID NO: 198 and its
homologs, through SEQ ID NO: 394 and its homologs. Three major
factors affecting the sequence alignments dramatically are (1)
protein weight matrices; (2) gap open penalty; (3) gap extension
penalty. Protein weight matrices available for ClustalW program
include Blosum, Pam and Gonnet series. Those parameters with gap
open penalty and gap extension penalty were extensively tested. On
the basis of the test results, Blosum weight matrix, gap open
penalty of 10 and gap extension penalty of 1 were chosen for
multiple sequence alignment. The consensus sequence of SEQ ID NO:
227 and its 25 homologs were derived according to the procedure
described above and is displayed in FIG. 1.
Example 4
Identification of Amino Acid Domain by Pfam Analysis
[0199] This example illustrates the identification of amino acid
domain by Pfam analysis.
[0200] The amino acid sequence of the expressed proteins that were
shown to be associated with an enhanced trait were analyzed for
Pfam protein family against the current Pfam collection of multiple
sequence alignments and hidden Markov models using the HMMER
software in the appended computer listing. The Pfam protein
families for the proteins of SEQ ID NO: 198 through 394 are shown
in Table 16. The Hidden Markov model databases for the identified
patent families are also in the appended computer listing allowing
identification of other homologous proteins and their cognate
encoding DNA to enable the full breadth of the invention for a
person of ordinary skill in the art. Certain proteins are
identified by a single Pfam domain and others by multiple Pfam
domains. For instance, the protein with amino acids of SEQ ID NO:
207 is characterized by two Pfam domains, i.e. "TFIIS_M" and
"TFIIS_C". See also the protein with amino acids of SEQ ID NO: 213
which is characterized by three copies of the Pfam domain "WD40".
In Table 16 "score" is the gathering score for the Hidden Markov
Model of the domain which exceeds the gathering cutoff reported in
Table 17.
TABLE-US-00016 TABLE 16 PEP SEQ Pfam domain ID NO GENE ID name
begin stop score E-value 198 CGPG106 CMAS 69 306 -149.8 5.00E-05
198 CGPG106 Methyltransf_11 131 229 109.1 1.20E-29 198 CGPG106
Methyltransf_12 131 227 54.6 3.10E-13 200 CGPG117 SNF5 14 252 66.9
5.90E-17 202 CGPG1288 Cupin_3 57 131 130.8 3.40E-36 204 CGPG1458
Phosphorylase 180 958 1454.7 0 205 CGPG1542 LMBR1 11 488 715.1
4.40E-212 206 CGPG170 Metallophos 54 249 140.4 4.50E-39 207
CGPG1828 TFIIS_M 206 327 203.5 4.50E-58 207 CGPG1828 TFIIS_C 338
376 83.3 7.00E-22 209 CGPG2217 ORMDL 14 156 356.8 3.20E-104 210
CGPG2292 Tim17 52 186 16.1 4.20E-05 210 CGPG2292 SAM_1 192 253 13.8
0.019 212 CGPG2499 Glycolytic 4 339 588.8 4.50E-174 213 CGPG2653
WD40 135 172 40.3 6.20E-09 213 CGPG2653 WD40 235 271 40.7 4.60E-09
213 CGPG2653 WD40 276 313 38 3.00E-08 214 CGPG2813 HLH 204 251 34.1
4.60E-07 215 CGPG3002 eIF-4B 5 451 -50.3 1.90E-10 216 CGPG3154 C2
14 102 101.8 1.80E-27 216 CGPG3154 C2 198 272 57.8 3.20E-14 217
CGPG3235 B_lectin 66 176 156.8 5.30E-44 217 CGPG3235 S_locus_glycop
189 315 227 3.90E-65 217 CGPG3235 PAN_2 332 402 103.9 4.20E-28 217
CGPG3235 Pkinase_Tyr 488 757 75.6 2.10E-20 217 CGPG3235 Pkinase 488
757 109 1.30E-29 218 CGPG3274 Allene_ox_cyc 74 253 401.6 1.10E-117
219 CGPG3275 Auxin_inducible 50 155 135.6 1.30E-37 220 CGPG3363
zf-AN1 21 58 10.5 0.0033 220 CGPG3363 zf-AN1 103 148 12.4 0.0019
221 CGPG3367 TFIIA 9 375 556 3.40E-164 222 CGPG3375 Tryp_alpha_amyl
33 111 55.2 1.90E-13 224 CGPG3534 Ank 42 74 13.6 0.31 224 CGPG3534
Ank 75 107 36.8 6.90E-08 224 CGPG3534 Ank 108 140 1.6 17 224
CGPG3534 Pkinase_Tyr 160 413 202.4 9.90E-58 224 CGPG3534 Pkinase
160 413 198.5 1.40E-56 225 CGPG3638 Ribonuclease_T2 30 219 367.3
2.20E-107 226 CGPG3918 Metallophos 60 255 159.1 1.10E-44 229
CGPG3972 14-3-3 8 243 501.1 1.20E-147 230 CGPG3990 zf-C3HC4 259 299
47.3 4.60E-11 231 CGPG3994 Pkinase 554 802 207 4.10E-59 231
CGPG3994 Pkinase_Tyr 554 802 233 5.90E-67 232 CGPG4026 CMAS 59 320
-174.5 0.003 232 CGPG4026 Methyltransf_11 122 220 96.3 8.10E-26 232
CGPG4026 Methyltransf_12 122 218 37 5.90E-08 232 CGPG4026
Sterol_MT_C 229 358 301.1 1.80E-87 233 CGPG4048 Pkinase 21 275
342.1 8.80E-100 233 CGPG4048 Pkinase_Tyr 21 275 65.1 1.20E-19 233
CGPG4048 NAF 304 364 104.5 2.80E-28 234 CGPG4052 DUF298 127 242
222.4 9.20E-64 236 CGPG4058 Asp 99 437 -128.4 4.60E-06 237 CGPG4069
adh_short 31 218 -16.7 4.80E-05 239 CGPG4088 SWIB 324 399 96.4
7.80E-26 241 CGPG4121 Cyclin_N 58 190 133.7 4.80E-37 241 CGPG4121
Cyclin_C 192 314 44.3 3.70E-10 242 CGPG4122 zf-C3HC4 100 141 27.6
4.10E-05 243 CGPG4140 p450 48 484 140.8 3.30E-39 244 CGPG4154
zf-CCCH 90 115 22.6 0.0012 245 CGPG4311 FBPase 70 395 347.9
1.50E-101 247 CGPG4369 BoIA 10 79 92.1 1.60E-24 248 CGPG442
2-Hacid_dh 85 394 140.9 3.20E-39 248 CGPG442 2-Hacid_dh_C 187 362
293 5.20E-85 248 CGPG442 ACT 551 621 39.9 8.10E-09 249 CGPG4454
p450 45 447 -35.5 2.10E-09 250 CGPG4456 p450 75 502 103.6 5.30E-28
252 CGPG4588 Auxin_inducible 1 102 151.5 2.00E-42 253 CGPG4765
DUF868 28 304 175.5 1.20E-49 255 CGPG4912 WD40 243 281 31 3.80E-06
256 CGPG4926 WD40 42 79 22.8 0.0012 256 CGPG4926 WD40 126 163 25.8
0.00014 257 CGPG4967 Asp 71 424 -94.7 4.70E-08 258 CGPG4977 Usp 3
157 85.3 1.80E-22 259 CGPG5001 adh_short 5 181 6.4 1.40E-06 260
CGPG5025 adh_short 30 212 5.7 1.50E-06 261 CGPG5041 DUF26 77 132
84.3 3.40E-22 261 CGPG5041 DUF26 188 242 100.7 4.00E-27 261
CGPG5041 Pkinase 333 558 60.3 5.70E-15 262 CGPG5116 ArfGap 15 137
174.1 3.20E-49 262 CGPG5116 C2 182 261 101.4 2.40E-27 263 CGPG5144
p450 61 535 157.9 2.40E-44 264 CGPG5171 p450 35 496 137 4.60E-38
265 CGPG5194 DUF1191 25 308 616.2 2.60E-182 268 CGPG5221 p450 30
499 135.4 1.40E-37 269 CGPG5269 PCI 297 401 108.9 1.30E-29 270
CGPG5404 Peptidase_S10 68 480 683.3 1.70E-202 271 CGPG5432 MtN3_slv
12 99 145.4 1.40E-40 271 CGPG5432 MtN3_slv 133 219 140.4 4.40E-39
272 CGPG5518 Ribosomal_S8e 1 238 325.9 6.30E-95 273 CGPG5535 WD40
335 371 28.8 1.80E-05 273 CGPG5535 WD40 434 471 37.1 5.50E-08 273
CGPG5535 WD40 476 513 47.2 5.20E-11 273 CGPG5535 WD40 518 555 35.6
1.60E-07 273 CGPG5535 WD40 567 604 36.1 1.10E-07 273 CGPG5535 WD40
621 658 34.9 2.50E-07 273 CGPG5535 WD40 663 706 35.5 1.60E-07 274
CGPG5540 ESCRT-III 21 207 265.7 8.40E-77 275 CGPG5568 AA_permease
69 521 -67.1 0.00013 276 CGPG5577 SMC_N 21 1049 -2.8 1.50E-11 277
CGPG5587 Thioredoxin 78 199 0.1 0.00015 278 CGPG5594 Histone 27 100
99.6 8.80E-27 279 CGPG5633 PGAM 91 277 153.2 6.30E-43 280 CGPG5640
Aminotran_1_2 30 385 362.1 8.00E-106 281 CGPG5646 iPGM_N 2 363
876.8 9.10E-261 281 CGPG5646 Metalloenzyme 373 488 173.3 5.40E-49
282 CGPG5656 Gln-synt_N 17 99 85.7 1.30E-22 282 CGPG5656 Gln-synt_C
105 358 528.1 8.50E-156 283 CGPG5659 Aminotran_3 35 361 321.7
1.20E-93 284 CGPG5661 PK 1 345 702 3.80E-208 284 CGPG5661 PK_C 355
469 115.4 1.50E-31 285 CGPG5684 Glycolytic 4 339 578.6 5.60E-171
286 CGPG5694 TIM 4 246 465.9 4.50E-137 287 CGPG5704 NDK 2 136 329
7.30E-96 288 CGPG5714 NDK 4 138 266.4 5.20E-77 289 CGPG5721
Rib_5-P_isom_A 47 215 354.4 1.70E-103 290 CGPG5728 zf-CCCH 80 106
39.3 1.20E-08 291 CGPG5757 Sad1_UNC 203 330 195.6 1.00E-55 292
CGPG5764 Actin 11 140 36.4 6.60E-08 293 CGPG5783 TPT 272 442 71.5
2.40E-18 294 CGPG5791 AA_permease 114 588 704.9 5.40E-209 295
CGPG5799 Aa_trans 206 601 453 3.60E-133 296 CGPG5856 Pkinase 79 353
159.5 8.00E-45 296 CGPG5856 Pkinase_Tyr 79 353 149.4 9.00E-42 297
CGPG5927 AAA 58 248 252.4 8.70E-73 297 CGPG5927 AAA 322 510 290.8
2.40E-84 298 CGPG5941 PfkB 42 336 86.2 9.00E-23 299 CGPG5957 CBM_20
86 178 23 5.70E-07 300 CGPG5967 DUF822 2 147 307.1 2.90E-89 301
CGPG6040 LEA_3 1 88 178.7 1.30E-50 302 CGPG607 PurA 28 275 44.4
5.80E-12 303 CGPG6178 DUF1336 53 267 427.9 1.30E-125 304 CGPG6185
UQ_con 7 148 197.9 2.10E-56 305 CGPG6306 APC8 1 161 401.5 1.10E-117
305 CGPG6306 TPR_1 339 372 34.7 2.90E-07 305 CGPG6306 TPR_2 339 372
23.8 0.00058 305 CGPG6306 TPR_2 373 406 23.7 0.00062 305 CGPG6306
TPR_1 373 406 34.1 4.40E-07 305 CGPG6306 TPR_2 407 440 22 0.0019
305 CGPG6306 TPR_1 407 440 24.3 0.00039 306 CGPG6318 MFS_1 31 496
35.3 1.90E-07 306 CGPG6318 PTR2 92 484 208.6 1.30E-59 307 CGPG6326
Kelch_1 34 79 45 2.40E-10 307 CGPG6326 Kelch_2 34 79 43.5 6.80E-10
307 CGPG6326 Kelch_1 152 198 26.7 7.70E-05 307 CGPG6326 Kelch_2 152
198 32.1 1.80E-06 307 CGPG6326 Kelch_2 203 249 20.2 0.0067 307
CGPG6326 Kelch_1 204 248 7.9 0.45 308 CGPG6370 Gp_dh_N 3 151 326
5.80E-95 308 CGPG6370 Gp_dh_C 156 313 362 8.80E-106 309 CGPG6429
ADH_N 25 135 159.4 8.40E-45 309 CGPG6429 ADH_zinc_N 166 305 105.1
1.90E-28 310 CGPG6440 PK 1 345 789.4 1.90E-234 310 CGPG6440 PK_C
357 471 167.5 3.10E-47 310 CGPG6440 PEP-utilizers 486 575 134
3.60E-37 311 CGPG6516 Aldedh 19 478 778.3 4.10E-231 312 CGPG6653
LRRNT_2 23 66 49.3 1.20E-11 312 CGPG6653 LRR_1 71 93 12.1 1.4 312
CGPG6653 LRR_1 95 117 10.5 2.8 312 CGPG6653 LRR_1 119 142 13 0.93
312 CGPG6653 LRR_1 144 166 19.5 0.011 312 CGPG6653 LRR_1 168 190
10.6 2.7 312 CGPG6653 LRR_1 192 214 8.8 5.7 312 CGPG6653 LRR_1 289
311 17.4 0.046 312 CGPG6653 LRR_1 313 335 10.8 2.4 312 CGPG6653
LRR_1 337 359 10.7 2.5 312 CGPG6653 LRR_1 361 384 12 1.5 312
CGPG6653 LRR_1 409 431 10.4 2.9 312 CGPG6653 LRR_1 457 479 11.9 1.5
312 CGPG6653 LRR_1 481 503 10.4 3 312 CGPG6653 LRR_1 505 527 10.5
2.8 312 CGPG6653 LRR_1 529 551 11.1 2.2 312 CGPG6653 LRR_1 553 575
9.3 4.7 312 CGPG6653 LRR_1 577 598 11.1 2.1 312 CGPG6653 Pkinase
695 966 134.8 2.20E-37 312 CGPG6653 Pkinase_Tyr 695 966 134.5
2.60E-37 313 CGPG6712 PGAM 91 277 154.6 2.30E-43 314 CGPG6737 PGAM
92 253 173 6.80E-49 315 CGPG6747 FBPase 106 429 448.6 7.20E-132 316
CGPG6796 Alpha-amylase 14 452 199.5 7.00E-57 318 CGPG6810 GH3 12
561 1261.5 0 320 CGPG6953 Ank 121 153 50.4 5.60E-12 321 CGPG7121
L51_S25_CI-B8 20 93 108.9 1.40E-29 322 CGPG7163 Prenylcys_lyase 149
500 788 5.20E-234 324 CGPG7206 Aldo_ket_red 14 298 389.4 4.80E-114
325 CGPG7225 Subtilisin_N 48 125 84.3 3.30E-22 326 CGPG7267 DUF588
34 164 154.6 2.40E-43 327 CGPG7272 DUF1005 50 254 524.8 8.50E-155
328 CGPG7281 FA_hydroxylase 86 229 361.9 9.30E-106 329 CGPG7308
CoA_binding 1 100 -10.8 0.04 329 CGPG7308 NAD_Gly3P_dh_N 4 147 -0.6
2.00E-06 329 CGPG7308 F420_oxidored 5 251 282.5 7.50E-82 330
CGPG7316 Anti-silence 1 155 419.9 3.20E-123 331 CGPG7371
Response_reg 29 157 94.8 2.30E-25 332 CGPG7457 PfkB 114 408 146.6
6.00E-41 335 CGPG7636 LSM 13 81 78.2 2.40E-20 338 CGPG7804 FAR1 62
279 364.9 1.20E-106 338 CGPG7804 SWIM 556 589 37.3 4.90E-08 339
CGPG7823 Rotamase 104 188 91.8 1.90E-24 339 CGPG7823 Rhodanese 203
298 44.1 4.40E-10 340 CGPG7828 DnaJ 12 81 66.1 1.00E-16 340
CGPG7828 zf-CSL 96 174 25.2 0.00021 343 CGPG7986 F-box 48 96 31.2
3.40E-06 343 CGPG7986 LRR_1 189 216 9 5.4 343 CGPG7986 LRR_1 428
451 8.2 7.7 343 CGPG7986 LRR_1 561 584 8.4 6.9 345 CGPG8015 zf-CCHC
18 35 24.2 7.90E-05 349 CGPG8083 Tryp_alpha_amyl 28 105 37.6
3.90E-08 350 CGPG8106 BURP 56 280 380 3.20E-111 353 CGPG8152
PAP_fibrillin 9 124 45.9 1.20E-10 354 CGPG8166 PBD 118 166 49.9
7.80E-12 355 CGPG8377 Oleosin 30 109 55 2.20E-13 356 CGPG8976
Ceramidase_alk 50 795 1545.5 0 357 CGPG8987 FH2 439 839 552.9
3.10E-163 358 CGPG9013 NAD_binding_1 234 350 138.6 1.50E-38 359
CGPG9080 EGF_CA 315 357 42.2 1.60E-09 359 CGPG9080 Pkinase 433 716
120.3 4.90E-33 359 CGPG9080 Pkinase_Tyr 433 704 113.1 7.40E-31 360
CGPG9081 DUF676 30 247 319 7.50E-93 361 CGPG9130 MMR_HSR1 266 369
71.9 1.80E-18 362 CGPG9133 PPR 125 159 13.2 0.19 362 CGPG9133 PPR
161 195 2.8 3.2 362 CGPG9133 PPR 196 230 22.7 0.0012 362 CGPG9133
PPR 232 266 42.1 1.70E-09 362 CGPG9133 PPR 267 301 29.4 1.20E-05
362 CGPG9133 PPR 302 336 49.4 1.10E-11 362 CGPG9133 PPR 337 371
32.9 1.00E-06 362 CGPG9133 PPR 372 407 7.9 0.8 362 CGPG9133 PPR 408
442 49.7 8.80E-12 362 CGPG9133 PPR 443 477 22.6 0.0013 362 CGPG9133
PPR 478 512 37.8 3.40E-08 362 CGPG9133 PPR 513 547 42.9 1.00E-09
362 CGPG9133 PPR 548 582 30.2 6.70E-06 362 CGPG9133 PPR 583 617
38.4 2.20E-08 363 CGPG9134 HD 91 232 46.2 1.00E-10 364 CGPG9137
RrnaAD 65 337 122.2 1.30E-33 365 CGPG9141 Pantoate_transf 40 306
402.9 4.40E-118 366 CGPG9145 Lung_7-TM_R 168 423 385.2 8.80E-113
367 CGPG9147 DNA_pol_E_B 178 389 249.9 5.00E-72 368 CGPG9148 p450
36 502 128.8 1.40E-35 369 CGPG9155 Pkinase 86 347 56.9 6.10E-14 369
CGPG9155 Pkinase_Tyr 86 351 73.7 2.90E-20 370 CGPG9163
Na_H_Exchanger 12 378 280.1 3.80E-81 370 CGPG9163 TrkA_N 416 531
118.8 1.50E-32 371 CGPG9170 Complex1_30 kDa 90 158 103.8
4.70E-28
371 CGPG9170 Complex1_49 kDa 298 537 2.8 6.10E-13 373 CGPG9183
HTH_11 1 56 72.4 1.30E-18 373 CGPG9183 BPL_LipA_LipB 84 182 94.5
2.90E-25 373 CGPG9183 BPL_C 275 322 43.3 7.80E-10 374 CGPG9186
DHBP_synthase 8 203 370.6 2.20E-108 374 CGPG9186 GTP_cyclohydro2
208 366 -2.3 3.80E-10 375 CGPG9205 NTP_transferase 4 288 421
1.50E-123 375 CGPG9205 MannoseP_isomer 299 465 350.8 2.00E-102 375
CGPG9205 Cupin_2 380 450 55.3 1.80E-13 376 CGPG9207 HTH_11 6 59
50.9 3.90E-12 376 CGPG9207 BPL_LipA_LipB 83 180 104.5 2.80E-28 376
CGPG9207 BPL_C 271 317 46.5 8.50E-11 377 CGPG9219 Complex1_30 kDa
107 175 99.8 7.60E-27 377 CGPG9219 Complex1_49 kDa 311 537 -11.6
5.90E-12 378 CGPG9220 GDC-P 3 443 700.9 8.30E-208 379 CGPG9230
Peptidase_S10 88 488 657.2 1.20E-194 381 CGPG9238 Tryp_alpha_amyl
36 114 56 1.20E-13 382 CGPG9259 Mit_rib_S27 14 93 135.3 1.50E-37
383 CGPG9271 NPH3 215 418 189.9 5.60E-54 384 CGPG9275 ETC_C1_NDUFA5
35 91 112.8 9.00E-31 386 CGPG9283 DUF1195 6 161 180.3 4.30E-51 387
CGPG9309 MAP65_ASE1 38 575 52.8 1.10E-12 389 CGPG9322 Pkinase 103
383 170 5.40E-48 389 CGPG9322 Pkinase_Tyr 103 383 158.7 1.30E-44
390 CGPG9335 Sugar_tr 7 726 210.6 3.30E-60 390 CGPG9335 MFS_1 11
685 107.7 3.00E-29 391 CGPG9341 RMMBL 531 573 35.9 1.20E-07 392
CGPG9344 TPR_2 531 564 23.9 0.00051 393 CGPG9345 UPF0261 5 432
483.7 2.00E-142 394 CGPG976 Glyco_transf_8 216 533 401.3
1.30E-117
TABLE-US-00017 TABLE 17 Pfam domain accession gathering name number
cutoff domain description 14-3-3 PF00244.9 25 14-3-3 protein
2-Hacid_dh PF00389.19 11.2 D-isomer specific 2-hydroxyacid
dehydrogenase, catalytic domain 2-Hacid_dh_C PF02826.7 -82.2
D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain
AAA PF00004.18 12.3 ATPase family associated with various cellular
activities (AAA) AA_permease PF00324.10 -120.8 Amino acid permease
ACT PF01842.13 0 ACT domain ADH_N PF08240.2 -14.5 Alcohol
dehydrogenase GroES-like domain ADH_zinc_N PF00107.16 23.8
Zinc-binding dehydrogenase APC8 PF04049.3 -19.8 Anaphase promoting
complex subunit 8/ Cdc23 Aa_trans PF01490.7 -128.4 Transmembrane
amino acid transporter protein Actin PF00022.8 -30 Actin Aldedh
PF00171.11 -209.3 Aldehde dehydrogenase family Aldo_ket_red
PF00248.10 -97 Aldo/keto reductase family Allene_ox_cyc PF06351.2
25 Allene oxide cyclase Alpha-amylase PF00128.12 -93 Alpha amylase,
catalytic domain Aminotran_1_2 PF00155.10 -57.5 Aminotransferase
class I and II Aminotran_3 PF00202.10 -207.6 Aminotransferase
class-III Ank PF00023.18 0 Ankyrin repeat Anti-silence PF04729.4 25
Anti-silencing protein, ASF1-like ArfGap PF01412.8 -17 Putative
GTPase activating protein for Arf Asp PF00026.13 -186.1 Eukaryotic
aspartyl protease Auxin_inducible PF02519.4 -15 Auxin responsive
protein BPL_C PF02237.6 16 Biotin protein ligase C terminal domain
BPL_LipA_LipB PF03099.8 -0.2 Biotin/lipoate A/B protein ligase
family BURP PF03181.5 -52 BURP domain B_lectin PF01453.14 28.2
D-mannose binding lectin BolA PF01722.7 23 BolA-like protein C2
PF00168.18 3.7 C2 domain CBM_20 PF00686.9 -3 Starch binding domain
CMAS PF02353.10 -177.9 Cyclopropane-fatty-acyl-phospholipid
synthase Ceramidase_alk PF04734.3 25 Neutral/alkaline non-lysosomal
ceramidase CoA_binding PF02629.8 -12.8 CoA binding domain
Complex1_30kDa PF00329.8 -3 Respiratory-chain NADH dehydrogenase,
30 Kd subunit Complex1_49kDa PF00346.8 -108 Respiratory-chain NADH
dehydrogenase, 49 Kd subunit Cupin_2 PF07883.1 16.6 Cupin domain
Cupin_3 PF05899.2 4.4 Protein of unknown function (DUF861) Cyclin_C
PF02984.8 -13 Cyclin, C-terminal domain Cyclin_N PF00134.13 -14.7
Cyclin, N-terminal domain DHBP_synthase PF00926.10 -116
3,4-dihydroxy-2-butanone 4-phosphate synthase DNA_pol_E_B PF04042.5
-47.5 DNA polymerase alpha/epsilon subunit B DUF1005 PF06219.2 25
Protein of unknown function (DUF1005) DUF1191 PF06697.2 25 Protein
of unknown function (DUF1191) DUF1195 PF06708.1 25 Protein of
unknown function (DUF1195) DUF1336 PF07059.2 -78.2 Protein of
unknown function (DUF1336) DUF26 PF01657.7 0 Domain of unknown
function DUF26 DUF298 PF03556.6 25 Domain of unknown function
(DUF298) DUF588 PF04535.2 25 Domain of unknown function (DUF588)
DUF676 PF05057.4 -60.7 Putative serine esterase (DUF676) DUF822
PF05687.3 25 Plant protein of unknown function (DUF822) DUF868
PF05910.2 25 Plant protein of unknown function (DUF868) DnaJ
PF00226.19 -8 DnaJ domain EGF_CA PF07645.4 24.5 Calcium binding EGF
domain ESCRT-III PF03357.10 -35.4 ESCRT-III complex subunit
ETC_C1_NDUFA5 PF04716.3 25 ETC complex I subunit conserved region
F-box PF00646.21 13.6 F-box domain F420_oxidored PF03807.6 -34.5
NADP oxidoreductase coenzyme F420- dependent FAR1 PF03101.4 0 FAR1
family FA_hydroxylase PF04116.2 -64.1 Fatty acid hydroxylase FBPase
PF00316.10 -170.3 Fructose-1-6-bisphosphatase FH2 PF02181.13 -98.3
Formin Homology 2 Domain GDC-P PF02347.5 -306.2 Glycine cleavage
system P-protein GH3 PF03321.3 -336 GH3 auxin-responsive promoter
GTP_cyclohydro2 PF00925.11 -49 GTP cyclohydrolase II Gln-synt_C
PF00120.14 -124 Glutamine synthetase, catalytic domain Gln-synt_N
PF03951.9 9 Glutamine synthetase, beta-Grasp domain Glyco_transf_8
PF01501.9 -43.2 Glycosyl transferase family 8 Glycolytic PF00274.9
-174.5 Fructose-bisphosphate aldolase class-I Gp_dh_C PF02800.9
-64.1 Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain
Gp_dh_N PF00044.12 -74.2 Glyceraldehyde 3-phosphate dehydrogenase,
NAD binding domain HD PF01966.11 18 HD domain HLH PF00010.15 8.2
Helix-loop-helix DNA-binding domain HTH_11 PF08279.1 11.3 HTH
domain Histone PF00125.13 17.4 Core histone H2A/H2B/H3/H4 Kelch_1
PF01344.14 7.8 Kelch motif Kelch_2 PF07646.4 14 Kelch motif
L51_S25_CI-B8 PF05047.5 25 Mitochondrial ribosomal protein L51/S25/
CI-B8 domain LEA_3 PF03242.3 25 Late embryogenesis abundant protein
LMBR1 PF04791.5 -116.3 LMBR1-like membrane protein LRRNT_2
PF08263.2 18.6 Leucine rich repeat N-terminal domain LRR_1
PF00560.21 7.7 Leucine Rich Repeat LSM PF01423.12 13.7 LSM domain
Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptor
MAP65_ASE1 PF03999.2 -134.8 Microtubule associated protein
(MAP65/ASE1 family) MFS_1 PF07690.5 23.5 Major Facilitator
Superfamily MMR_HSR1 PF01926.11 31.2 GTPase of unknown function
MannoseP_isomer PF01050.8 -70 Mannose-6-phosphate isomerase
Metalloenzyme PF01676.7 -14.4 Metalloenzyme superfamily Metallophos
PF00149.17 22 Calcineurin-like phosphoesterase Methyltransf_11
PF08241.1 17.1 Methyltransferase domain Methyltransf_12 PF08242.1
21.4 Methyltransferase domain Mit_rib_S27 PF08293.1 25
Mitochondrial ribosomal subunit S27 MtN3_slv PF03083.5 -0.8
MtN3/saliva family NAD_Gly3P_dh_N PF01210.12 -44 NAD-dependent
glycerol-3-phosphate dehydrogenase N-terminus NAD_binding_1
PF00175.10 -3.9 Oxidoreductase NAD-binding domain NAF PF03822.4 4.5
NAF domain NDK PF00334.9 -59.9 Nucleoside diphosphate kinase NPH3
PF03000.4 25 NPH3 family NTP_transferase PF00483.12 -90.5
Nucleotidyl transferase Na_H_Exchanger PF00999.10 -67.9
Sodium/hydrogen exchanger family ORMDL PF04061.4 25 ORMDL family
Oleosin PF01277.7 -27 Oleosin PAN_2 PF08276.2 -4.9 PAN-like domain
PAP_fibrillin PF04755.2 25 PAP_fibrillin PBD PF00786.17 12.2
P21-Rho-binding domain PCI PF01399.15 25 PCI domain PEP-utilizers
PF00391.12 10 PEP-utilising enzyme, mobile domain PGAM PF00300.12
-3 Phosphoglycerate mutase family PK PF00224.10 -244 Pyruvate
kinase, barrel domain PK_C PF02887.5 -44 Pyruvate kinase,
alpha/beta domain PPR PF01535.11 0 PPR repeat PTR2 PF00854.12 -50
POT family Pantoate_transf PF02548.5 -93 Ketopantoate
hydroxymethyltransferase Peptidase_S10 PF00450.11 -198 Serine
carboxypeptidase PfkB PF00294.13 -67.8 pfkB family carbohydrate
kinase Phosphorylase PF00343.9 -601.1 Carbohydrate phosphorylase
Pkinase PF00069.14 -70.8 Protein kinase domain Pkinase_Tyr
PF07714.5 65 Protein tyrosine kinase Prenylcys_lyase PF07156.3
-164.1 Prenylcysteine lyase PurA PF04845.3 25 PurA ssDNA and
RNA-binding protein RMMBL PF07521.1 18.5 RNA-metabolising
metallo-beta-lactamase Response_reg PF00072.12 4 Response regulator
receiver domain Rhodanese PF00581.9 25 Rhodanese-like domain
Rib_5-P_isom_A PF06026.4 25 Ribose 5-phosphate isomerase A
(phosphoriboisomerase A) Ribonuclease_T2 PF00445.8 -53 Ribonuclease
T2 family Ribosomal_S8e PF01201.11 25 Ribosomal protein S8e
Rotamase PF00639.10 4 PPIC-type PPIASE domain RrnaAD PF00398.9
-73.3 Ribosomal RNA adenine dimethylase SAM_1 PF00536.18 11.3 SAM
domain (Sterile alpha motif) SMC_N PF02463.8 -95.8 RecF/RecN/SMC N
terminal domain SNF5 PF04855.3 25 SNF5/SMARCB1/INI1 SWIB PF02201.9
-7 SWIB/MDM2 domain SWIM PF04434.7 10 SWIM zinc finger
S_locus_glycop PF00954.11 -12.7 S-locus glycoprotein family
Sad1_UNC PF07738.2 -20.4 Sad1/UNC-like C-terminal Sterol_MT_C
PF08498.1 30.5 Sterol methyltransferase C-terminal Subtilisin_N
PF05922.6 26.1 Subtilisin N-terminal Region Sugar_tr PF00083.13 -85
Sugar (and other) transporter TFIIA PF03153.4 25 Transcription
factor IIA, alpha/beta subunit TFIIS_C PF01096.9 15 Transcription
factor S-II (TFIIS) TFIIS_M PF07500.3 7.4 Transcription factor S-II
(TFIIS), central domain TIM PF00121.8 -97 Triosephosphate isomerase
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat TPR_2 PF07719.5 20.1
Tetratricopeptide repeat TPT PF03151.7 -15.3 Triose-phosphate
Transporter family Thioredoxin PF00085.9 -25.7 Thioredoxin Tim17
PF02466.8 2.7 Tim17/Tim22/Tim23 family TrkA_N PF02254.7 4.7 TrkA-N
domain Tryp_alpha_amyl PF00234.10 -4 Protease inhibitor/seed
storage/LTP family UPF0261 PF06792.1 25 Uncharacterised protein
family (UPF0261) UQ_con PF00179.16 -30 Ubiquitin-conjugating enzyme
Usp PF00582.16 36.1 Universal stress protein family WD40 PF00400.20
21.5 WD domain, G-beta repeat adh_short PF00106.14 -17 short chain
dehydrogenase elF-4B PF06273.1 -205.7 Plant specific eukaryotic
initiation factor 4B iPGM_N PF06415.3 -263.4 BPG-independent PGAM
N-terminus (iPGM_N) p450 PF00067.11 -105 Cytochrome P450 zf-AN1
PF01428.6 0 AN1-like Zinc finger zf-C3HC4 PF00097.13 16.9 Zinc
finger, C3HC4 type (RING finger) zf-CCCH PF00642.14 0 Zinc finger
C-x8-C-x5-C-x3-H type (and similar) zf-CCHC PF00098.12 17.9 Zinc
knuckle zf-CSL PF05207.3 2.9 CSL zinc finger
Example 5
Plasmid Contraction for Transferring Recombinant DNA
[0201] This example illustrates the construction of plasmids for
transferring recombinant DNA into the nucleus of a plant cell which
can be regenerated into a transgenic crop plant of this invention.
Primers for PCR amplification of protein coding nucleotides of
recombinant DNA are designed at or near the start and stop codons
of the coding sequence, in order to eliminate most of the 5' and 3'
untranslated regions. DNA of interest, i.e. each DNA identified in
Table 1 and the DNA for the identified homologous genes, are cloned
and amplified by PCR prior to insertion into the insertion site the
base vector.
[0202] A. Corn Transformation Vector
[0203] Elements of an exemplary common expression vector, pMON93093
are illustrated in Table 18. The exemplary base vector which is
especially useful for corn transformation is illustrated in FIG. 2
and assembled using technology known in the art. The DNA of
interest are inserted in a expression vector at the insertion site
between the intron1 of rice act 1 gene and the termination sequence
of PinII gene.
TABLE-US-00018 TABLE 18 pMON93093 Coordinates of SEQ ID NO:
function name annotation 19940 Agro B-AGRtu.right border Agro right
border 11364-11720 transforamtion sequence, essential for transfer
of T-DNA. Gene of E-Os.Act1 upstream promoter 19-775 interest
region of the rice actin expression 1 gene cassette
E-CaMV.35S.2xA1-B3 duplicated35S A1-B3 788-1120 domain without TATA
box P-Os.Act1 promoter region of the 1125-1204 rice actin 1 gene
L-Ta.Lhcb1 5' untranslated leader 1210-1270 of wheat major
chlorophyll a/b binding protein I-Os.Act1 first intron and flanking
1287-1766 UTR exon sequences from the rice actin 1 gene T-St.Pis4
3' non-translated region 1838-2780 of the potato proteinase
inhibitor II gene which functions to direct polyadenylation of the
mRNA Plant P-Os.Act1 Promoter from the rice 2830-3670 selectable
actin 1 gene marker L-Os.Act1 first exon of the rice 3671-3750
expression actin 1 gene cassette I-Os.Act1 first intron and
flanking 3751-4228 UTR exon sequences from the rice actin 1 gene
TS-At.ShkG-CTP2 Transit peptide region 4238-4465 of Arabidopsis
EPSPS CR-AGRtu.aroA- Synthetic CP4 coding 4466-5833 CP4.nat region
with dicot preferred codon usage. T-AGRtu.nos A 3' non-translated
5849-6101 region of the nopaline synthase gene of Agrobacterium
tumefaciens Ti plasmid which functions to direct polyadenylation of
the mRNA. Agro B-AGRtu.left border Agro left border 6168-6609
transformation sequence, essential for transfer of T-DNA.
Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092 in E.
coli replication from plasmid RK2. CR-Ec.rop Coding region for
8601-8792 repressor of primer from the ColE1 plasmid. Expression of
this gene product interferes with primer binding at the origin of
replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The
minimal origin of 9220-9808 replication from the E. coli plasmid
ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 10339-10380
adenylyltransferase (AAD(3'')) CR-Ec.aadA-SPC/STR Coding region for
Tn7 10381-11169 adenylyltransferase (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR
from the Tn7 11170-11227 adenylyltransferase (AAD(3'')) gene of E.
coli.
[0204] B. Soybean Transformation Vector
[0205] Plasmids for use in transformation of soybean are also
prepared. Elements of an exemplary common expression vector plasmid
pMON82053 are shown in Table 19 below. This exemplary soybean or
canola transformation base vector illustrated in FIG. 3 is
assembled using the technology known in the art. Recombinant DNA of
interest, i.e. each DNA identified in Table 1 and the DNA for the
identified homologous genes, is cloned and amplified by PCR prior
to insertion into the insertion site the base vector at the
insertion site between the enhanced 35S CaMV promoter and the
termination sequence of cotton E6 gene.
TABLE-US-00019 TABLE 19 pMON82053 Coordinates of SEQ ID NO:
function name annotation 19941 Agro B-AGRtu.left Agro left border
sequence, essential 6144-6585 transforamtion border for transfer of
T-DNA. Plant P-At.Act7 Promoter from the arabidopsis actin
6624-7861 selectable 7 gene marker L-At.Act7 5'UTR of Arabidopsis
Act7 gene expression I-At.Act7 Intron from the Arabidopsis actin7
cassette gene TS-At.ShkG- Transit peptide region of Arabidopsis
7864-8091 CTP2 EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region
with 8092-9459 CP4.nno_At dicot preferred codon usage. T-AGRtu.nos
A 3' non-translated region of the 9466-9718 nopaline synthase gene
of Agrobacterium tumefaciens Ti plasmid which functions to direct
polyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for
35S RNA from CaMV 1-613 interest containing a duplication of the
-90 to expression -350 region. cassette T-Gb.E6-3b 3' untranslated
region from the fiber 688-1002 protein E6 gene of sea-island
cotton; Agro B-AGRtu.right Agro right border sequence, essential
1033-1389 transformation border for transfer of T-DNA. Maintenance
OR-Ec.oriV-RK2 The vegetative origin of replication 5661-6057 in E.
coli from plasmid RK2. CR-Ec.rop Coding region for repressor of
primer 3961-4152 from the ColE1 plasmid. Expression of this gene
product interferes with primer binding at the origin of
replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The
minimal origin of replication 2945-3533 from the E. coli plasmid
ColE1. P-Ec.aadA- romoter for Tn7 adenylyltransferase 2373-2414
SPC/STR (AAD(3'')) CR-Ec.aadA- Coding region for Tn7 1584-2372
SPC/STR adenylyltransferase (AAD(3'')) conferring spectinomycin and
streptomycin resistance. T-Ec.aadA- 3' UTR from the Tn7 1526-1583
SPC/STR adenylyltransferase (AAD(3'')) gene of E. coli.
[0206] C. Cotton Transformation Vector
[0207] Plasmids for use in transformation of cotton are also
prepared. Elements of an exemplary common expression vector plasmid
pMON99053 are shown in Table 20 below and FIG. 4. Primers for PCR
amplification of protein coding nucleotides of recombinant DNA are
designed at or near the start and stop codons of the coding
sequence, in order to eliminate most of the 5' and 3' untranslated
regions. Each recombinant DNA coding for a protein identified in
Table 1 is amplified by PCR prior to insertion into the insertion
site within the gene of interest expression cassette of one of the
base.
TABLE-US-00020 TABLE 20 Coordinates of SEQ ID function name
annotation NO: 19942 Agro B-AGRtu.right border Agro right border
sequence, 11364-11720 transforamtion essential for transfer of T-
DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S
7794-8497 expression enh + ph.DnaK RNA promoter from CaMV cassette
plus the petunia hsp70 5' untranslated region T-Ps.RbcS2-E9 The 3'
non-translated region 67-699 of the pea RbcS2 gene which functions
to direct polyadenylation of the mRNA. Plant selectable
Exp-CaMV.35S Promoter and 5' untranslated 730-1053 marker region of
the 35S RNA from expression CaMV cassette CR-Ec.nptII-Tn5 Neomycin
1087-1881 Phosphotransferase II gene that confers resistance to
neomycin and kanamycin T-AGRtu.nos A 3' non-translated region of
1913-2165 the nopaline synthase gene of Agrobacterium tumefaciens
Ti plasmid which functions to direct polyadenylation of the mRNA.
Agro B-AGRtu.left border Agro left border sequence, 2211-2652
transformation essential for transfer of T- DNA. Maintenance in
OR-Ec.oriV-RK2 The vegetative origin of 2739-3135 E. coli
replication from plasmid RK2. CR-Ec.rop Coding region for repressor
4644-4835 of primer from the ColE1 plasmid. Expression of this gene
product interferes with primer binding at the origin of
replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The
minimal origin of 5263-5851 replication from the E. coli plasmid
ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 6382-6423
adenylyltransferase (AAD(3'')) CR-Ec.aadA-SPC/STR Coding region for
Tn7 6424-7212 adenylyltransferase (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR
from the Tn7 7213-7270 adenylyltransferase (AAD(3'')) gene of E.
coli.
Example 6
Corn Plant Transformation
[0208] This example illustrates the production and identification
of transgenic corn cells in seed of transgenic corn plants having
an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency,
increased yield, enhanced water use efficiency, enhanced tolerance
to cold and/or improved seed compositions as compared to control
plants. Transgenic corn cells are prepared with recombinant DNA
expressing each of the protein encoding DNAs listed in Table 1 by
Agrobacterium-mediated transformation using the corn transformation
vectors pMON93093 as disclosed in Example 6. Corn transformation is
effected using methods disclosed in U.S. Patent Application
Publication 2004/0344075 A1 where corn embryos are inoculated and
co-cultured with the Agrobacterium tumefaciens strain ABI and the
corn transformation vector. To regenerate transgenic corn plants
the transgenic callus resulting from transformation is placed on
media to initiate shoot development in plantlets which are
transferred to potting soil for initial growth in a growth chamber
followed by a mist bench before transplanting to pots where plants
are grown to maturity. The plants are self fertilized and seed is
harvested for screening as seed, seedlings or progeny R2 plants or
hybrids, e.g., for yield trials in the screens indicated above.
[0209] Many transgenic events which survive to fertile transgenic
plants that produce seeds and progeny plants do not exhibit an
enhanced agronomic trait. The transgenic plants and seeds having
the transgenic cells of this invention which have recombinant DNA
imparting the enhanced agronomic traits are identified by screening
for nitrogen use efficiency, yield, water use efficiency, cold
tolerance and improved seed composition.
Example 7
Soybean Plant Transformation
[0210] This example illustrates the production and identification
of transgenic soybean cells in seed of transgenic soybean plants
having an enhanced agronomic trait, i.e. enhanced nitrogen use
efficiency, increased yield, enhanced water use efficiency,
enhanced tolerance to cold and/or improved seed compositions as
compared to control plants. Transgenic soybean cells are prepared
with recombinant DNA expressing each of the protein encoding DNAs
listed in Table 1 by Agrobacterium-mediated transformation using
the soybean transformation vectors pMON82053 disclosed in Example
7. Soybean transformation is effected using methods disclosed in
U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded
then inoculated and co-cultured with the soybean transformation
vector, then transferred to selection media for 6-8 weeks to allow
selection and growth of transgenic shoots.
[0211] The transformation is repeated for each of the protein
encoding DNAs identified in Table 1.
[0212] Transgenic shoots producing roots are transferred to the
greenhouse and potted in soil. Many transgenic events which survive
to fertile transgenic plants that produce seeds and progeny plants
do not exhibit an enhanced agronomic trait. The transgenic plants
and seeds having the transgenic cells of this invention which have
recombinant DNA imparting the enhanced agronomic traits are
identified by screening for nitrogen use efficiency, yield, water
use efficiency, cold tolerance and improved seed composition.
Example 8
Canola Transformation
[0213] This example illustrates plant transformation useful in
producing the transgenic canola plants of this invention and the
production and identification of transgenic seed for transgenic
canola having enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil.
[0214] Tissues from in vitro grown canola seedlings are prepared
and inoculated with overnight-grown Agrobacterium cells containing
plasmid DNA with the gene of interest cassette and a plant
selectable marker cassette. Following co-cultivation with
Agrobacterium, the infected tissues are allowed to grow on
selection to promote growth of transgenic shoots, followed by
growth of roots from the transgenic shoots. The selected plantlets
are then transferred to the greenhouse and potted in soil.
Molecular characterization are performed to confirm the presence of
the gene of interest, and its expression in transgenic plants and
progenies. Progeny transgenic plants are selected from a population
of transgenic canola events under specified growing conditions and
are compared with control canola plants. Control canola plants are
substantially the same canola genotype but without the recombinant
DNA, for example, either a parental canola plant of the same
genotype that is not transformed with the identical recombinant DNA
or a negative isoline of the transformed plant
[0215] Transgenic canola plant cells are transformed with
recombinant DNA from each of the genes identified in Table 1.
Transgenic progeny plants and seed of the transformed plant cells
are screened for enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil as reported in Example
9.
Example 9
Selection of Transgenic Plants with Enhanced Agronomic Trait(s)
[0216] This example illustrates identification of nuclei of the
invention by screening derived plants and seeds for an enhanced
trait identified below.
[0217] Many transgenic events which survive to fertile transgenic
plants that produce seeds and progeny plants will not exhibit an
enhanced agronomic trait. Populations of transgenic seed and plants
prepared in Examples 6 and 7 are screened to identify those
transgenic events providing transgenic plant cells with a nucleus
having recombinant DNA imparting an enhanced trait. Each population
is screened for enhanced nitrogen use efficiency, increased yield,
enhanced water use efficiency, enhanced tolerance to cold and heat,
increased level of oil and protein in seed using assays described
below. Plant cell nuclei having recombinant DNA with each of the
genes identified in Table 1 and the identified homologs are
identified in plants and seeds with at least one of the enhanced
traits.
A. Selection for Enhanced Nitrogen Use Efficiency
[0218] Transgenic corn plants with nuclei of the invention are
planted in fields with three levels of nitrogen (N) fertilizer
being applied, i.e. low level (0 pounds per acre N), medium level
(80 pounds per acre N) and high level (180 pounds per acre N).
Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N
source and apply by broadcast boom and incorporate with a field
cultivator with rear rolling basket in the same direction as
intended crop rows. Although there is no N applied in the low level
treatment, the soil should still be disturbed in the same fashion
as the treated area. Transgenic plants and control plants can be
grouped by genotype and construct with controls arranged randomly
within genotype blocks. For improved statistical analysis each type
of transgenic plant can be tested by 3 replications and across 4
locations. Nitrogen levels in the fields are analyzed before
planting by collecting sample soil cores from 0-24'' and 24 to 48''
soil layer. Soil samples are analyzed for nitrate-nitrogen,
phosphorus (P), potassium (K), organic matter and pH to provide
baseline values. P, K and micronutrients are applied based upon
soil test recommendations.
[0219] Transgenic corn plants prepared in Example 6 and which
exhibit a 2 to 5% yield increase as compared to control plants when
grown in the high nitrogen field are selected as having nuclei of
the invention. Transgenic corn plants which have at least the same
or higher yield as compared to control plants when grown in the
medium nitrogen field are selected as having nuclei of the
invention. Transgenic corn plants having a nucleus with DNA
identified in Table 3 as imparting nitrogen use efficiency (LN) and
homologous DNA are selected from a nitrogen use efficiency screen
as having a nucleus of this invention.
B. Selection for Increased Yield
[0220] Many transgenic plants of this invention exhibit improved
yield as compared to a control plant. Improved yield can result
from enhanced seed sink potential, i.e. the number and size of
endosperm cells or kernels and/or enhanced sink strength, i.e. the
rate of starch biosynthesis. Sink potential can be established very
early during kernel development, as endosperm cell number and size
are determined within the first few days after pollination.
[0221] Much of the increase in corn yield of the past several
decades has resulted from an increase in planting density. During
that period, corn yield has been increasing at a rate of 2.1
bushels/acre/year, but the planting density has increased at a rate
of 250 plants/acre/year. A characteristic of modern hybrid corn is
the ability of these varieties to be planted at high density. Many
studies have shown that a higher than current planting density
should result in more biomass production, but current germplasm
does not perform. well at these higher densities. One approach to
increasing yield is to increase harvest index (HI), the proportion
of biomass that is allocated to the kernel compared to total
biomass, in high density plantings.
[0222] Effective yield selection of enhanced yielding transgenic
corn events uses hybrid progeny of the transgenic event over
multiple locations with plants grown under optimal production
management practices, and maximum pest control. A useful target for
improved yield is a 5% to 10% increase in yield as compared to
yield produced by plants grown from seed for a control plant.
Selection methods may be applied in multiple and diverse geographic
locations, for example up to 16 or more locations, over one or more
planting seasons, for example at least two planting seasons to
statistically distinguish yield improvement from natural
environmental effects. It is to plant multiple transgenic plants,
positive and negative control plants, and pollinator plants in
standard plots, for example 2 row plots, 20 feet long by 5 feet
wide with 30 inches distance between rows and a 3 foot alley
between ranges. Transgenic events can be grouped by recombinant DNA
constructs with groups randomly placed in the field. A pollinator
plot of a high quality corn line is planted for every two plots to
allow open pollination when using male sterile transgenic events. A
useful planting density is about 30,000 plants/acre. High planting
density is greater than 30,000 plants/acre, preferably about 40,000
plants/acre, more preferably about 42,000 plants/acre, most
preferably about 45,000 plants/acre. Each of the transgenic corn
plants and soybean plants with a nucleus of the invention prepared
in Examples 6 and 7 are screened for yield enhancement. At least
one event from each of the corn and soybean plants is selected as
having at least between 3 and 5% increase in yield as compared to a
control plant as having a nucleus of this invention.
C. Selection for Enhanced Water Use Efficiency (WUE)
[0223] The following is a high-throughput method for screening for
water use efficiency in a greenhouse to identify the transgenic
corn plants with a nucleus of this invention. This selection
process imposes 3 drought/re-water cycles on plants over a total
period of 15 days after an initial stress free growth period of 11
days. Each cycle consists of 5 days, with no water being applied
for the first four days and a water quenching on the 5th day of the
cycle. The primary phenotypes analyzed by the selection method are
the changes in plant growth rate as determined by height and
biomass during a vegetative drought treatment. The hydration status
of the shoot tissues following the drought is also measured. The
plant height are measured at three time points. The first is taken
just prior to the onset drought when the plant is 11 days old,
which is the shoot initial height (SIH). The plant height is also
measured halfway throughout the drought/re-water regimen, on day 18
after planting, to give rise to the shoot mid-drought height (SMH).
Upon the completion of the final drought cycle on day 26 after
planting, the shoot portion of the plant is harvested and measured
for a final height, which is the shoot wilt height (SWH) and also
measured for shoot wilted biomass (SWM). The shoot is placed in
water at 40 degree Celsius in the dark. Three days later, the shoot
is weighted to give rise to the shoot turgid weight (STM). After
drying in an oven for four days, the shoots are weighted for shoot
dry biomass (SDM). The shoot average height (SAH) is the mean plant
height across the 3 height measurements. The procedure described
above may be adjusted for +/- .about.one day for each step given
the situation.
[0224] To correct for slight differences between plants, a size
corrected growth value is derived from SIH and SWH. This is the
Relative Growth Rate (RGR). Relative Growth Rate (RGR) is
calculated for each shoot using the formula [RGR
%=(SWH-SIH)/((SWH+SIH)/2).times.100]. Relative water content (RWC)
is a measurement of how much (%) of the plant was water at harvest.
Water Content (RWC) is calculated for each shoot using the formula
[RWC %=(SWM-SDM)/(STM-SDM).times.100]. Fully watered corn plants of
this age run around 98% RWC.
[0225] Transgenic corn plants and soybean plants prepared in
Examples 6 and 7 are screened for water use efficiency. Transgenic
plants having at least a 1% increase in RGR and RWC as compared to
control plants are identified as having enhanced water used
efficiency and are selected as having a nucleus of this invention.
Transgenic corn and soybean plants having in their nucleus DNA
identified in Table 3 as imparting drought tolerance improvement
(DS) and homologous DNA are identified as showing increased water
use efficiency as compared to control plants and are selected as
having a nucleus of this invention.
D. Selection for Growth Under Cold Stress
[0226] Cold germination assay--Three sets of seeds are used for the
assay. The first set consists of positive transgenic events (F1
hybrid) where the genes of the present invention are expressed in
the seed. The second seed set is nontransgenic, wild-type negative
control made from the same genotype as the transgenic events. The
third set consisted of two cold tolerant and one cold sensitive
commercial check lines of corn. All seeds are treated with a
fungicide "Captan" (MAESTRO.RTM. 80DF Fungicide, Arvesta
Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied
per 45 g of corn seeds by mixing it well and drying the fungicide
prior to the experiment.
[0227] Corn kernels are placed embryo side down on blotter paper
within an individual cell (8.9.times.8.9 cm) of a germination tray
(54.times.36 cm). Ten seeds from an event are placed into one cell
of the germination tray. Each tray can hold 21 transgenic events
and 3 replicates of wildtype (LH244SDms+LH59), which is randomized
in a complete block design. For every event there are five
replications (five trays). The trays are placed at 9.7 C for 24
days (no light) in a Convrion.RTM. growth chamber (Conviron Model
PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and
fifty milliliters of deionized water are added to each germination
tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th,
17th, 19th, 21st, and 24th day after start date of the experiment.
Seeds are considered germinated if the emerged radicle size is 1
cm. From the germination counts germination index is
calculated.
[0228] The germination index is calculated as per:
Germination
index=(.SIGMA.([T+1-n.sub.i]*[P.sub.i-P.sub.i-1]))/T
where T is the total number of days for which the germination assay
is performed. The number of days after planting is defined by n.
"i" indicated the number of times the germination had been counted,
including the current day. P is the percentage of seeds germinated
during any given rating. Statistical differences are calculated
between transgenic events and wild type control. After statistical
analysis, the events that show a statistical significance at the p
level of less than 0.1 relative to wild-type controls will advance
to a secondary cold selection. The secondary cold screen is
conducted in the same manner of the primary selection only
increasing the number of repetitions to ten. Statistical analysis
of the data from the secondary selection is conducted to identify
the events that show a statistical significance at the p level of
less than 0.05 relative to wild-type controls.
[0229] Transgenic corn plants and soybean plants prepared in
Examples 6 and 7 are screened for water use efficiency. Transgenic
plants having at least a 5% increase in germination index as
compared to control plants are identified as having enhanced cold
stress tolerance and are selected as having a nucleus of this
invention. Transgenic corn and soybean plants having in their
nucleus DNA identified in Table 3 as imparting cold tolerance
improvement (CK or CS) and homologous DNA are identified as showing
increased cold stress tolerance as compared to control plants and
are selected as having a nucleus of this invention.
E. Screens for Transgenic Plant Seeds with Increased Protein and/or
Oil Levels
[0230] The following is a high-throughput selection method for
identifying plant seeds with improvement in seed composition using
the Infratec.RTM. 1200 series Grain Analyzer, which is a
near-infrared transmittance spectrometer used to determine the
composition of a bulk seed sample. Near infrared analysis is a
non-destructive, high-throughput method that can analyze multiple
traits in a single sample scan. An NIR calibration for the analytes
of interest is used to predict the values of an unknown sample. The
NIR spectrum is obtained for the sample and compared to the
calibration using a complex chemometric software package that
provides a predicted values as well as information on how well the
sample fits in the calibration.
[0231] Infratec.RTM. Model 1221, 1225, or 1227 analyzer with
transport module by Foss North America is used with cuvette, item
#1000-4033, Foss North America or for small samples with small cell
cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and
soy check samples of varying composition maintained in check cell
cuvettes are supplied by Leon Girard Co. NIT collection software is
provided by Maximum Consulting Inc. Calculations are performed
automatically by the software. Seed samples are received in packets
or containers with barcode labels from the customer. The seed is
poured into the cuvettes and analyzed as received.
TABLE-US-00021 TABLE 21 Typical sample(s): Whole grain corn and
soybean seeds Analytical time to run Less than 0.75 min per sample
method: Total elapsed time per run: 1.5 minute per sample Typical
and minimum sample Corn typical: 50 cc; minimum 30 cc size: Soybean
typical: 50 cc; minimum 5 cc Typical analytical range: Determined
in part by the specific calibration. Corn - moisture 5-15%, oil
5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean
- moisture 5-15%, oil 15-25%, and protein 35-50%.
Transgenic corn plants and soybean plants prepared in Examples 6
and 7 are screened for increased protein and oil in seed.
Transgenic inbred corn and soybean plants having an increase of at
least 1 percentage point in the total percent seed protein or at
least 0.3 percentage point in total seed oil and transgenic hybrid
corn plants having an increase of at least 0.4 percentage point in
the total percent seed protein as compared to control plants are
identified as having enhanced seed protein or enhanced seed oil and
are selected as having a nucleus of this invention.
Example 10
Cotton Transgenic Plants with Enhanced Agronomic Traits
[0232] Cotton transformation is performed as generally described in
WO0036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants
containing each of the recombinant DNA having a sequence of SEQ ID
NO: 1 through SEQ ID NO: 197 are obtained by transforming with
recombinant DNA from each of the genes identified in Table 1.
Progeny transgenic plants are selected from a population of
transgenic cotton events under specified growing conditions and are
compared with control cotton plants. Control cotton plants are
substantially the same cotton genotype but without the recombinant
DNA, for example, either a parental cotton plant of the same
genotype that was not transformed with the identical recombinant
DNA or a negative isoline of the transformed plant. Additionally, a
commercial cotton cultivar adapted to the geographical region and
cultivation conditions, i.e. cotton variety ST474, cotton variety
FM 958, and cotton variety Siokra L-23, are used to compare the
relative performance of the transgenic cotton plants containing the
recombinant DNA. The specified culture conditions are growing a
first set of transgenic and control plants under "wet" conditions,
i.e. irrigated in the range of 85 to 100 percent of
evapotranspiration to provide leaf water potential of -14 to -18
bars, and growing a second set of transgenic and control plants
under "dry" conditions, i.e. irrigated in the range of 40 to 60
percent of evapotranspiration to provide a leaf water potential of
-21 to -25 bars. Pest control, such as weed and insect control is
applied equally to both wet and dry treatments as needed. Data
gathered during the trial includes weather records throughout the
growing season including detailed records of rainfall; soil
characterization information; any herbicide or insecticide
applications; any gross agronomic differences observed such as leaf
morphology, branching habit, leaf color, time to flowering, and
fruiting pattern; plant height at various points during the trial;
stand density; node and fruit number including node above white
flower and node above crack boll measurements; and visual wilt
scoring. Cotton boll samples are taken and analyzed for lint
fraction and fiber quality. The cotton is harvested at the normal
harvest timeframe for the trial area. Enhanced water use efficiency
is indicated by increased yield, improved relative water content,
enhanced leaf water potential, increased biomass, enhanced leaf
extension rates, and improved fiber parameters.
[0233] The transgenic cotton plants of this invention are
identified from among the transgenic cotton plants by agronomic
trait screening as having increased yield and enhanced water use
efficiency.
Example 11
Monocot and Dicot Plant Transformation for the Suppression of
Endogeneous Protein
[0234] This example illustrates monocot and dicot plant
transformation to produce nuclei of this invention in cells of a
transgenic plant by transformation where the recombinant DNA
suppresses the expression of an endogenous protein identified by
Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf.sub.--8. Corn
callus and soybean tissue are transformed as describe in Examples 6
and 7 using recombinant DNA in the nucleus with DNA that
transcribes to RNA that forms double-stranded RNA targeted to an
endogenous gene with DNA encoding the protein. The genes for which
the double-stranded RNAs are targeted are the native gene in corn
and soybean that are homolog of the genes encoding the protein with
an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and
394.
[0235] Populations of transgenic corn plants and soybean plants
prepared in Examples 6 and 7 with DNA for suppressing a gene
identified in Table 3 as providing an enhanced trait by gene
suppression are screened to identify an event from those plants
with a nucleus of the invention by selecting the trait identified
in this specification.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080295196A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080295196A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References