U.S. patent application number 13/021410 was filed with the patent office on 2011-08-04 for cry1f and cry1ac transgenic cotton lines and event-specific identification thereof.
This patent application is currently assigned to Dow AgroSciences LLC. Invention is credited to John William Pellow, Ping Song, Laura Ann Tagllani.
Application Number | 20110191900 13/021410 |
Document ID | / |
Family ID | 34959539 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110191900 |
Kind Code |
A1 |
Song; Ping ; et al. |
August 4, 2011 |
CRY1F AND CRY1AC TRANSGENIC COTTON LINES AND EVENT-SPECIFIC
IDENTIFICATION THEREOF
Abstract
This invention relates to plant breeding and the protection of
plants from insects. More specifically, this invention includes
novel transformation events of cotton plants comprising one or more
polynucleotide sequences, as described herein, inserted into
specific site(s) within the genome of a cotton cell. In highly
preferred embodiments, said polynucleotide sequences encode
"stacked" Cry1F and Cry1Ac lepidopteran insect inhibitory proteins.
However, the subject invention includes plants having single cry1F
or cry1Ac events, as described herein. Additionally, the invention
is related to cotton plants derived from that transformation event
and to assays for detecting the presence of the event in a sample.
More specifically, the present invention provides DNA and related
assays for detecting the presence of certain insect-resistance
events in cotton. The assays are based on the DNA sequences of
recombinant constructs inserted into the cotton genome and of the
genomic sequences flanking the insertion sites. These sequences are
unique. Based on these insert and border sequences, event-specific
primers were generated. PCR analysis demonstrated that these cotton
lines can be identified in different cotton genotypes by analysis
of the PCR amplicons generated with these event-specific primer
sets. Thus, these and other related procedures can be used to
uniquely identify these cotton lines. Kits and conditions useful in
conducting the assays are also provided. These materials and
methods can also be used to assist breeding programs to further
develop traits in cotton.
Inventors: |
Song; Ping; (Carmel, IN)
; Tagllani; Laura Ann; (Zionsville, IN) ; Pellow;
John William; (Corcoran, CA) |
Assignee: |
Dow AgroSciences LLC
Indianapolis
IN
|
Family ID: |
34959539 |
Appl. No.: |
13/021410 |
Filed: |
February 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10964838 |
Oct 13, 2004 |
7179965 |
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13021410 |
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60556586 |
Mar 26, 2004 |
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60613851 |
Sep 27, 2004 |
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Current U.S.
Class: |
800/278 ;
800/314 |
Current CPC
Class: |
C12N 15/8286 20130101;
Y02A 40/162 20180101; C07K 14/325 20130101; C12Q 1/6895
20130101 |
Class at
Publication: |
800/278 ;
800/314 |
International
Class: |
A01H 1/00 20060101
A01H001/00; A01H 5/00 20060101 A01H005/00; A01H 5/10 20060101
A01H005/10 |
Claims
1. A transgenic cotton plant comprising an insert that disrupts a
genomic sequence selected from the group consisting of a. a first
DNA sequence comprising a 5' end comprising nucleotides 1-2074 of
SEQ ID NO:1, a first interior segment comprising SEQ ID NO:15, and
a 3' end comprising nucleotides 12,749-15,490 of SEQ ID NO:1, and
b. a second DNA sequence comprising a 5' end comprising nucleotides
1-527 of SEQ ID NO:2, a second interior segment comprising SEQ ID
NO:16, and a 3' end comprising nucleotides 8,901-9,382 of SEQ ID
NO:2.
2. A cotton plant comprising at least one insert that disrupts a
genomic sequence selected from the group consisting of a. a first
DNA sequence comprising a 5' end comprising nucleotides 1-2074 of
SEQ ID NO:1, and a 3' end comprising nucleotides 12,749-15,490 of
SEQ ID NO:1; and b. a second DNA sequence comprising a 5' end
comprising nucleotides 1-527 of SEQ ID NO:2, and a 3' end
comprising nucleotides 8,901-9,382 of SEQ ID NO:2.
3. The plant of claim 1 wherein said insert is a non-cry1F and
non-cry1Ac insert.
4. The plant of claim 2 wherein said insert is a non-cry1F and
non-cry1Ac insert.
5. A method for targeted homologous recombination, said method
comprising targeting a genomic cotton sequence for insertion of a
transgene genetic insert, and introducing said transgene genetic
insert into said genomic cotton sequence, wherein said genomic
sequence is selected from the group consisting of a. a first DNA
sequence comprising a 5' end comprising nucleotides 1-2074 of SEQ
ID NO:1, and a 3' end comprising nucleotides 12,749-15,490 of SEQ
ID NO:1; and b. a second DNA sequence comprising a 5' end
comprising nucleotides 1-527 of SEQ ID NO:2, and a 3' end
comprising nucleotides 8,901-9,382 of SEQ ID NO:2.
6. A seed of the plant of claim 1, wherein said seed comprises said
insert.
7. A seed of the plant of claim 2, wherein said seed comprises said
insert.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
11/704,418, filed Feb. 9, 2007, which was a divisional of U.S. Ser.
No. 10/964,838, filed Oct. 13, 2004, now U.S. Pat. No. 7,179,965,
which claims the benefit of provisional application Ser. No.
60/556,586, filed Mar. 26, 2004, and to provisional application
Ser. No. 60/613,851, filed Sep. 27, 2004.
BACKGROUND OF THE INVENTION
[0002] Cotton is an important fiber crop. Breeding and
biotechnology have been applied to cotton to improve its agronomic
traits and the quality of the product. One such agronomic trait is
resistance to insects, the advantages of which are readily
apparent. Genes encoding insecticidal proteins have been introduced
into cotton plants. In order to alleviate any concern that a given
type of insect could develop resistance to a single type of
insecticidal protein, plants are often developed that produce two
different types of insecticidal proteins. Thus, the odds of an
insect being hypothetically capable of developing resistance to two
different insecticidal proteins are extremely low.
[0003] Cry1Ac insecticidal proteins and genes are known in the art.
See, e.g., U.S. Pat. Nos. 6,114,138; 5,710,020; 6,251,656; and
6,229,004. Cry1F insecticidal proteins and genes are also known in
the art. See, e.g., U.S. Pat. Nos. 5,188,960; 5,691,308; 6,096,708;
and 6,573,240.
[0004] The expression of foreign genes in plants is influenced by
where the foreign gene is inserted in the chromosome. This could be
due to chromatin structure (e.g., heterochromatin) or the proximity
of transcriptional regulation elements (e.g., enhancers) close to
the integration site (Weising et al., Ann. Rev. Genet 22:421-477,
1988). For example, the same gene in the same type of transgenic
plant (or other organism) can exhibit a wide variation in
expression level amongst different events. There may also be
differences in spatial or temporal patterns of expression. For
example, differences in the relative expression of a transgene in
various plant tissues may not correspond to the patterns expected
from transcriptional regulatory elements present in the introduced
gene construct.
[0005] Thus, it is necessary to create and screen a large number of
events in order to identify an event that optimally expresses an
introduced gene of interest. For commercial purposes, it is common
to produce hundreds to thousands of different events and to screen
those events for a single event that has desired transgene
expression levels and patterns. An event that has desired levels
and/or patterns of transgene expression is useful for introgressing
the transgene into other genetic backgrounds by sexual outcrossing
using conventional breeding methods. Progeny of such crosses
maintain the transgene expression characteristics of the original
transformant. This strategy is used to ensure reliable gene
expression in a number of varieties that are well adapted to local
growing conditions.
[0006] It would be advantageous to be able to detect the presence
of a particular event in order to determine whether progeny of a
sexual cross contain a transgene of interest. In addition, a method
for detecting a particular event would be helpful for complying
with regulations requiring the pre-market approval and labeling of
foods derived from recombinant crop plants, for example. It is
possible to detect the presence of a transgene by any well-known
nucleic acid detection method such as polymerase chain reaction
(PCR) or DNA hybridization using nucleic acid probes. These
detection methods generally focus on frequently used genetic
elements, such as promoters, terminators, marker genes, and the
like. As a result, such methods may not be useful for
discriminating between different events, particularly those
produced using the same DNA construct, unless the sequence of
chromosomal DNA adjacent to the inserted DNA ("flanking DNA") is
known. An event-specific PCR assay is discussed, for example, by
Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459462,
1999). This related to the identification of glyphosate tolerant
soybean event 40-3-2 by PCR using a primer set spanning the
junction between the insert and flanking DNA. More specifically,
one primer included sequence from the insert and a second primer
included sequence from flanking DNA.
[0007] U.S. Patent Apps. 20020120964 A1 and 20040009504 A1 relate
to cotton event PV-GHGT07(1445) and compositions and methods for
the detection thereof. WO 02/100163 relates to cotton event
MONI5985 and compositions and methods for the detection thereof. WO
2004/011601 relates to corn event MON863 plants and compositions
and methods for the detection thereof. WO 2004/072235 relates to
cotton event MON 88913 and compositions and methods for the
detection thereof.
[0008] However, no such procedures and materials were specifically
known, heretofore, that could be used to specifically identify
Cry1F and/or Cry1Ac stacked cotton as discussed below.
BRIEF SUMMARY OF THE INVENTION
[0009] This invention relates to plant breeding and the protection
of plants from insects. More specifically, this invention includes
novel transformation events of cotton plants comprising one or more
polynucleotide sequences, as described herein, inserted into
specific site(s) within the genome of a cotton cell. In highly
preferred embodiments, said polynucleotide sequences encode
"stacked" Cry1F and Cry1Ac lepidopteran insect inhibitory proteins.
However, the subject invention includes plants having single Cry1F
or Cry1Ac events, as described herein.
[0010] Additionally, the subject invention provides assays for
detecting the presence of one or more of the subject events in a
sample. The present invention provides DNA and related assays for
detecting the presence of certain insect-resistance events in
cotton. The assays are based on the DNA sequences of recombinant
constructs inserted into the cotton genome and of the genomic
sequences flanking the insertion sites. Kits and conditions useful
in conducting the assays are also provided.
[0011] Thus, the subject invention relates in part to the cloning
and analysis of the DNA sequences of a whole cry1F insert, whole
cry1Ac inserts, and the border regions thereof (in transgenic
cotton lines). These sequences are unique. Based on these insert
and border sequences, event-specific primers were generated. PCR
analysis demonstrated that these events can be identified by
analysis of the PCR amplicons generated with these event-specific
primer sets. Thus, these and other related procedures can be used
to uniquely identify cotton lines comprising one or more events of
the subject invention.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 illustrates the inserted cry1F transgene and flanking
sequences for cotton event 281-24-236. This Figure also shows
amplicons and primers as described herein.
[0013] FIG. 2 illustrates an inserted cry1Ac transgene and flanking
sequences for cotton event 3006-210-23. This Figure also shows
amplicons and primers as described herein.
BRIEF DESCRIPTION OF THE SEQUENCES
[0014] SEQ ID NO:1 is the DNA sequence for the cry1F event
281-24-236 insert and its border sequences.
[0015] SEQ ID NO:2 is the DNA sequence for the cry1Ac event
3006-210-23 insert and its border sequences.
[0016] SEQ ID NO:3 is the sequence of forward primer "281-14" used
with reverse primer "281-15" to amplify a 603 bp amplicon that
spans the 5' junction between the flanking and insert regions of
cry1F event 281-24-236.
[0017] SEQ ID NO:4 is the sequence of the reverse primer "281-15"
used with forward primer "281-14" to amplify a 603 bp amplicon that
spans the 5' junction between the flanking and insert regions of
cry1F event 281-24-236.
[0018] SEQ ID NO:5 is the 603 bp sequence of the amplicon produced
using the primers of SEQ ID NOS:3 and 4.
[0019] SEQ ID NO:6 is the sequence of forward primer "281-9" used
with reverse primer "281-10" to amplify a 562 bp amplicon that
spans the 3' junction between the insert and flanking regions of
cry1F event 281-24-236.
[0020] SEQ ID NO:7 is the sequence of the reverse primer "281-10"
used with forward primer "281-9" to amplify a 562 bp amplicon that
spans the 3' junction between the flanking and insert regions of
cry1F event 281-24-236.
[0021] SEQ ID NO:8 is the 562 bp sequence of the amplicon produced
using the primers of SEQ ID NOS:6 and 7.
[0022] SEQ ID NO:9 is the sequence of forward primer "3006-20" used
with reverse primer "3006-22" to amplify a 614 bp amplicon that
spans the 5' junction between the flanking and insert regions of
cry1Ac event 3006-210-23.
[0023] SEQ ID NO:10 is the sequence of the reverse primer "3006-22"
used with forward primer "3006-20" to amplify a 614 bp amplicon
that spans the 5' junction between the flanking and insert regions
of cry1Ac event 3006-210-23.
[0024] SEQ ID NO:11 is the 614 bp sequence of the amplicon produced
using the primers of SEQ ID NOS:9 and 10.
[0025] SEQ ID NO:12 is the sequence of forward primer "3006-9" used
with reverse primer "3006-12" to amplify a 662 bp amplicon that
spans the 3' junction between the insert and flanking regions of
cry1Ac event 3006-210-23.
[0026] SEQ ID NO:13 is the sequence of the reverse primer "3006-12"
used with forward primer "3006-9" to amplify a 662 bp amplicon that
spans the 3' junction between the flanking and insert regions of
cry1Ac event 3006-210-23.
[0027] SEQ ID NO:14 is the 662 bp sequence of the amplicon produced
using the primers of SEQ ID NOS:12 and 13.
[0028] SEQ ID NO:15 is a segment of genomic cotton DNA for event
281-24-236 (53 missing bases).
[0029] SEQ ID NO:16 is a segment of genomic cotton DNA for event
3006-210-23 (16 missing bases).
DETAILED DESCRIPTION OF THE INVENTION
[0030] This invention relates to plant breeding and the protection
of plants from insects. More specifically, this invention includes
novel transformation events of cotton plants (e.g. Gossypium
hirsutum and Gossypium barbadense) comprising one or more
polynucleotide sequences, as described herein, inserted into
specific site(s) within the genome of a cotton cell. In highly
preferred embodiments, said polynucleotide sequences encode
"stacked" Cry1F and Cry1Ac lepidopteran insect inhibitory proteins.
However, the subject invention includes plants having single Cry1F
or Cry1Ac events, as described herein.
[0031] Additionally, the subject invention provides assays for
detecting the presence of one or more of the subject events in a
sample. Aspects of the subject invention include methods of
designing and/or producing any of the diagnostic nucleic acid
molecules exemplified or suggested herein, particularly those based
wholly or partially on the subject flanking sequences.
[0032] More specifically, the subject invention relates in part to
two transgenic cotton events (cry1F 281-24-236 and cry1Ac
3006-210-23), plant lines comprising these events, and the cloning
and analysis of the DNA sequences of this cry1F insert, these
cry1Ac inserts, and/or the border regions thereof. Plant lines of
the subject invention can be detected using sequences disclosed and
suggested herein.
[0033] In preferred embodiments, this invention relates to
insect-resistant cotton lines, and the identification thereof, that
produces two "stacked" insecticidal proteins known as Cry1F and
Cry1Ac. In preferred embodiments, a plant line of the subject
invention comprises cry1F event 281-24-236 and cry1Ac event
3006-210-23. However, plants of the subject invention can comprise
any one or, preferably, both of the events discussed herein.
[0034] As alluded to above in the Background section, the
introduction and integration of a transgene into a plant genome
involves some random events (hence the name "event" for a given
insertion that is expressed). That is, with many transformation
techniques such as Agrobacterium transformation, the "gene gun,"
and WHISKERS, it is unpredictable where in the genome a transgene
will become inserted. Thus, identifying the flanking plant genomic
DNA on both sides of the insert can be important for identifying a
plant that has a given insertion event. For example, PCR primers
can be designed that generate a PCR amplicon across the junction
region of the insert and the host genome. This PCR amplicon can be
used to identify a unique or distinct type of insertion event.
[0035] As "events" are random events and generally cannot be
duplicated, as part of this disclosure at least 2500 seeds of a
cotton line, comprising the cry1F event 281-24-236 and cry1Ac event
3006-210-23, have been deposited, and made available to the public
without restriction (but subject to patent rights), with the
American Type Culture Collection (ATCC), Rockville, Md. 20852. The
deposit has been designated as ATCC Deposit No. PTA-6233. The
deposit will be maintained without restriction at the ATCC
depository, which is a public depository, for a period of 30 years,
or five years after the most recent request, or for the effective
life of the patent, whichever is longer, and will be replaced if it
becomes nonviable during that period.
[0036] The deposited seeds are part of the subject invention.
Clearly, cotton plants can be grown from these seeds, and such
plants are part of the subject invention. The subject invention
also relates to DNA sequences contained in these cotton plants that
are useful for detecting these plants and progeny thereof.
Detection methods and kits of the subject invention can be directed
to identifying any one, two, or even all three of these events,
depending on the ultimate purpose of the test.
[0037] Definitions and examples are provided herein to help
describe the present invention and to guide those of ordinary skill
in the art to practice the invention. Unless otherwise noted, terms
are to be understood according to conventional usage by those of
ordinary skill in the relevant art. The nomenclature for DNA bases
as set forth at 37 CFR .sctn.1.822 is used.
[0038] A transgenic "event" is produced by transformation of plant
cells with heterologous DNA, i.e., a nucleic acid construct that
includes a transgene of interest, regeneration of a population of
plants resulting from the insertion of the transgene into the
genome of the plant, and selection of a particular plant
characterized by insertion into a particular genome location. The
term "event" refers to the original transformant and progeny of the
transformant that include the heterologous DNA. The term "event"
also refers to progeny produced by a sexual outcross between the
transformant and another variety that includes the
genomic/transgene DNA. Even after repeated back-crossing to a
recurrent parent, the inserted transgene DNA and flanking genomic
DNA (genomic/transgene DNA) from the transformed parent is present
in the progeny of the cross at the same chromosomal location. The
term "event" also refers to DNA from the original transformant and
progeny thereof comprising the inserted DNA and flanking genomic
sequence immediately adjacent to the inserted DNA that would be
expected to be transferred to a progeny that receives inserted DNA
including the transgene of interest as the result of a sexual cross
of one parental line that includes the inserted DNA (e.g., the
original transformant and progeny resulting from selfing) and a
parental line that does not contain the inserted DNA.
[0039] A "junction sequence" spans the point at which DNA inserted
into the genome is linked to DNA from the cotton native genome
flanking the insertion point, the identification or detection of
one or the other junction sequences in a plant's genetic material
being sufficient to be diagnostic for the event. Included are the
DNA sequences that span the insertions in herein-described cotton
events and similar lengths of flanking DNA. Specific examples of
such diagnostic sequences are provided herein; however, other
sequences that overlap the junctions of the insertions, or the
junctions of the insertions and the genomic sequence, are also
diagnostic and could be used according to the subject
invention.
[0040] The subject invention relates to the identification of such
flanking, junction, and insert sequences. Related PCR primers and
amplicons are included in the invention. According to the subject
invention, PCR analysis methods using amplicons that span across
inserted DNA and its borders (of Cry1F 281-24-236 and/or Cry1Ac
3006-210-23) can be used to detect or identify commercialized
transgenic cotton varieties or lines derived from the subject
proprietary transgenic cotton lines.
[0041] The entire sequences of each of these inserts, together with
the respective flanking sequences, are provided herein as SEQ ID
NO:1 (cry1F 281-24-236) and SEQ ID NO:2 (cry1Ac 3006-210-23). Table
1 provides the coordinates of the insert and flanking sequences for
these events.
TABLE-US-00001 TABLE 1 For indicated SEQ ID NO:, residue location
of: Event 5' Flanking Insert 3' Flanking cry1F 1-2074 2,075-12,748
12,749-15,490 281-24-236 (SEQ ID NO: 1) cry1Ac 1-527 528-8,900
8,901-9,382 3006-210-23 (SEQ ID NO: 2)
[0042] These insertion events, and further components thereof, are
further illustrated in FIGS. 1 and 2. These sequences (particularly
the flanking sequences) are unique. Based on these insert and
border sequences, event-specific primers were generated. PCR
analysis demonstrated that these cotton lines can be identified in
different cotton genotypes by analysis of the PCR amplicons
generated with these event-specific primer sets. Thus, these and
other related procedures can be used to uniquely identify these
cotton lines. The sequences identified herein are unique. For
example, BLAST searches against GENBANK databases did not reveal
any significant homology between the cloned border sequences and
sequences in the database.
[0043] Detection techniques of the subject invention are especially
useful in conjunction with plant breeding, to determine which
progeny plants comprise a given event, after a parent plant
comprising an event of interest is crossed with another plant line
in an effort to impart one or more additional traits of interest in
the progeny. These PCR analysis methods benefit cotton breeding
programs as well as quality control, especially for commercialized
transgenic cottonseeds. PCR detection kits for these transgenic
cotton lines can also now be made and used. This can also benefit
product registration and product stewardship.
[0044] Furthermore, flanking cotton sequences can be used to
specifically identify the genomic location of each insert. This
information can be used to make molecular marker systems specific
to each event. These can be used for accelerated breeding
strategies and to establish linkage data.
[0045] Still further, the flanking sequence information can be used
to study and characterize transgene integration processes, genomic
integration site characteristics, event sorting, stability of
transgenes and their flanking sequences, and gene expression
(especially related to gene silencing, transgene methylation
patterns, position effects, and potential expression-related
elements such as MARS [matrix attachment regions], and the
like).
[0046] In light of all the subject disclosure, it should be clear
that the subject invention includes seeds available under ATCC
Deposit No. PTA-6233. The subject invention also includes an
insect-resistant cotton plant grown from a seed deposited with the
ATCC under accession number PTA-6233. The subject invention further
includes parts of said plant, such as leaves, tissue samples, seeds
produced by said plant, pollen, and the like.
[0047] Still further, the subject invention includes descendant
and/or progeny plants of plants grown from the deposited seed,
preferably an insect-resistant cotton plant wherein said plant has
a genome comprising a detectable wild-type genomic DNA/insert DNA
junction sequence as described herein. As used herein, the term
"cotton" means Gossypium hirsutum and includes all plant varieties
that can be bred with cotton, including Gossypium barbadense.
[0048] This invention further includes processes of making crosses
using a plant of the subject invention as at least one parent. For
example, the subject invention includes an F.sub.1 hybrid plant
having as one or both parents any of the plants exemplified herein.
Also within the subject invention is seed produced by such F.sub.1
hybrids of the subject invention. This invention includes a method
for producing an F.sub.1 hybrid seed by crossing an exemplified
plant with a different (e.g. in-bred parent) plant and harvesting
the resultant hybrid seed. The subject invention includes an
exemplified plant that is either a female parent or a male parent.
Characteristics of the resulting plants may be improved by careful
consideration of the parent plants.
[0049] An insect-resistant cotton plant can be bred by first
sexually crossing a first parental cotton plant consisting of a
cotton plant grown from seed of any one of the lines referred to
herein, and a second parental cotton plant, thereby producing a
plurality of first progeny plants; and then selecting a first
progeny plant that is resistant to insects (or that possesses at
least one of the events of the subject invention); and selfing the
first progeny plant, thereby producing a plurality of second
progeny plants; and then selecting from the second progeny plants a
plant that is resistant to insects (or that possesses at least one
of the events of the subject invention). These steps can further
include the back-crossing of the first progeny plant or the second
progeny plant to the second parental cotton plant or a third
parental cotton plant. A cotton crop comprising cotton seeds of the
subject invention, or progeny thereof, can then be planted.
[0050] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes. Back-crossing to a parental plant and
out-crossing with a non-transgenic plant are also contemplated, as
is vegetative propagation. Other breeding methods commonly used for
different traits and crops are known in the art. Backcross breeding
has been used to transfer genes for a simply inherited, highly
heritable trait into a desirable homozygous cultivar or inbred
line, which is the recurrent parent. The source of the trait to be
transferred is called the donor parent. The resulting plant is
expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent. After the initial cross, individuals possessing the
phenotype of the donor parent are selected and repeatedly crossed
(backcrossed) to the recurrent parent. The resulting parent is
expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent.
[0051] The DNA molecules of the present invention can be used as
molecular markers in a marker assisted breeding (MAB) method. DNA
molecules of the present invention can be used in methods (such as,
AFLP markers, RFLP markers, RAPD markers, SNPs, and SSRs) that
identify genetically linked agronomically useful traits, as is
known in the art. The insect-resistance trait can be tracked in the
progeny of a cross with a cotton plant of the subject invention (or
progeny thereof and any other cotton cultivar or variety) using the
MAB methods. The DNA molecules are markers for this trait, and MAB
methods that are well known in the art can be used to track the
insect-resistance trait(s) in cotton plants where at least one
cotton line of the subject invention, or progeny thereof, was a
parent or ancestor. The methods of the present invention can be
used to identify any cotton variety having the insect-resistance
event from cotton line 281-24-236 (cry1F) and/or 3006-210-23
(cry1Ac).
[0052] Methods of the subject invention include a method of
producing an insect-resistant cotton plant wherein said method
comprises breeding with a plant of the subject invention. More
specifically, said methods can comprise crossing two plants of the
subject invention, or one plant of the subject invention and any
other plant. Preferred methods further comprise selecting progeny
of said cross by analyzing said progeny for an event detectable
according to the subject invention.
[0053] A preferred plant, or a seed, of the subject invention
comprises in its genome at least one of the insert sequences, as
identified in Table 1, together with at least 20-500 or more
contiguous flanking nucleotides on both sides of the insert, as
identified in Table 1. Unless indicated otherwise, "cry1F cotton
event 281-24-236" refers to DNA of SEQ ID NO:1 that includes the
heterologous DNA inserted in the original transformant (nucleotides
2075-12,748 of SEQ ID NO:1) and all or part of both of the flanking
genomic sequences of SEQ ID NO:1 (nucleotide residues 1-2074 and
12,749-15,490) immediately adjacent to the inserted DNA that would
be expected to be transferred to progeny that receives the inserted
DNA as a result of a sexual cross of a parental line that includes
the event. Similarly, unless indicated otherwise, "cry1Ac cotton
event 3006-210-23" refers to DNA of SEQ ID NO:2 that includes the
heterologous DNA inserted in the original transformant (nucleotides
528-8900 of SEQ ID NO:2) and all or part of both of the flanking
genomic sequences of SEQ ID NO:2 (residues 1-527 and 8901-9382)
immediately adjacent to the inserted DNA that would be expected to
be transferred to progeny that receives the inserted DNA as a
result of a sexual cross of a parental line that includes the
event.
[0054] The subject invention includes tissue cultures of
regenerable cells of a plant of the subject invention. Also
included is a plant regenerated from such tissue culture,
particularly where said plant is capable of expressing all the
morphological and physiological properties of an exemplified
variety. Preferred plants of the subject invention have all the
physiological and morphological characteristics of a plant grown
from the deposited seed. This invention further comprises progeny
of such seed and seed possessing the quality traits of
interest.
[0055] Manipulations (such as mutation, further transfection, and
further breeding) of plants or seeds, or parts thereof, may lead to
the creation of what may be termed "essentially derived" varieties.
The International Union for the Protection of New Varieties of
Plants (UPOV) has provided the following guideline for determining
if a variety has been essentially derived from a protected
variety:
[0056] [A] variety shall be deemed to be essentially derived from
another variety ("the initial variety") when
[0057] (i) it is predominantly derived from the initial variety, or
from a variety that is itself predominantly derived from the
initial variety, while retaining the expression of the essential
characteristics that result from the genotype or combination of
genotypes of the initial variety;
[0058] (ii) it is clearly distinguishable from the initial variety;
and
[0059] (iii) except for the differences which result from the act
of derivation, it conforms to the initial variety in the expression
of the essential characteristics that result from the genotype or
combination of genotypes of the initial variety.
[0060] UPOV, Sixth Meeting with International Organizations,
Geneva, Oct. 30, 1992; document prepared by the Office of the
Union.
[0061] As used herein, a "line" is a group of plants that display
little or no genetic variation between individuals for at least one
trait. Such lines may be created by several generations of
self-pollination and selection, or vegetative propagation from a
single parent using tissue or cell culture techniques.
[0062] As used herein, the terms "cultivar" and "variety" are
synonymous and refer to a line which is used for commercial
production.
[0063] "Stability" or "stable" means that with respect to the given
component, the component is maintained from generation to
generation and, preferably, at least three generations at
substantially the same level, e.g., preferably .+-.15%, more
preferably .+-.10%, most preferably .+-.5%. The stability may be
affected by temperature, location, stress and the time of planting.
Comparison of subsequent generations under field conditions should
produce the component in a similar manner.
[0064] "Commercial Utility" is defined as having good plant vigor
and high fertility, such that the crop can be produced by farmers
using conventional farming equipment, and the oil with the
described components can be extracted from the seed using
conventional crushing and extraction equipment. To be commercially
useful, the yield, as measured by seed weight, oil content, and
total oil produced per acre, is within 15% of the average yield of
an otherwise comparable commercial canola variety without the
premium value traits grown in the same region.
[0065] "Agronomically elite" means that a line has desirable
agronomic characteristics such as yield, maturity, disease
resistance, and the like, in addition to the insect resistance due
to the subject event(s).
[0066] As one skilled in the art will recognize in light of this
disclosure, preferred embodiments of detection kits, for example,
can include probes and/or primers directed to and/or comprising
"junction sequences" or "transition sequences" (where the cotton
genomic flanking sequence meets the insert sequence). For example,
this includes a polynucleotide probe, primer, or amplicon
comprising a sequence including residues 2074-2075 or 12,748-12,749
of SEQ ID NO:1, or residues 527-528 or 8,900-8,901 of SEQ ID NO:2,
as indicated in Table 1. To be diagnostic for these particular
events, preferred "junction primers" should include at least
.about.15 residues of the adjacent flanking sequence and at least
.about.15 residues of the adjacent insert sequence. With this
arrangement, another primer in either the flanking or insert region
can be used to generate a detectable amplicon that indicates the
presence of an event of the subject invention. In preferred
embodiments, however, one primer binds in the flanking region and
one binds in the insert, and these primers can be used to generate
an amplicon that spans (and includes) a junction sequence as
indicated above.
[0067] One skilled in the art will also recognize that primers and
probes can be designed to hybridize, under a range of standard
hybridization and/or PCR conditions, to a segment of SEQ ID NO:1,
SEQ ID NO:2, and complements thereof, wherein the primer or probe
is not perfectly complementary to the exemplified sequence. That
is, some degree of mismatch can be tolerated. For an approximately
20 nucleotide primer, for example, typically one or two or so
nucleotides do not need to bind with the opposite strand if the
mismatched base is internal or on the end of the primer that is
opposite the amplicon. Various appropriate hybridization conditions
are provided below. Synthetic nucleotide analogs, such as inosine,
can also be used in probes. Peptide nucleic acid (PNA) probes, as
well as DNA and RNA probes, can also be used. What is important is
that such probes and primers are diagnostic for (able to uniquely
identify and distinguish) the presence of an event of the subject
invention.
[0068] It should be further noted that errors in PCR amplification
can occur which might result in minor sequencing errors, for
example. That is, unless otherwise indicated, the sequences listed
herein were determined by generating long amplicons from cotton
genomic DNAs, and then cloning and sequencing the amplicons. It is
not unusual to find slight differences and minor discrepancies in
sequences generated and determined in this manner, given the many
rounds of amplification that are necessary to generate enough
amplicon for sequencing from genomic DNAs. For example, the
following differences between the determined sequences of the
event-flanking DNAs and the corresponding, known/wild-type/genomic
DNAs are noted. In the 5' flank for the subject cry1F event,
residue 2037 of SEQ ID NO:1 was determined to be/is listed as "G"
whereas the corresponding residue of the 281-24-236 locus of the
known genomic sequence is "A" (R can be used in a consensus
sequence, according to standard IUPAC-IUB conventions). In the 3'
flank of this event, residue 12,781 of SEQ ID NO:1 is listed herein
as T whereas C is provided in the published genomic sequence at the
corresponding location (Y is the consensus code). Position 12,811
of SEQ ID NO:1 is C whereas T is provided for the genome (Y would
be the consensus). Position 12,866 is listed as C in SEQ ID NO:1
whereas T appears in the genome (Y is the consensus). Position
12,882 is listed as G in SEQ ID NO:1 whereas A appears for the
genome (R is the consensus). Position 12,918 is listed as A in SEQ
ID NO:1 whereas G appears in the genome (R is the consensus).
Residue 13,129 is listed as G in SEQ ID NO:1 whereas A appears in
the genome (R is the consensus). Residue 13,222 is listed as C in
SEQ ID NO:1 whereas T appears in the genomic sequence (Y is the
consensus). At position 13,441 in SEQ ID NO:1, a T appears whereas
there is no corresponding residue in the genomic listing. Thus,
this apparent insertion would shift the downstream numbering of SEQ
ID NO:1 accordingly, as compared to the genomic sequence. One
skilled in the art should recognize and be put on notice than any
adjustments needed due to these types of common sequencing errors
or discrepancies are within the scope of the subject invention.
[0069] Similar differences also appear in the 5' flank for the
subject cry1Ac event. At positions 149, 153, 159, 165, and 244 of
SEQ ID NO:2, the following residues are listed, respectively: C, G,
C, C, and C. In the genomic sequence at the 3006-210-23 locus, the
following residues appear, respectively, at corresponding
locations: A, A, A, A, and A. Consensus codes for these
substitutions are, respectively, M, R, M, M, and M. Adjustments to
probes and primers can be made accordingly, and corresponding
differences might be noted in amplicons that span or include any of
the above residues.
[0070] It should also be noted that it is not uncommon for some
genomic sequence to be deleted when a sequence is inserted during
the creation of an event. This was the case for both events of the
subject invention. That is, SEQ ID NO:1 provides a 53-base segment
of genomic cotton DNA for event 281-24-236 that was deleted during
the insertion. This "interior segment" occurs between residues 2074
and 12,749 of SEQ ID NO:1 in the non-transformed cotton genome.
Similarly, SEQ ID NO:2 provides a 16-base segment of genomic cotton
DNA for event 3006-210-23 that was deleted during the insertion.
This "interior segment" occurs between residues 527 and 8,901 of
SEQ ID NO:2 in the non-transformed cotton genome.
[0071] As illustrated in FIGS. 1 and 2, the components of each of
the "inserts" are as follows. The transgene genetic element DNA
molecules contained in the subject event Cry1F 281-24-236 consists
of the maize ubiquitin 1 promoter, operably connected to the
phosphinothricin N-acetyltransferase (PAT) from Streptomyces
viridochromogenes, operably connected to the ORF25 polyadenylation
sequences (Baker et al., Plant Molecular Biology 2:335-350, 1983);
the chimeric promoter [(4OCS).delta.MAS] containing a partially
deleted mannopines synthase promoter with 4 enhancer elements from
the octopine synthase promoter, operably connected to the
Cry1F(synpro) from Bacillus thuringiensis var. aizawai, operably
connected to ORF25 polyadenylation sequences (Baker et al., Plant
Molecular Biology 2:335-350, 1983); and the maize ubiquitin 1
promoter unoperably connected to a partial pat sequence. The DNA
polynucleotide sequences or fragments of these components can be
used as DNA primers or probes in the methods of the present
invention.
[0072] The transgene genetic element DNA molecules contained in the
subject event Cry1Ac 3006-210-23 consists of the (4OCS).delta.MAS
promoter operably connected to the PAT (as described above),
operably connected to the ORF25; and the maize ubiquitin 1 promoter
operably connected to the Cry1Ac (synpro) from Bacillus
thuringiensis var. kurstaki, operably connected to the ORF25
polyadenylation sequences. The DNA polynucleotide sequences of
these components, or fragments thereof, can be used as DNA primers
or probes in the methods of the present invention.
[0073] In some embodiments of the invention, compositions and
methods are provided for detecting the presence of the
transgene/genomic insertion region, in plants and seeds and the
like, from a cotton plant designated WIDESTRIKE comprising Cry1F
event 281-24-236 and Cry1Ac event 3006-210-23. DNA sequences are
provided that comprise at least one transgene/genomic insertion
region junction sequence provided herein in SEQ ID NO:1, SEQ ID
NO:2, segments thereof, and complements of the exemplified
sequences and any segments thereof. The insertion region junction
sequence spans the junction between heterologous DNA inserted into
the genome and the DNA from the cotton cell flanking the insertion
site. Such sequences are diagnostic for one or more of the given
events.
[0074] Based on these insert and border sequences, event-specific
primers were generated. PCR analysis demonstrated that these cotton
lines (Cry1F 281-24-236 and Cry1Ac 3006-210-23) can be identified
in different cotton genotypes by analysis of the PCR amplicons
generated with these event-specific primer sets. These and other
related procedures can be used to uniquely identify these cotton
lines. Thus, PCR amplicons derived from such primer pairs are
unique and can be used to identify these cotton lines.
[0075] In some embodiments, DNA sequences that comprise at least
one of the novel transgene/genomic insertion regions are an aspect
of this invention. Included are DNA sequences that comprise a
sufficient length of polynucleotides of transgene insert sequence
and a sufficient length of polynucleotides of cotton genomic
sequence from one or more of the three aforementioned cotton plants
and/or sequences that are useful as primer sequences for the
production of an amplicon product diagnostic for one or more of
these cotton plants.
[0076] Related embodiments pertain to DNA sequences that comprise
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, or more contiguous nucleotides of a
transgene portion of a DNA sequence selected from the group
consisting of SEQ ID NO:1 and SEQ ID NO:2, or complements thereof,
and a similar length of flanking cotton DNA sequence from these
sequences, or complements thereof. Such sequences are useful as DNA
primers in DNA amplification methods. The amplicons produced using
these primers are diagnostic for any of the cotton events referred
to herein. Therefore, the invention also includes the amplicons
produced by such DNA primers and homologous primers.
[0077] Following is a table that summarizes specific embodiments of
the subject invention:
TABLE-US-00002 TABLE 2 List of Primers and Their Sequences Used in
Event-Specific PCR Amplification Forward Reverse Amplicon Sequence
Sequence Size Target Event (5'-3) (5'-3') (bp) Region 281-
tgtcggctgaa ccggacatga 603 5' insert 24- ggtagggagg agccatttac (SEQ
ID junction 236 (281-14) (281-15) NO: 5) (SEQ ID (SEQ ID NO: 3) NO:
4) tctctagagag Cgagctggaga 562 3' insert gggcacgacc gaccggtgac (SEQ
ID junction (281-9) (281-10) NO: 8) (SEQ ID (SEQ ID NO: 6) NO: 7)
3006- ttccaacctttaa gctgcggaca 614 5' insert 210- ctattatcctgc
tctacatttt (SEQ ID junction 23 (3006-20) (3006-22) NO: 11) (SEQ ID
(SEQ ID NO: 9) NO: 10) gacatgcaatgc Aagtctctgcc 662 3' insert
tcattatctcta ttctaccctgg (SEQ ID junction (3006-9) (3006-12) NO:
14) (SEQ ID (SEQ ID NO: 12) NO: 13)
[0078] This invention also includes methods of detecting the
presence of DNA, in a sample, that corresponds to at least one of
the cotton events referred to herein. Such methods can comprise:
(a) contacting the sample comprising DNA with a primer set that,
when used in a nucleic acid amplification reaction with DNA from at
least one of these cotton events, produces an amplicon that is
diagnostic for said event(s); (b) performing a nucleic acid
amplification reaction, thereby producing the amplicon; and (c)
detecting the amplicon.
[0079] Further detection methods of the subject invention include a
method of detecting the presence of a DNA, in a sample,
corresponding to at least one of said events, wherein said method
comprises: (a) contacting the sample comprising DNA with a probe
that hybridizes under stringent hybridization conditions with DNA
from at least one of said cotton events and which does not
hybridize under the stringent hybridization conditions with a
control cotton plant (non-event-of-interest DNA); (b) subjecting
the sample and probe to stringent hybridization conditions; and (c)
detecting hybridization of the probe to the DNA.
[0080] In still further embodiments, the subject invention includes
methods of producing a cotton plant comprising a cry1F and/or a
cry1Ac event of the subject invention, wherein said method
comprises the steps of: (a) sexually crossing a first parental
cotton line (comprising an expression cassettes of the present
invention, which confers said insect resistance trait to plants of
said line) and a second parental cotton line (that lacks this
insect tolerance trait) thereby producing a plurality of progeny
plants; and (b) selecting a progeny plant by the use of molecular
markers. Such methods may optionally comprise the further step of
back-crossing the progeny plant to the second parental cotton line
to producing a true-breeding cotton plant that comprises said
insect tolerance trait.
[0081] According to another aspect of the invention, methods of
determining the zygosity of progeny of a cross with any one (or
more) of said three events are provided. Said methods can comprise
contacting a sample, comprising cotton DNA, with a primer set of
the subject invention. Said primers, when used in a nucleic-acid
amplification reaction with genomic DNA from at least one of said
cotton events, produce a first amplicon that is diagnostic for at
least one of said cotton events. Such methods further comprise
performing a nucleic acid amplification reaction, thereby producing
the first amplicon; detecting the first amplicon; and contacting
the sample comprising cotton DNA with said primer set (said primer
set, when used in a nucleic-acid amplification reaction with
genomic DNA from cotton plants, produces a second amplicon
comprising the native cotton genomic DNA homologous to the cotton
genomic region of a transgene insertion identified as one of said
cotton events); and performing a nucleic acid amplification
reaction, thereby producing the second amplicon. The methods
further comprise detecting the second amplicon, and comparing the
first and second amplicons in a sample, wherein the presence of
both amplicons indicates that the sample is heterozygous for the
transgene insertion.
[0082] DNA detection kits can be developed using the compositions
disclosed herein and methods well known in the art of DNA
detection. The kits are useful for identification of the subject
cotton event DNA in a sample and can be applied to methods for
breeding cotton plants containing this DNA. The kits contain DNA
sequences homologous or complementary to the amplicons, for
example, disclosed herein, or to DNA sequences homologous or
complementary to DNA contained in the transgene genetic elements of
the subject events. These DNA sequences can be used in DNA
amplification reactions or as probes in a DNA hybridization method.
The kits may also contain the reagents and materials necessary for
the performance of the detection method.
[0083] A "probe" is an isolated nucleic acid molecule to which is
attached a conventional detectable label or reporter molecule (such
as a radioactive isotope, ligand, chemiluminescent agent, or
enzyme). Such a probe is complementary to a strand of a target
nucleic acid, in the case of the present invention, to a strand of
genomic DNA from one of said cotton events, whether from a cotton
plant or from a sample that includes DNA from the event. Probes
according to the present invention include not only
deoxyribonucleic or ribonucleic acids but also polyamides and other
probe materials that bind specifically to a target DNA sequence and
can be used to detect the presence of that target DNA sequence.
[0084] "Primers" are isolated nucleic acids that are annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, then
extended along the target DNA strand by a polymerase, e.g., a DNA
polymerase. Primer pairs of the present invention refer to their
use for amplification of a target nucleic acid sequence, e.g., by
the polymerase chain reaction (PCR) or other conventional
nucleic-acid amplification methods.
[0085] Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,
177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,
242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,
268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,
281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,
294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,
307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,
320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,
333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,
346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,
359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,
372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,
385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,
398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,
411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,
424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,
437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,
450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,
463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,
476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,
489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500
polynucleotides or more in length. Such probes and primers
hybridize specifically to a target sequence under high stringency
hybridization conditions. Preferably, probes and primers according
to the present invention have complete sequence similarity with the
target sequence, although probes differing from the target sequence
and that retain the ability to hybridize to target sequences may be
designed by conventional methods.
[0086] Methods for preparing and using probes and primers are
described, for example, in Molecular Cloning: A Laboratory Manual,
2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. PCR-primer pairs
can be derived from a known sequence, for example, by using
computer programs intended for that purpose.
[0087] Primers and probes based on the flanking DNA and insert
sequences disclosed herein can be used to confirm (and, if
necessary, to correct) the disclosed sequences by conventional
methods, e.g., by re-cloning and sequencing such sequences.
[0088] The nucleic acid probes and primers of the present invention
hybridize under stringent conditions to a target DNA sequence. Any
conventional nucleic acid hybridization or amplification method can
be used to identify the presence of DNA from a transgenic event in
a sample. Nucleic acid molecules or fragments thereof are capable
of specifically hybridizing to other nucleic acid molecules under
certain circumstances. As used herein, two nucleic acid molecules
are said to be capable of specifically hybridizing to one another
if the two molecules are capable of forming an anti-parallel,
double-stranded nucleic acid structure. A nucleic acid molecule is
said to be the "complement" of another nucleic acid molecule if
they exhibit complete complementarity. As used herein, molecules
are said to exhibit "complete complementarity" when every
nucleotide of one of the molecules is complementary to a nucleotide
of the other. Two molecules are said to be "minimally
complementary" if they can hybridize to one another with sufficient
stability to permit them to remain annealed to one another under at
least conventional "low-stringency" conditions. Similarly, the
molecules are said to be "complementary" if they can hybridize to
one another with sufficient stability to permit them to remain
annealed to one another under conventional "high-stringency"
conditions. Conventional stringency conditions are described by
Sambrook et al., 1989. Departures from complete complementarity are
therefore permissible, as long as such departures do not completely
preclude the capacity of the molecules to form a double-stranded
structure. In order for a nucleic acid molecule to serve as a
primer or probe it need only be sufficiently complementary in
sequence to be able to form a stable double-stranded structure
under the particular solvent and salt concentrations employed.
[0089] As used herein, a substantially homologous sequence is a
nucleic acid sequence that will specifically hybridize to the
complement of the nucleic acid sequence to which it is being
compared under high stringency conditions. The term "stringent
conditions" is functionally defined with regard to the
hybridization of a nucleic-acid probe to a target nucleic acid
(i.e., to a particular nucleic-acid sequence of interest) by the
specific hybridization procedure discussed in Sambrook et al.,
1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52
and 9.56-9.58. Accordingly, the nucleotide sequences of the
invention may be used for their ability to selectively form duplex
molecules with complementary stretches of DNA fragments.
[0090] Depending on the application envisioned, one can use varying
conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications
requiring high selectivity, one will typically employ relatively
stringent conditions to form the hybrids, e.g., one will select
relatively low salt and/or high temperature conditions, such as
provided by about 0.02 M to about 0.15 M NaCl at temperatures of
about 50.degree. C. to about 70.degree. C. Stringent conditions,
for example, could involve washing the hybridization filter at
least twice with high-stringency wash buffer (0.2.times.SSC, 0.1%
SDS, 65.degree. C.). Appropriate stringency conditions which
promote DNA hybridization, for example, 6.0.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times.SSC at 50.degree. C. are known to those skilled
in the art, 6.3.1-6.3.6. For example, the salt concentration in the
wash step can be selected from a low stringency of about
2.0.times.SSC at 50.degree. C. to a high stringency of about
0.2.times.SSC at 50.degree. C. In addition, the temperature in the
wash step can be increased from low stringency conditions at room
temperature, about 22.degree. C., to high stringency conditions at
about 65.degree. C. Both temperature and salt may be varied, or
either the temperature or the salt concentration may be held
constant while the other variable is changed. Such selective
conditions tolerate little, if any, mismatch between the probe and
the template or target strand. Detection of DNA sequences via
hybridization is well-known to those of skill in the art, and the
teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 are exemplary
of the methods of hybridization analyses.
[0091] In a particularly preferred embodiment, a nucleic acid of
the present invention will specifically hybridize to one or more of
the primers (or amplicons or other sequences) exemplified or
suggested herein, including complements and fragments thereof,
under high stringency conditions. In one aspect of the present
invention, a marker nucleic acid molecule of the present invention
has the nucleic acid sequence set forth in SEQ ID NOS:3-14, or
complements and/or fragments thereof.
[0092] In another aspect of the present invention, a marker nucleic
acid molecule of the present invention shares between 80% and 100%
or 90% and 100% sequence identity with such nucleic acid sequences.
In a further aspect of the present invention, a marker nucleic acid
molecule of the present invention shares between 95% and 100%
sequence identity with such sequence. Such sequences may be used as
markers in plant breeding methods to identify the progeny of
genetic crosses. The hybridization of the probe to the target DNA
molecule can be detected by any number of methods known to those
skilled in the art, these can include, but are not limited to,
fluorescent tags, radioactive tags, antibody based tags, and
chemiluminescent tags.
[0093] Regarding the amplification of a target nucleic acid
sequence (e.g., by PCR) using a particular amplification primer
pair, "stringent conditions" are conditions that permit the primer
pair to hybridize only to the target nucleic-acid sequence to which
a primer having the corresponding wild-type sequence (or its
complement) would bind and preferably to produce a unique
amplification product, the amplicon.
[0094] The term "specific for (a target sequence)" indicates that a
probe or primer hybridizes under stringent hybridization conditions
only to the target sequence in a sample comprising the target
sequence.
[0095] As used herein, "amplified DNA" or "amplicon" refers to the
product of nucleic-acid amplification of a target nucleic acid
sequence that is part of a nucleic acid template. For example, to
determine whether the cotton plant resulting from a sexual cross
contains transgenic event genomic DNA from the cotton plant of the
present invention, DNA extracted from a cotton plant tissue sample
may be subjected to nucleic acid amplification method using a
primer pair that includes a primer derived from flanking sequence
in the genome of the plant adjacent to the insertion site of
inserted heterologous DNA, and a second primer derived from the
inserted heterologous DNA to produce an amplicon that is diagnostic
for the presence of the event DNA. The amplicon is of a length and
has a sequence that is also diagnostic for the event. The amplicon
may range in length from the combined length of the primer pairs
plus one nucleotide base pair, and/or the combined length of the
primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,
256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,
282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,
295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,
308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,
321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,
360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,
399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,
412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,
425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,
438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,
451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,
464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,
477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750,
1000, 1250, 1500, 1750, 2000, or more nucleotide base pairs (plus
or minus any of the increments listed above). Alternatively, a
primer pair can be derived from flanking sequence on both sides of
the inserted DNA so as to produce an amplicon that includes the
entire insert nucleotide sequence. A member of a primer pair
derived from the plant genomic sequence may be located a distance
from the inserted DNA sequence. This distance can range from one
nucleotide base pair up to about twenty thousand nucleotide base
pairs. The use of the term "amplicon" specifically excludes primer
dimers that may be formed in the DNA thermal amplification
reaction.
[0096] Nucleic-acid amplification can be accomplished by any of the
various nucleic-acid amplification methods known in the art,
including the polymerase chain reaction (PCR). A variety of
amplification methods are known in the art and are described, inter
alia, in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202. PCR
amplification methods have been developed to amplify up to 22 kb of
genomic DNA. These methods as well as other methods known in the
art of DNA amplification may be used in the practice of the present
invention. The sequence of the heterologous transgene DNA insert or
flanking genomic sequence from a subject cotton event can be
verified (and corrected if necessary) by amplifying such sequences
from the event using primers derived from the sequences provided
herein followed by standard DNA sequencing of the PCR amplicon or
of the cloned DNA.
[0097] The amplicon produced by these methods may be detected by a
plurality of techniques. Agarose gel electrophoresis and staining
with ethidium bromide is a common well known method of detecting
DNA amplicons. Another such method is Genetic Bit Analysis where an
DNA oligonucleotide is designed which overlaps both the adjacent
flanking genomic DNA sequence and the inserted DNA sequence. The
oligonucleotide is immobilized in wells of a microwell plate.
Following PCR of the region of interest (using one primer in the
inserted sequence and one in the adjacent flanking genomic
sequence), a single-stranded PCR product can be hybridized to the
immobilized oligonucleotide and serve as a template for a single
base extension reaction using a DNA polymerase and labelled ddNTPs
specific for the expected next base. Readout may be fluorescent or
ELISA-based. A signal indicates presence of the insert/flanking
sequence due to successful amplification, hybridization, and single
base extension.
[0098] Another method is the Pyrosequencing technique as described
by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method an
oligonucleotide is designed that overlaps the adjacent genomic DNA
and insert DNA junction. The oligonucleotide is hybridized to
single-stranded PCR product from the region of interest (one primer
in the inserted sequence and one in the flanking genomic sequence)
and incubated in the presence of a DNA polymerase, ATP,
sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and
luciferin. DNTPs are added individually and the incorporation
results in a light signal that is measured. A light signal
indicates the presence of the transgene insert/flanking sequence
due to successful amplification, hybridization, and single or
multi-base extension.
[0099] Fluorescence Polarization is another method that can be used
to detect an amplicon of the present invention. Following this
method, an oligonucleotide is designed which overlaps the genomic
flanking and inserted DNA junction. The oligonucleotide is
hybridized to single-stranded PCR product from the region of
interest (one primer in the inserted DNA and one in the flanking
genomic DNA sequence) and incubated in the presence of a DNA
polymerase and a fluorescent-labeled ddNTP. Single base extension
results in incorporation of the ddNTP. Incorporation can be
measured as a change in polarization using a fluorometer. A change
in polarization indicates the presence of the transgene
insert/flanking sequence due to successful amplification,
hybridization, and single base extension.
[0100] TAQMAN (PE Applied Biosystems, Foster City, Calif.) is a
method of detecting and quantifying the presence of a DNA sequence.
Briefly, a FRET oligonucleotide probe is designed that overlaps the
genomic flanking and insert DNA junction. The FRET probe and PCR
primers (one primer in the insert DNA sequence and one in the
flanking genomic sequence) are cycled in the presence of a
thermostable polymerase and dNTPs. Hybridization of the FRET probe
results in cleavage and release of the fluorescent moiety away from
the quenching moiety on the FRET probe. A fluorescent signal
indicates the presence of the flanking/transgene insert sequence
due to successful amplification and hybridization.
[0101] Molecular Beacons have been described for use in sequence
detection. Briefly, a FRET oligonucleotide probe is designed that
overlaps the flanking genomic and insert DNA junction. The unique
structure of the FRET probe results in it containing secondary
structure that keeps the fluorescent and quenching moieties in
close proximity. The FRET probe and PCR primers (one primer in the
insert DNA sequence and one in the flanking genomic sequence) are
cycled in the presence of a thermostable polymerase and dNTPs.
Following successful PCR amplification, hybridization of the FRET
probe to the target sequence results in the removal of the probe
secondary structure and spatial separation of the fluorescent and
quenching moieties. A fluorescent signal results. A fluorescent
signal indicates the presence of the flanking genomic/transgene
insert sequence due to successful amplification and
hybridization.
[0102] Having disclosed two general locations in the cotton genome
that are excellent for insertions, the subject invention also
comprises a cotton seed and/or a cotton plant comprising at least
one non-cry1F and non-cry1Ac insert in the general vicinity of one
or both of these locations. One option is to substitute a different
insert in place of the cry1F and/or cry1Ac insert exemplified
herein. In these generally regards, targeted homologous
recombination, for example, can be used according to the subject
invention. This type of technology is the subject of, for example,
WO 03/080809 A2 and the corresponding published U.S. application
(USPA 20030232410).
[0103] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety to the extent they are not inconsistent
with the explicit teachings of this specification.
[0104] The following examples are included to illustrate procedures
for practicing the invention and to demonstrate certain preferred
embodiments of the invention. These examples should not be
construed as limiting. It should be appreciated by those of skill
in the art that the techniques disclosed in the following examples
represent specific approaches used to illustrate preferred modes
for its practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many changes can
be made in these specific embodiments while still obtaining like or
similar results without departing from the spirit and scope of the
invention. Unless otherwise indicated, all percentages are by
weight and all solvent mixture proportions are by volume unless
otherwise noted.
Example 1
Production of Deposited Seed
[0105] WideStrike.TM. brand insect resistance for cotton is a
transgenic trait developed by Dow AgroSciences that provides
in-plant insect resistance against Lepidoptera. It contains two
insect tolerance genes, cry1Ac and cry1F, which were derived from
Bacillus thuringiensis subspecies kurstaki and Bacillus
thuringiensis subspecies aizawai, respectively. Bacillus
thuringiensis (B.t.) is a common, gram-positive, soil-borne
bacterium. In its spore-forming stage, it produces several
insecticidal protein crystals (known as delta-endotoxins) including
Cry1Ac and Cry1F. These proteins are toxic to certain lepidopteran
insects. In susceptible insects, they bind to specific receptors
present on midgut epithelial cells, forming pores that disrupt
osmotic balance and eventually result in cell lysis and death.
Cry1Ac and Cry1F have been shown to be non-toxic to humans,
livestock, and beneficial insects, which do not have binding sites
for the delta-endotoxin. Using two delta-endotoxins rather than one
will provide improved insect resistance because the two Cry
proteins provide a greater spectrum of control than either does
alone and have differential activity against the lepidopteran pests
that they are effective against. More importantly, it may help
delay the development of resistant insects.
[0106] The cry1Ac and cry1F genes in WideStrike were introduced
using Agrobacterium mediated transformation into GC-510 cotton
(Gossypium hirsutum L.) plants in two separate transformation
events, 3006-210-23 and 281-24-236. Following crossing into an
elite cotton variety, these events were combined by conventional
breeding to produce cotton bearing the WideStrike insect-resistance
trait. WideStrike also contains the pat gene from Streptomyces
viridochromogenes, a common aerobic soil bacteria. The pat gene
codes for the Phosphinothricin Acetyl Transferase (PAT) enzyme,
which detoxifies glufosinate ammonium into an inactive compound by
acetylation. The pat gene was included to allow for selection of
transformed cotton plants.
Example 2
Diagnostic Test for Cry1F Cotton Event 281-24-236
[0107] DNA from Cry1F event 281-24-236 and Cry1Ac events
3006-210-23, and non-transgenic cotton PCS355 was extracted from
cotton leaves using QIAGEN's Plant DNeasy kit (catalog #69181,
Qiagen, Valencia, Calif., USA). The manufacturer's suggested
protocol was followed. In brief, leaf discs were disrupted in an
RNAse supplemented preheated buffer using a tungsten carbide bead
(0.125 mm diameter) and a Retsch MM3000 Mixer Mill. The mixture was
centrifuged at room temperature, and the supernatant was
subsequently captured by running through a DNeasy 96 plate. DNA was
eluted in an elution buffer and stored frozen until use.
[0108] The DNA extracted from the cotton leaf tissue was used in a
PCR DNA amplification of the 5' genomic/transgene insert sequences
in Cry1F event 281-24-236 using primer 281-14 (SEQ ID NO:3,
5'TGTCGGCTGAAGGTAGGGAGG3') and primer 281-15 (SEQ ID NO:4, 5'
CCGGACATGAAGCCATTTAC3'), and the 3' genomic/transgene insert
sequences flanking using primer 281-9 (SEQ ID NO:6,
5'TCTCTAGAGAGGGGCACGACC3') and primer 281-10 (SEQ ID NO:7,
5'CGAGCTGGAGAGACCGGTGAC3'). The PCR DNA amplification analyses were
conducted using genomic DNA extracted from cotton event Cry1F
281-24-236 and non-transgenic cotton line PCS355. The amplification
reaction for the 5' flanking genomic sequence was conducted using
QIAGEN HotStarTaq PCR kit (catalog #203203 or 203205, QIAGEN,
Valencia, Calif., USA) with a final concentration of 0.4 .mu.M for
Primer 281-14 and Primer 281-15 in a 50 .mu.l reaction volume. The
reactions were performed using a GenAmp PCR System 9600 (Applied
Biosystem, Foster City, Calif.) under the following cycling
conditions: 1 cycle at 95.degree. C. for 15 minute; 35 cycles of
94.degree. C. for 30 seconds, 57.degree. C. for 30 seconds,
72.degree. C. for 60 seconds; 1 cycle at 72.degree. C. for 10
minutes. The PCR for the 3' flanking genomic sequence was conducted
using Takara ExTaq PCR kit (Catalog #RR001A, Panvera, Madison,
Wis.) in a 50 .mu.l reaction volume containing a final
concentration of 0.4 .mu.M of Primer 281-9 and Primer 281-10. The
reactions were performed using a GenAmp PCR System 9600 (Applied
Biosystem, Foster City, Calif.) under the following cycling
conditions: 1 cycle at 95.degree. C. for 5 minute; 35 cycles of
94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds,
72.degree. C. for 60 seconds; 1 cycle at 72.degree. C. for 10
minutes. The PCR products were separated using 1.0% agarose gel
electrophoresis at 100 V for about 1 hour and visualized by
ethidium bromide staining.
[0109] The 5' PCR product DNA sequence was determined resulting in
a 603 nucleotide base pair sequence representing the 5'
genomic/transgene insert sequence of cotton Cry1F event 281-24-236
and identified as SEQ ID NO:5. The 3' PCR product DNA sequence was
determined resulting in a 562 nucleotide base pair sequence
representing the 3' genomic/transgene insert sequence of cotton
Cry1F event 281-24-236 and identified in SEQ ID NO:8.
[0110] The genomic/transgene junction sequences, SEQ ID NO:5 and
SEQ ID NO:8 are novel DNA sequences in Cry1F event 281-24-236 that
are diagnostic for cotton plant Cry1F event 281-24-236 and its
progeny.
Example 3
Diagnostic Test for Cry1Ac Cotton Event 3006-210-23
[0111] The DNA extracted from the cotton leaf tissue was used in a
PCR DNA amplification of the 5' genomic/transgene insert sequences
in Cry1Ac event 3006-210-23 using primer 3006-20 (SEQ ID NO:9,
5'TTCCAACCTTTAACTATTATCCTGC3') and primer 3006-22 (SEQ ID NO:10,
5'GCTGCGGACATCTACATTTT3'), and the 3' genomic/transgene insert
sequences flanking using primer 3006-9 (SEQ ID NO:12,
5'GACATGCAATGCTCATTATCTCTA3') and primer 3006-12 (SEQ ID NO:13,
5'AAGTCTCTGCCTTCTACCCTGG3'). The PCR DNA amplification analyses
were conducted using genomic DNA extracted from cotton event Cry1Ac
3006-210-23 and non-transgenic cotton line PCS355. The
amplification reaction for the 5' flanking genomic sequence was
conducted using QIAGEN HotStarTaq PCR kit (catalog #203203 or
203205, QIAGEN, Valencia, Calif., USA) with a final concentration
of 0.4 .mu.M for Primer 3006-20 and Primer 3006-22 in a 50 .mu.l
reaction volume. The reactions were performed using a GenAmp PCR
System 9600 (Applied Biosystem, Foster City, Calif.) under the
following cycling conditions: 1 cycle at 95.degree. C. for 15
minute; 35 cycles of 94.degree. C. for 30 seconds, 53.degree. C.
for 30 seconds, 72.degree. C. for 60 seconds; 1 cycle at 72.degree.
C. for 10 minutes. The PCR for the 3' flanking genomic sequence was
conducted using QIAGEN HotStarTaq PCR kit (catalog #203203 or
203205, QIAGEN, Valencia, Calif., USA) in a 50 .mu.l reaction
volume containing a final concentration of 0.4 .mu.M of Primer
3006-9 and Primer 3006-12. The reactions were performed using a
GenAmp PCR System 9600 (Applied Biosystem, Foster City, Calif.)
under the following cycling conditions: 1 cycle at 95.degree. C.
for 5 minutes; 30 cycles of 94.degree. C. for 30 seconds,
56.degree. C. for 30 seconds, 72.degree. C. for 60 seconds; 1 cycle
at 72.degree. C. for 10 minutes. The PCR products were separated
using 1.0% agarose gel electrophoresis at 100 V for about. 1 hour
and visualized by ethidium bromide staining.
[0112] The 5' PCR product DNA sequence was determined resulting in
a 614 nucleotide base pair sequence representing the 5'
genomic/transgene insert sequence of cotton Cry1Ac event
3006-210-23 (identified here as SEQ ID NO:11). The 3' PCR product
DNA sequence was determined resulting in a 662 nucleotide base pair
sequence representing the 3' genomic/transgene insert sequence of
cotton Cry1Ac event 3006-210-23 (identified here as SEQ ID
NO:14).
[0113] The genomic/transgene junction sequences, SEQ ID NO:11 and
SEQ ID NO:14 are novel DNA sequences in Cry1Ac event 3006-210-23
that are diagnostic for cotton plant Cry1Ac event 3006-210-23 and
its progeny.
Example 4
Further Diagnostic Tests
[0114] DNA event primer pairs are used to produce an amplicon
diagnostic for Cry1F event 281-24-23 and Cry1Ac event 3006-210-23.
These event primer pairs include, but are not limited to, SEQ ID
NO:3 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:12, and SEQ ID NO:13. When used in a DNA
amplification method (PCR), these primers produce an amplicon
diagnostic for Cry1F event 281-24-236 and/or Cry1Ac event
3006-210-23, and their progenies. In addition to these primer
pairs, further aspects of the subject invention include any primer
pair derived from the amplicon product of SEQ ID NO:5, SEQ ID NO:9,
SEQ ID NO:11, and/or SEQ ID NO:14 that, in a DNA amplification
reaction, produces an amplicon diagnostic for Cry1F event
281-24-236, Cry1Ac event 3006-210-23, and their progenies. Any
modification involving the use of DNA primers to produce an
amplicon diagnostic for Cry1F event 281-24-236, Cry1Ac event
3006-210-23, and their progenies is within the ordinary skill of
the art, given the benefit of the subject disclosure. The analysis
of plant tissue sample from Cry1F event 281-24-236, Cry1Ac event
3006-210-23, and their progenies should include a positive tissue
control from these events, a negative control from a cotton plant
that is not any of these events, and a negative control that
contains no template cotton DNA. Additional primer sequences can be
derived from SEQ ID NO:1 and/or SEQ ID NO:2 by those skilled in the
art of DNA amplification methods. Conditions optimized for the
production of an amplicon may differ from the methods described in
the Examples above. The use of these DNA primer sequences with
modifications to the methods described in these Examples is within
the scope of the invention. Amplicons and primers derived from SEQ
ID NO:1 and/or SEQ ID NO:2 that are diagnostic for Cry1F event
281-24-236 and/or Cry1Ac event 3006-210-23, and their progenies are
aspects of the invention. The assay for amplicons of the Cry1F
event 281-24-236, Cry1Ac event 3006-210-23, and their progenies can
be performed by using a Stratagene Robocycler, MJ, Engine, or
Eppendorf Mastercycler Gradient thermocycler, or by methods and
apparatus known to those skilled in the art.
[0115] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. We claim all modifications
that are within the spirit and scope of the appended claims.
Sequence CWU 1
1
21115490DNAArtificial SequenceCry1F event 281-24-236 insert and its
border sequences 1aagcttgctt aaaagtatca caagccatga tccttataaa
aatgatatct gacactatgc 60ttctttgcac attcttcact atgctttctc atttgagcaa
tggtgggatt tgctctcaaa 120tttggtggcc ctatgtcagt attcaaaaat
tttcataggt caaaggcttg aagataagcc 180ttcattttta ccacccatat
gtagtaaatt tcaccgatga atacaagagg tggaggtggt 240gagaaactag
atgaatacat tcttgcagaa aacgtccctc aaagatcaaa atggctctca
300ataccaattc ttggtgtttt cagactaaga gagaaagcaa aacgagacaa
tgaacccaca 360agatgaatta atttcaatac atttttttaa atttcatttc
aaacggttac aatatatacc 420ttttgttttc caaacaactc aactgatcca
actaacatct tggaaatagg aaaacttaac 480ttgtacacaa actgattgtg
aaatcaacac ctaaacacat caagtcatta ccaacttagt 540ttattcctag
ctaagtatct taacataagg attgaacaaa aggttaaacc aaaactttga
600tttttttgct ctaggaagac agcttgcact tcatcataca gttcgcctta
ttgttaatca 660actcacactt tggctgtccg ttgatatgct ggcagttaaa
atgttcacca atgtgcttag 720acaatccgcc aagcctatcg tgtaaaaact
ctttgcatga cagttcacct attgacagtc 780cgccagtctt ttaatgctca
gactgtctac catgtgactt agagacggaa ttgtttgcca 840tatgtgttcg
ccaactagat tgttcaccat ttgtgttgtt taccatgtgt gttcgctaat
900gcatggttgg ccatgtcgca aaacaaattt ttggatgggc caaaattgga
attttttcat 960ttgaccaaat ataaaaaaac gaattgaaca aattactttt
agagggatta aaaattaaaa 1020ttatactatt tatcgagggg agtcaaggct
cctatcttct tcgcttcttc tattgtttag 1080attaagacta aaattttaaa
atttatagaa attaaaattg atgaaattaa aatacaaaat 1140taaatatata
attcagttag gtttaaccat tttttaatgt tgcttagctt taatgtttgg
1200gatttggcta ctttcagtcg ttatgcagtt atgctcagac aaatttgttc
tctttctgtc 1260ttatcaacta ctcaaaatct cagtatagtt atgtcattta
atctcttcat cgtagatgtt 1320atattggtga aaatggggcc aagaaatcca
cattcaatga ctttgaaaga atatatattg 1380ttagttgcac attcccttat
tcaatcacag ttgcttgttt ctgagtctat agaatcatga 1440tatttgtaaa
tcttatataa agtaagagta tatggctaga cagtctggcc ctgtcggctg
1500aaggtaggga ggaattaatc aatcacagtt gcttgtttct gagtctatag
aatcatgaat 1560tttaaattta tggaatgcat tttttcgaag atattgtatg
cattaagtgt aattttagtt 1620tcaatatgaa atttgagatt tatatatata
cttacataaa accctccttt actgaattag 1680tgccatggat aaaagaccaa
ttaagcaatc cttccaacac gtgcatgcac tggattttca 1740tcgcctcgtc
cattgttaaa ttgataggtt aataagaaca attagttggc tactgattat
1800atggattctg ggttaaaagt atttaggttt actgttacat acatggagga
tctacatcta 1860ttttcacttt tgtttaatta atttaagtta gttttgatga
gtttaaggat tgtactagcc 1920aatagtagta cataaaggag atagagtacc
aaaacaaaga aaaagccgaa aggtgttaat 1980gctaaattgt aaaagaaagt
taaaataaga gactcgaatt ataatatgat tctctggcgc 2040actaattaag
ctactatata ttgtcaatag tattgtaaat ggcttcatgt ccgggaaatc
2100tacatggatc agcaatgagt atgatggtca atatggagaa aaagaaagag
taattaccaa 2160ttttttttca attcaaaaat gtagatgtcc gcagcgttat
tataaaatga aagtacattt 2220tgataaaacg acaaattacg atccgtcgta
tttataggcg aaagcaataa acaaattatt 2280ctaattcgga aatctttatt
tcgacgtgtc tacattcacg tccaaatggg ggccacttgg 2340ctgcagtgca
gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta
2400agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc
agtttatcta 2460tctttataca tatatttaaa ctttactcta cgaataatat
aatctatagt actacaataa 2520tatcagtgtt ttagagaatc atataaatga
acagttagac atggtctaaa ggacaattga 2580gtattttgac aacaggactc
tacagtttta tctttttagt gtgcatgtgt tctccttttt 2640tttgcaaata
gcttcaccta tataatactt catccatttt attagtacat ccatttaggg
2700tttagggtta atggttttta tagactaatt tttttagtac atctatttta
ttctatttta 2760gcctctaaat taagaaaact aaaactctat tttagttttt
ttatttaata atttagatat 2820aaaatagaat aaaataaagt gactaaaaat
taaacaaata ccctttaaga aattaaaaaa 2880actaaggaaa catttttctt
gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 2940gagtctaacg
gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac
3000ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc
caccgttgga 3060cttgctccgc tgtcggcatc cagaaattgc gtggcggagc
ggcagacgtg agccggcacg 3120gcaggcggcc tcctcctcct ctcacggcac
cggcagctac gggggattcc tttcccaccg 3180ctccttcgct ttcccttcct
cgcccgccgt aataaataga caccccctcc acaccctctt 3240tccccaacct
cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac
3300ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc
cctctctacc 3360ttctctagat cggcgttccg gtccatggtt agggcccggt
agttctactt ctgttcatgt 3420ttgtgttaga tccgtgtttg tgttagatcc
gtgctgctag cgttcgtaca cggatgcgac 3480ctgtacgtca gacacgttct
gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 3540gatggctcta
gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat
3600agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt
ttgtcgggtc 3660atcttttcat gctttttttt gtcttggttg tgatgatgtg
gtctggttgg gcggtcgttc 3720tagatcggag tagaattctg tttcaaacta
cctggtggat ttattaattt tggatctgta 3780tgtgtgtgcc atacatattc
atagttacga attgaagatg atggatggaa atatcgatct 3840aggataggta
tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt
3900cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta
gatcggagta 3960gaatactgtt tcaaactacc tggtgtattt attaattttg
gaactgtatg tgtgtgtcat 4020acatcttcat agttacgagt ttaagatgga
tggaaatatc gatctaggat aggtatacat 4080gttgatgtgg gttttactga
tgcatataca tgatggcata tgcagcatct attcatatgc 4140tctaaccttg
agtacctatc tattataata aacaagtatg ttttataatt attttgatct
4200tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta
gccctgcctt 4260catacgctat ttatttgctt ggtactgttt cttttgtcga
tgctcaccct gttgtttggt 4320gttacttctg caggtcgaca tgtctccgga
gaggagacca gttgagatta ggccagctac 4380agcagctgat atggccgcgg
tttgtgatat cgttaaccat tacattgaga cgtctacagt 4440gaactttagg
acagagccac aaacaccaca agagtggatt gatgatctag agaggttgca
4500agatagatac ccttggttgg ttgctgaggt tgagggtgtt gtggctggta
ttgcttacgc 4560tgggccctgg aaggctagga acgcttacga ttggacagtt
gagagtactg tttacgtgtc 4620acataggcat caaaggttgg gcctaggatc
cacattgtac acacatttgc ttaagtctat 4680ggaggcgcaa ggttttaagt
ctgtggttgc tgttataggc cttccaaacg atccatctgt 4740taggttgcat
gaggctttgg gatacacagc ccggggtaca ttgcgcgcag ctggatacaa
4800gcatggtgga tggcatgatg ttggtttttg gcaaagggat tttgagttgc
cagctcctcc 4860aaggccagtt aggccagtta cccagatctg agtcgacgga
tccccgacat atgccccggt 4920ttcgttgcga ctaacatgag ttcttggaca
aatttgattg gacctgatga gatgatccaa 4980cccgaggata tagcaaagct
cgttcgtgca gcaatggaac ggccaaaccg tgcttttgtc 5040cccaagaatg
aggtgctatg catgaaggaa tctacccgtt gatgtccaac agtctcaggg
5100ttaatgtcta tgtatcttaa ataatgttgt cggtattttg taatctcata
tagattttca 5160ctgtgcgacg caaaaatatt aaataaatat tattattatc
tacgttttga ttgagatatc 5220atcaatatta taataaaaat atccattaaa
cacgatttga tacaaatgac agtcaataat 5280ctgatttgaa tatttattaa
ttgtaacgaa ttacataaag atcgaataga aaatactgca 5340ctgcaaatga
aaattaacac atactaataa atgcgtcaaa tatctttgcc aagatcaagc
5400ggagtgaggg cctcatatcc ggtctcagtt acaagcacgg tatccccgaa
gcgcgctcca 5460ccaatgccct cgacatagat gccgggctcg acgctgagga
cattgcctac cttgagcatg 5520gtctcagcgc cggctttaag ctcaatccca
tcccaatctg aatatcctat cccgcgccca 5580gtccggtgta agaacgggtc
tgtccatcca cctctgttgg gaattctgat cttggcggta 5640cccggggatc
ctcattcctc catcagaagt aactccacgc tatcaacaat gaatgttcct
5700tccgtttctc caatctcaat ccaaaccttg tcggtttctg gaaagtactc
taactctttg 5760gtgacatagc cggctggtaa cggtgtgtag tccccatagc
ctctgttaga ttcgcaagga 5820ttgtccctac gtccatcggt gtaagccttc
tcctcatagg ctgatgcata gtcagcgggt 5880acagaagagt tgctctcata
ggctccatcg tatcctcgat tgcgagaagt gtaagtaccc 5940tcatactcct
cttgagtcgc agtgtagtca ttgcaagtta cggtgttgtt tgggtagact
6000tcctcctcga cgcagttgct gaacttcagc tcgtcggtgt tgttctcaat
ctcgtgtatg 6060gtgacgcaac cttctccgta tccttctttg tacgcggtaa
cacgaagaat gtagccacga 6120ccaggacaga cacgaacttc ttgtgaaact
tctgcttccc actcaggaac aacaaggaca 6180gagcggtgat tgttctgttc
ttctacatct acgtgccctt tcacattcca gcaggatagg 6240ccattgttga
agtcaccatt cttgatgaca ttcctcgcat catacaagga gaatgcagtg
6300aagatgcgcc cttctaactc ttcaaagata gcagcattga cacccggaat
cacgctaagt 6360tcaggaaggt aagcttcccg aatgctatga acgcgtttgt
ctgcagcatg aatcatagct 6420atgttggtat cagcttggag cctatcatac
tgagagttca caaacagagc gtcaacgctt 6480tctttggctt ctttgtacac
aatgtttgtt tcccattcca acttctctct cttgtccctc 6540cacttcttct
cagccctctt cactctagcg agggcttctc caacaagtgg tttctcttct
6600agaaactcca gattgcctag cctggcatgg ccatcttgag tcttgatctt
gaagatcacc 6660cacacaccga ggtcttcgtt caggtcggta cagccaacgt
ctatgtccaa ggagaagtgg 6720tgtgagtgat gggcacactt gccgatggga
cttggggctg aaagtggcca gagtgaaccc 6780gtcccaggca cattgactgt
ctcatgtttg gcgttgtatc tgatgaggta gatctcaagg 6840tcttgactgt
cctcgatgta acctctcaac tggtatcttg tgtaggcttt gagtttcgat
6900tcatctatct tctggtacag gtatgttgga tagcactcat caaaggtacc
caagagcgta 6960acatagttct ccttgaacac atcatcacct ccttgaatgg
tgatgtccgt acttcccctc 7020catccacgat ctagttgcct gttgatcccg
cgaaagttgg gatcttgaag caagttccgc 7080tcatcactaa gtcgcttagc
atgtttgacc ttctcggaca actccttctt ctcatccaaa 7140cagaactcat
cagagaggca ctcaacaagg ttggaaacgc gatcgatgtg atagtcagtc
7200acatctgtct tgagcccaat ctgattggac gaagtgaaca gagcattcac
cgccttctgt 7260gctctttcca agtcagactc tgcctcgagt gtggcagtaa
ctggaatcaa ctcaaacctg 7320tcaatgtaca cttcgttgcc tgaactgaag
gtatcagcac ctactgtgaa actgctctgg 7380ctcattggaa aggtgaacgc
ggtgttgata gtggcgtagg agaaagattg gaatgtaagt 7440ggatcaccgg
tatccattgt cttgttgaac tgaccagcaa agatccgttc acctgcaacc
7500gtaacgtaga ttcttagatt ggtagtagag gcatagcgta tcctggcacg
atacctttgg 7560ggaagttgcc cattgatgtt gacaatggtg tacgcgaatg
gtcctccact agtgcgtcga 7620agaatgtctc ctcccgtgaa ccccggccct
cttacaactg tagttcctga ctgaagtgtg 7680tgtgccttca ccaagggaat
ctgagtgatt ctctctggat caatggtgtt tgtgggggta 7740gcgctacgat
gcgtccaaga gaacattggt gctctccatg agtcggaacc tgagatctca
7800cctggccagc gcacaaaggt aacatgattc agcacatggg agtagtcatt
ccaaggtgcg 7860ccgctgttgt cttgaggtgg tatctcatct agagagtcaa
tggtcccgga gttcctgaat 7920gtgcgggtgt gattcgtacc agtttgttga
aaggccactc ccctaagacc gagtacatag 7980tgaggattgc caaagcctcc
tcggacgaag acaggatctg acaaggtccg atagaaagga 8040cgtggatctt
catctgcaat ccagatggcg cccccgggat tgaagacccc gtaactagga
8100aagttgatac gattgccagc cgtgttgcgt gagctaacta agtgtcctcc
ccacacagtt 8160tgggatctaa cagtctctgc agtcacaaac aaagagttca
tgaagtccat gagatggggt 8220ggtctgactc caaactcagc cctgttgaaa
ccattgggta tgttcgcaga aactggagag 8280tcttcaatga ctgaactggt
gtagatctcc cttgtaagtt gggatgacgt ttgaatcgga 8340taggtacgaa
catcgtagtt cggaaagaga gcaactatgt ctaacacagt aagtgtaagg
8400tctctcctga actgattgaa cctggcccat tggcgagtgt tagtacctct
caggttctcc 8460aatccctgat tgtaggtatc caaacaatgt ttcgtgtatc
gatgaatcag attgatgagt 8520ctgttgtagt gattgttgac agtagctatg
tccagtcccc aaccttgccc aaacgacaca 8580gcgtcgcgca gtagtgacaa
gtgcaggtta gcagcttgaa catagaccga gagaagaggg 8640atctcgaagc
tggtaagggt gaagttgttg atggctgtga tcaaagcatc atctgtgtta
8700gcaaagcgta tacgcacatc ttctctcagt tgggcattgt taggattggc
ttcccactct 8760cttagtgctt caatgtagat ctcatagctg tctgctaagc
cacgaagggt agtgatggcc 8820cgattccttt ccaaggtctc aatcctttgt
tcaatcaact gttcaatctg gagaagaaag 8880aggctccaat cagatggagt
gatgaagccc cagatgaggt cgaagaggcc aaacgcaact 8940cccacacctg
gaacaaactc agacaacagg agacgtgtaa gggacaggga gatgtctaac
9000ggcaatctgc cagtcgacct ctcttcgttg agaatctcta cttcaggatt
gttgaggcag 9060ttgtagggga cgcactgatt ctgtatgttg ttctccattg
ttggatccgc gatttggtgt 9120atcgagattg gttatgaaat tcagatgcta
gtgtaatgta ttggtaattt gggaagatat 9180aataggaagc aaggctattt
atccatttct gaaaaggcga aatggcgtca ccgcgagcgt 9240cacgctctag
tcgaccatgt acgtaagcgc ttacgttttt ggtggacccc ctcgaccatg
9300tacgtaagcg cttacgtttt tggtggaccc cctcgaccat gtacgtaagc
gcttacgttt 9360ttggtggacc ccctcgacca tgtacgtaag cgcttacgtt
tttggtggac cccctcgacg 9420gatcccccct cgaccctaga cgtatctatt
caaaagtcgt taatggctgc ggatcaagaa 9480aaagttggaa tagaaacaga
atacccgcga aattcaggcc cggttgccat gtcctacacg 9540ccgaaataaa
cgaccaaatt agtagaaaaa taaaaactga ctcggatact tacgtcacgt
9600cttgcgcact gatttgaaaa atctcaatat aaacaaagac ggccacaaga
aaaaaccaaa 9660acaccgatat tcattaatct tatctagttt ctcaaaaaaa
ttcatatctt ccacaccctc 9720gagatctaga tatcgatgaa ttttggcgcg
ccttaattaa ggaattcctc gagtttaaac 9780ggatccctga aagcgacgtt
ggatgttaac atctgcaaat tgccttttct tatcgaccat 9840gtacgtaagc
gcttacgttt ttggtggacc cttgaggaaa ctggtagctg ttgtgggcct
9900gtggtctcaa gatggatcat taatttccac cttcacctac gatggggggc
atcgcaccgg 9960tgagtaatat tgtacggcta agagcgaatt tggcctgtag
acctcaattg cgagctttct 10020aatttcaaac tattcgagct ttctaattga
atatatccag ggcccagcgt aagcaatacc 10080agccacaaca ccctcaacct
cagcaaccaa ccaagggtat ctatcttgca acctctctag 10140atcatcaatc
cactcttgtg gtgtttgtgg ctctgtccta aagttcactg tagacgtctc
10200aatgtaatgg ttaacgatat cacaaaccgc ggccatatca gctgctgtag
ctggcctaat 10260ctcaactggt ctcctctccg gagacatgtc gacctgcaga
agtaacacca aacaacaggg 10320tgagcatcga caaaagaaac agtaccaagc
aaataaatag cgtatgaagg cagggctaaa 10380aaaatccaca tatagctgct
gcatatgcca tcatccaagt atatcaagat caaaataatt 10440ataaaacata
cttgtttatt ataatagata ggtactcaag gttagagcat atgaatagat
10500gctgcatatg ccatcatgta tatgcatcag taaaacccac atcaacatgt
atacctatcc 10560tagatcgata tttccatcca tcttaaactc gtaactatga
agatgtatga cacacacata 10620cagttccaaa attaataaat acaccaggta
gtttgaaaca gtattctact ccgatctaga 10680acgaatgaac gaccgcccaa
ccacaccaca tcatcacaac caagcgaaca aaaagcatct 10740ctgtatatgc
atcagtaaaa cccgcatcaa catgtatacc tatcctagat cgatatttcc
10800atccatcatc ttcaattcgt aactatgaat atgtatggca cacacataca
gatccaaaat 10860taataaatcc accaggtagt ttgaaacaga attctactcc
gatctagaac gaccgcccaa 10920ccagaccaca tcatcacaac caagacaaaa
aaaagcatga aaagatgacc cgacaaacaa 10980gtgcacggca tatattgaaa
taaaggaaaa gggcaaacca aaccctatgc aacgaaacaa 11040aaaaaatcat
gaaatcgatc ccgtctgcgg aacggctaga gccatcccag gattccccaa
11100agagaaacac tggcaagtta gcaatcagaa cgtgtctgac gtacaggtcg
catccgtgta 11160cgaacgctag cagcacggat ctaacacaaa cacggatcta
acacaaacat gaacagaagt 11220agaactaccg ggccctaacc atggaccgga
acgccgatct agagaaggta gagagggggg 11280gggggggggg aggacgagcg
gctgtacctt gaagcggagg tgccgacggg tggatttggg 11340ggagatctgg
ttgtgtgtgt gtgcgctccg aacaacacga ggttggggaa agagggtgtg
11400gagggggtgt ctatttatta cggcgggcga ggaagggaaa gcgaaggagc
ggtgggaaag 11460gaatcccccg tagctgccgg tgccgtgaga ggaggaggag
gccgcctgcc gtgccggctc 11520acgtctgccg ctccgccacg caatttctgg
atgccgacag cggagcaagt ccaacggtgg 11580agcggaactc tcgagagggg
tccagaggca gcgacagaga tgccgtgccg tctgcttcgc 11640ttggcccgac
gcgacgctgc tggttcgctg gttggtgtcc gttagactcg tcgacggcgt
11700ttaacaggct ggcattatct actcgaaaca agaaaaatgt ttccttagtt
tttttaattt 11760cttaaagggt atttgcttaa tttttagtca ctttatttta
ttctatttta tatctaaatt 11820attaaataaa aaaactaaaa tagagtttta
gttttcttaa tttagaggct aaaatagaat 11880aaaatagatg tactaaaaaa
attagtctat aaaaaccatt aaccctaaac cctaaatgga 11940tgtactaata
aaatggatga agtattatat aggtgaagct atttgcaaaa aaaaaggaga
12000acacatgcac actaaaaaga taaaactgta gagtcctgtt gtcaaaatac
tcaattgtcc 12060tttagaccat gtctaactgt tcatttatat gattctctaa
aacactgata ttattgtagt 12120actatagatt atattattcg tagagtaaag
tttaaatata tgtataaaga tagataaact 12180gcacttcaaa caagtgtgac
aaaaaaaata tgtggtaatt ttttataact tagagatgca 12240atgctcatta
tctctagaga ggggcacgac cgggtcacgc tgcactgcag ccaagtggcc
12300cccatttgga cgtgaatgta gacacgtcga aataaagatt tccgaattag
aataatttgc 12360ttattgcttt cgcctataaa tacgacggat cgtaatttgt
cgctttatca gaatgtactt 12420tcattttata ataacgctgc ggacatctac
atttttgaat tgaaaaaaaa ttggtaatta 12480ctctttcttt ttctccatat
tgaccatcat actcattgct gatccatgta gatttccctt 12540acttgtctcc
ctctaatctg actttattaa cccaaagcaa ttgcttattt gttccccacg
12600cccacaaagc ccagcattgt ccctaaggta ttaatttgtt gttcgattct
tgttcttgaa 12660cccatttgga gaatgcaaca agggttttca tgtcagcacg
gtaatggttc tgtgtaaatt 12720ccagtagtgc tgcccaagta aagtctgggt
atttcctcga atttgcggca ttaactaagc 12780tagctgctgg tgtcaccggt
ctctccagct cgggacatag aaagaatgca agtgattttc 12840tcaccgtttc
agtattcact actgcccaag cagctcttgt agataccgtt tgtcaaagcc
12900tgtattcaaa caccacaacc tcattttcgt taaaattttt tgtatatacg
tatgcatata 12960tgttcagaat gtttattacc atgaatgtat cgcaatgttg
acaacgaaag ctcctgggat 13020atgagcaacg gagtgccact tttcatcggc
aaacacttga aggcctccaa cttgatcctg 13080atgaaggatt gtcaaggaag
tgggatcggt gtggggaccg gttcccaggg tcaactcagg 13140cttttcgcat
ggagagtagt gattcagtct caggattgaa tcattttgtt cgaaaaagtc
13200tttgaagtag gcttggtcaa gccctagact tatccctagc agccccatta
tctctttgga 13260aaccttgttc ctggcttcac aatattcctg gtaaagcctc
ctgtcaagta cgaaaagcaa 13320aatcgagtgt tagattccta tcctatggta
aaacttgtcg caacattcta caaattaaca 13380taggttataa aagagaaacg
caaagtcaaa aaaggccatc cagttataat gtgtattttt 13440tgttttagga
gaaggggtat ttaagcaatc catgttgaat ggacttaggg ttcggtcaat
13500gcaaggaatg atgtctattg aatttgtcac cccatctctt tcaaacctaa
aaaagatgta 13560tatccaattg tcactatttg tacaaggctt gaaatacaca
tggggttaac aaatacgctg 13620aaactgcaac atgtattttt tgcattttgg
agactaaatc ttgacgtaaa aataagctga 13680tcatctcgta tattgactga
atgcaaatta gcatttctcc tatttaatta ggaatggaag 13740ggtgagaaga
atttatttac ccaaaatctc taaaatcttc agtgccgaaa agacagtgtt
13800tctttccatg gcaacttgga ggaaaacctg ccaacgaaac tgcttgcata
cccatagctt 13860tctcctactt ttctcttagc cttttgcttc tcagagagtt
gcaggctgaa gaagcggtcc 13920atgtactgat gagcttacaa ccaaaaaaaa
cccatgcttc ttgcatgcct cattcaccgc 13980ccctgccact tttgatacag
caagagagcc cccaagcaag aaagctccca agtcaatggc 14040ttgaattaca
agttccgggt catccaggca tggcttatcg tcgtcaggcc atatgaactg
14100agagggtata ttggattcag atccaaggat tgatgcatcg aaagctaaag
ggcgatgctc 14160attctttgcg acagcagcag ttggcggaag catcggcttc
tcttgaatga cagaaactat 14220tggcagacaa tgtaccattt tctgtttctt
gttttaggtg gtcggagagg aaaaagaaga 14280caagatatag aggagcaaaa
ctaagagggt ttaaataaga gaagtagaaa accaataata 14340tttggaatct
aattgtttgt tttgaactgt tgcctcattt cccctttaat ttgtgttgtg
14400ttggcagctt aacgcttcct ttgggttcga ttgcaatata gtgcgttgaa
atttttacct 14460tgattttcaa tctcactaca ccacagtgac ttgcttggtt
gtttggataa tttttacctt 14520gattttcaat ctcactacac cacagtgact
tgcttggttg tttggataat ttttaccttg 14580attttcaatc tcactacacc
acagtgactt gcctggttgt ttggatatct tggtttccag 14640cacagtgaga
caagcctgct gcactcgaca accttatctt cactggtgct aaaagctcca
14700gtcagctcta gctagggcaa taggtatttt tggatgacaa aaatacccat
actttaattt 14760attattttac cattttatcc ttagatgtta aactaatttc
tttcccaatc ttatttgttt 14820tcagatttgg gaataaaaca atagtttctc
ccttgttctt gctggtttct cccttgttct 14880tgctggtttc tcccctcttt
tttgctttgt tcttggttgt gggagcttag gatattcttt 14940ttcttcaatc
agactaacaa gttagagata tcttgtgttt tttcacttta
ttttctcatg 15000ctcaacattt accctttttc tcaattaaca ggggaagtct
acattaatta gtgcactctc 15060agatagtaat ctgtatagtg atgcaatgta
tatatattct ttaaaacagt ttttcctcga 15120agttaaattc tttgttaaaa
gtaaaaggct ggatgttttt acctaattgg aggtaatgtc 15180tttgtgtaga
ttgtttgcaa cattggatgg ttgattaaaa agtgttgttc ttccttcaag
15240gtgagatggt ttgctgtcac tcactattat tattgttgtt attgttattg
cttttccatt 15300gaatagcctg gttctaaatg atatacttac cttatcccat
aggcagcaac attttatctt 15360ttgatctttg gacctatcat ttagcatgct
ttacactcta tttagtaata ttattactaa 15420ctataatttt aagtcataca
caatgaaatt acctaaacat ctaaactcaa aaaattataa 15480cttaaagctt
1549029382DNAArtificial SequenceCry1Ac event 3006-210-23 insert and
its border sequences 2accaattatt atcgtctttt ttaattattc caacctttaa
ctattatcct gccttaaaat 60tcgaatacat ttattatcta taaactatcc gaatattatt
atctaaatcc taattaaata 120ctatttttta tcgagtattc gtatccgcca
aggaaatcca tctccaaatt ttcaattatt 180tttcagatat ctaaatctgt
aaaatttcaa attcaagtac gttacaattc tttataaata 240atccaaatta
taaatatttt ataactatta attcataaat taaaatttat tattcaaata
300ttcgaataat ctatttttaa gacgtaaagt attacatcga agggttactt
tcaaagggta 360gtgtatttcc atttcaatta ttcagaacgt tgtcgttttg
ttccggtcat agaaaagggc 420tctggaagag aagaaaatga cttgactttt
caatttcatg ctcatccact cgtttcaatt 480actgtttact aaaaaaataa
taaaataaaa tattaacaat gcattgagta tgatgtccgg 540gaaatctaca
tggatnagca atgagtatga tggtcaatat ggagaaaaag aaagagtaat
600taccaatttt ttttcaattc aaaaatgtag atgtccgcag cgttattata
aaatgaaagt 660acattttgat aaaacgacaa attacgatcc gtcgtattta
taggcgaaag caataaacaa 720attattctaa ttcggaaatc tttatttcga
cgtgtctaca ttcacgtcca aatgggggcc 780acttggctgc agccaagctt
tcgcgagctc gagatccccg acatatgccc cggtttcgtt 840gcgactaaca
tgagttcttg gacaaatttg attggacctg atgagatgat ccaacccgag
900gatatagcaa agctcgttcg tgcagcaatg gaacggccaa accgtgcttt
tgtccccaag 960aatgaggtgc tatgcatgaa ggaatctacc cgttgatgtc
caacagtctc agggttaatg 1020tctatgtatc ttaaataatg ttgtcggtat
tttgtaatct catatagatt ttcactgtgc 1080gacgcaaaaa tattaaataa
atattattat tatctacgtt ttgattgaga tatctagatc 1140tcgaggtgtg
gaagatatga atttttttga gaaactagat aagattaatg aatatcggtg
1200ttttggtttt ttcttgtggc cgtctttgtt tatattgaga tttttcaaat
cagtgcgcaa 1260gacgtgacgt aagtatccga gtcagttttt atttttctac
taatttggtc gtttatttcg 1320gcgtgtagga catggcaacc gggcctgaat
ttcgcgggta ttctgtttct attccaactt 1380tttcttgatc cgcagccatt
aacgactttt gaatagatac gtctagggtc gaggggggat 1440ccgtcgaggg
ggtccaccaa aaacgtaagc gcttacgtac atggtcgagg gggtccacca
1500aaaacgtaag cgcttacgta catggtcgag ggggtccacc aaaaacgtaa
gcgcttacgt 1560acatggtcga gggggtccac caaaaacgta agcgcttacg
tacatggtcg actagagcgt 1620gacgctcgcg gtgacgccat ttcgcctttt
cagaaatgga taaatagcct tgcttcctat 1680tatatcttcc caaattacca
atacattaca ctagcatctg aatttcataa ccaatctcga 1740tacaccaaat
cgcggatccg tcgacctgca ggtcgacatg tctccggaga ggagaccagt
1800tgagattagg ccagctacag cagctgatat ggccgcggtt tgtgatatcg
ttaaccatta 1860cattgagacg tctacagtga actttaggac agagccacaa
acaccacaag agtggattga 1920tgatctagag aggttgcaag atagataccc
ttggttggtt gctgaggttg agggtgttgt 1980ggctggtatt gcttacgctg
ggccctggaa ggctaggaac gcttacgatt ggacagttga 2040gagtactgtt
tacgtgtcac ataggcatca aaggttgggc ctaggatcca cattgtacac
2100acatttgctt aagtctatgg aggcgcaagg ttttaagtct gtggttgctg
ttataggcct 2160tccaaacgat ccatctgtta ggttgcatga ggctttggga
tacacagccc ggggtacatt 2220gcgcgcagct ggatacaagc atggtggatg
gcatgatgtt ggtttttggc aaagggattt 2280tgagttgcca gctcctccaa
ggccagttag gccagttacc cagatctgag tcgacggatc 2340cccgacatat
gccccggttt cgttgcgact aacatgagtt cttggacaaa tttgattgga
2400cctgatgaga tgatccaacc cgaggatata gcaaagctcg ttcgtgcagc
aatggaacgg 2460ccaaaccgtg cttttgtccc caagaatgag gtgctatgca
tgaaggaatc tacccgttga 2520tgtccaacag tctcagggtt aatgtctatg
tatcttaaat aatgttgtcg gtattttgta 2580atctcatata gattttcact
gtgcgacgca aaaatattaa ataaatatta ttattatcta 2640cgttttgatt
gagatatcat caatattata ataaaaatat ccattaaaca cgatttgata
2700caaatgacag tcaataatct gatttgaata tttattaatt gtaacgaatt
acataaagat 2760cgaatagaaa atactgcact gcaaatgaaa attaacacat
actaataaat gcgtcaaata 2820tctttgccaa gatcaagcgg agtgagggcc
tcatatccgg tctcagttac aagcacggta 2880tccccgaagc gcgctccacc
aatgccctcg acatagatgc cgggctcgac gctgaggaca 2940ttgcctacct
tgagcatggt ctcagcgccg gctttaagct caatcccatc ccaatctgaa
3000tatcctatcc cgcgcccagt ccggtgtaag aacgggtctg tccatccacc
tctgttggga 3060attctgatct tggcgcgcat gcggatcctc attcctccat
cagaagtaac tccacgctat 3120caacaatgaa tgttccttcc gtttctccaa
tctcaatcca aaccttgtcg gtttctggaa 3180agtactctaa ctctttggtg
acatagccgg ctggtaacgg tgtgtagtcc ccatagcctc 3240tgttagattc
gcaaggattg tccctacgtc catcggtgta agccttctcc tcataggctg
3300atgcatagtc agcgggtaca gaagagttgc tctcataggc tccatcgtat
cctcgattgc 3360gagaagtgta agtaccctca tactcctctt gagtcgcagt
gtagtcattg caagttacgg 3420tgttgtttgg gtagacttcc tcctcgacgc
agttgctgaa cttcagctcg tcggtgttgt 3480tctcaatctc gtgtatggtg
acgcaacctt ctccgtatcc ttctttgtac gcggtaacac 3540gaagaatgta
gccacgacca ggacagacac gaacttcttg tgaaacttct gcttcccact
3600caggaacaac aaggacagag cggtgattgt tctgttcttc tacatctacg
tgccctttca 3660cattccagca ggataggcca ttgttgaagt caccattctt
gatgacattc ctcgcatcat 3720acaaggagaa tgcagtgaag atgcgccctt
ctaactcttc aaagatagca gcattgacac 3780ccggaatcac gctaagttca
ggaaggtaag cttcccgaat gctatgaacg cgtttgtctg 3840cagcatgaat
catagctatg ttggtatcag cttggagcct atcatactga gagttcacaa
3900acagagcgtc aacgctttct ttggcttctt tgtacacaat gtttgtttcc
cattccaact 3960tctctctctt gtccctccac ttcttctcag ccctcttcac
tctagcgagg gcttctccaa 4020caagtggttt ctcttctaga aactccagat
tgcctagcct ggcatggcca tcttgagtct 4080tgatcttgaa gatcacccac
acaccgaggt cttcgttcag gtcggtacag ccaacgtcta 4140tgtccaagga
gaagtggtgt gagtgatggg cacacttgcc gatgggactt ggggctgaaa
4200gtggccagag tgaacccgtc ccaggcacat tgactgtctc atgtttggcg
ttgtatctga 4260tgaggtagat ctcaaggtct tgactgtcct cgatgtaacc
tctcaactgg tatcttgtgt 4320aggctttgag tttcgattca tctatcttct
ggtacaggta tgttggatag cactcatcaa 4380aggtacccaa gagcgtaaca
tagttctcct tgaacacatc atcacctcct tgaatggtga 4440tgtccgtact
tcccctccat ccacgatcta gttgcctgtt gatcccgcga aagttgggat
4500cttgaagcaa gttccgctca tcactaagtc gcttagcatg tttgaccttc
tcggacaact 4560ccttcttctc atccaaacag aactcatcag agaggcactc
aacaaggttg gaaacgcgat 4620cgatgtgata gtcagtcaca tctgtcttga
gcccaatctg attggacgaa gtgaacagag 4680cattcaccgc cttctgtgct
ctttccaagt cagactctgc ctcgagcgtt gcagtaacgg 4740gaatgaattc
gaagcggtcg attatcactc cggcggttcc ggagaaattt ctaacaccta
4800ctatgttacc tagggaagag gtgaaggcat tggcactttc gaagtaaccg
aaatcgctag 4860attggagatt atccaaggat gtagctgtcg ctggtactgt
attggaaaag atggaggaat 4920taccccaatt gacgttgagg tgaatagggg
taacagaggc ataccttaca cgaacacgat 4980atctggtaga tgtcgatggg
aagtgaatgg gcacttcaat ataccctcta ttctggatgt 5040tgttgccgga
agaattcagc ctaaccaagt cgcctccagt gaatcctggt cctgaaatga
5100cagaaccatt aaagagaaag ttccccttga cagctgggat ctgagtaatg
ctatcggatg 5160caattatgtt gttaaactca gcactacgat gtatccaaga
gaacatcgga gctctgatga 5220tactaacgct gctattacta aagcctgaac
ggaacatgga cacatggcta aggcgatggc 5280taaacccttg cctaggtgga
acgttgttgt tctgtggagg gatctcatcc aagctatcaa 5340ctgttccgct
ctttctgtag acagcggatg gcagatttga ggaggttcca taggcaaatt
5400ctgtcccgtc aagcacagac aattgttgat tgttgatgcc gatgttgaaa
ggtctcctat 5460atagagtgct ggacaaggtt ctatacacgc cctgaccgag
ttgagcaaca atacgttgtt 5520gtggagctgc attgcccata gtcccgtaaa
gtgggaaagt gaattctggt ccagagaacc 5580caacgggtga tgccatgatc
tgatgccctg accagtagta ataaccgcgg tgcgcatcgg 5640tgtagatcgt
gatactgttc aatatgtcca tcaggtgtgg agacctgatg cttctctcta
5700tgccctgagc cgagcctcga aagctaccgt cgaagttctc gaggactggg
tttgtgtaga 5760tttcccgggt caattgtgac acagtacgga ttgggtagcg
cctagagtcg tagttgggaa 5820agagagcgac aatgtctagg acagttagtg
tcaactctcg cctgaactgg ttgtacctga 5880cccaatctct agaatccggt
ccccagacac gttcgagacc cgtgttgtac cagcgaacag 5940cataatcggt
atagttgcca ataagcctag tcagatcatt ataacgacta ttgatagttg
6000cggcatcaaa gccccaccgt tgtccgaaca cggagacatc gcggagcacc
gacaagtgca 6060ggttggcagc ctgcacgtac acggataaaa gaggaacttg
gtaattctga acggcgaaga 6120gcggaattgc ggtcgtcagc gcgctgttca
tgtcattgaa ttgaatgcgc atctcctctc 6180ttaaggcagg attggtcggg
tctgcttccc actctcgaaa agattctgcg taaatctggt 6240aaaggttgct
gaggccttct aaccttgaga tggcttggtt cctagcgaat tcttctattc
6300tttggttaat taactgctct atctgtacaa gaaaggcgtc ccattgagag
ggaccaaaga 6360ttccccaaat gatatcgaca agtccaagca cgaatccagc
accgggcacg aactctgaca 6420aaaggaattg ggtaagtgac aacgagatgt
cgataggtgt gtaaccagtc tcaatccgtt 6480ctccacccag cacctcaacc
tcagggttgc tcaggcagtt gtaaggaatg cactcgttga 6540tgttgggatt
gttgtccatt gttggatcct ctagagtcga cctgcagaag taacaccaaa
6600caacagggtg agcatcgaca aaagaaacag taccaagcaa ataaatagcg
tatgaaggca 6660gggctaaaaa aatccacata tagctgctgc atatgccatc
atccaagtat atcaagatca 6720aaataattat aaaacatact tgtttattat
aatagatagg tactcaaggt tagagcatat 6780gaatagatgc tgcatatgcc
atcatgtata tgcatcagta aaacccacat caacatgtat 6840acctatccta
gatcgatatt tccatccatc ttaaactcgt aactatgaag atgtatgaca
6900cacacataca gttccaaaat taataaatac accaggtagt ttgaaacagt
attctactcc 6960gatctagaac gaatgaacga ccgcccaacc acaccacatc
atcacaacca agcgaacaaa 7020aagcatctct gtatatgcat cagtaaaacc
cgcatcaaca tgtataccta tcctagatcg 7080atatttccat ccatcatctt
caattcgtaa ctatgaatat gtatggcaca cacatacaga 7140tccaaaatta
ataaatccac caggtagttt gaaacagaat tctactccga tctagaacga
7200ccgcccaacc agaccacatc atcacaacca agacaaaaaa aagcatgaaa
agatgacccg 7260acaaacaagt gcacggcata tattgaaata aaggaaaagg
gcaaaccaaa ccctatgcaa 7320cgaaacaaaa aaaatcatga aatcgatccc
gtctgcggaa cggctagagc catcccagga 7380ttccccaaag agaaacactg
gcaagttagc aatcagaacg tgtctgacgt acaggtcgca 7440tccgtgtacg
aacgctagca gcacggatct aacacaaaca cggatctaac acaaacatga
7500acagaagtag aactaccggg ccctaaccat ggaccggaac gccgatctag
agaaggtaga 7560gagggggggg gggggaggac gagcggcgta ccttgaagcg
gaggtgccga cgggtggatt 7620tgggggagat ctggttgtgt gtgtgtgcgc
tccgaacaac acgaggttgg ggaaagaggg 7680tgtggagggg gtgtctattt
attacggcgg gcgaggaagg gaaagcgaag gagcggtggg 7740aaaggaatcc
cccgtagctg ccggtgccgt gagaggagga ggaggccgcc tgccgtgccg
7800gctcacgtct gccgctccgc cacgcaattt ctggatgccg acagcggagc
aagtccaacg 7860gtggagcgga actctcgaga ggggtccaga ggcagcgaca
gagatgccgt gccgtctgct 7920tcgcttggcc cgacgcgacg ctgctggttc
gctggttggt gtccgttaga ctcgtcgacg 7980gcgtttaaca ggctggcatt
atctactcga aacaagaaaa atgtttcctt agttttttta 8040atttcttaaa
gggtatttgt ttaattttta gtcactttat tttattctat tttatatcta
8100aattattaaa taaaaaaact aaaatagagt tttagttttc ttaatttaga
ggctaaaata 8160gaataaaata gatgtactaa aaaaattagt ctataaaaac
cattaaccct aaaccctaaa 8220tggatgtact aataaaatgg atgaagtatt
atataggtga agctatttgc aaaaaaaaag 8280gagaacacat gcacactaaa
aagataaaac tgtagagtcc tgttgtcaaa atactcaatt 8340gtcctttaga
ccatgtctaa ctgttcattt atatgattct ctaaaacact gatattattg
8400tagtactata gattatatta ttcgtagagt aaagtttaaa tatatgtata
aagatagata 8460aactgcactt caaacaagtg tgacaaaaaa aatatgtggt
aattttttat aacttagaca 8520tgcaatgctc attatctcta gagaggggca
cgaccgggtc acgctgcact gcaggcatgc 8580gcgccttaat taaggaattc
ctcgagttta aacggatccc tgaaagcgac gttggatgtt 8640aacatctaca
aattgccttt tcttatcgac catgtacgta agcgcttacg tttttggtgg
8700acccttgagg aaactggtag ctgttgtggg cctgtggtct caagatggat
cattaatttc 8760caccttcacc tacgatgggg ggcatcgcac cggtgagtaa
tattgtacgg ctaagagcga 8820atttggcctg tagacctcaa ttgcgagctt
tctaatttca aactattcgg gcctaacttt 8880tggtgtgatg atgctgactg
gcttacgtgt ggaaaaaatt tgcaatctat gtagtcttta 8940actaatgttt
ttttctttaa aaaaaaagtc attatttttg gtttgattaa tatatttggt
9000ttaaattaaa taaaatatta aaaagtttag ttaaatcatc tatttaaacg
atttgtactg 9060atttgtgatc tattaatttt ttaacttaat ctagaccagg
gtactagttg gtccgatccc 9120atcttgaaaa cactatcttt agcttgctgg
taggttccag ggtagaaggc agagactttt 9180ttggagggtt tttattatta
aatttatatt tttataattt ttaaatgatt aaaataaaaa 9240tttattattt
taagaggaga taaagtgcaa ttttaccata tattaattta aaattttata
9300aatttaaaaa agaaaaaaac taaaatttta attttatagg ttctaaaata
ataaatataa 9360cttactgagt ttttttaagc tt 9382310348DNAArtificial
SequenceCry1Ac event 3006-48-81 insert and its border sequences
3gaatcaatct tttacgggaa aactcaattg gaatgaaact tgtttccagt aaaagtatta
60aaaaatgtat aaactagtcc atgtatgata gtgtaaagag tatattggag ttaaaaattt
120catcccatgt tactctatat tacattataa aggaagctat aggttggctc
actgcaccac 180ccaatcaaga tactcatatg tattacttaa tatataatat
taaaagatat catttctttt 240tgttatcaga ttaaagagaa ctaagatatt
acaaacttgc aaaacaatag ttaggccaac 300ccatatttta aatattttct
tcttttttct actcaaacct attttgtttc caaatattga 360agagttctgt
tctgattgga tatactctcg gacaagttta actctaaatt ttccaaatat
420tgaaatatgg atttgctcac atggaaaaac ggttcttgaa caataaatca
aaatagcata 480ttctatcaat ggaatcaagg aattccatat tcatttcaaa
gttggagcca agcatgcatg 540ataatgattt atagcaattt tgtcaaaagg
aaaaaataag gtgattggat gacaagaaac 600aaagggaaaa tgaattaaca
gcctgcattt aatatcattc atgtccggga aatctacatg 660gatnagcaat
gagtatgatg gtcaatatgg agaaaaagaa agagtaatta ccaatttttt
720ttcaattcaa aaatgtagat gtccgcagcg ttattataaa atgaaagtac
attttgataa 780aacgacaaat tacgatccgt cgtatttata ggcgaaagca
ataaacaaat tattctaatt 840cggaaatctt tatttcgacg tgtctacatt
cacgtccaaa tgggggccac ttggctgcag 900ccaagctttc gcgagctcga
gatccccgac atatgccccg gtttcgttgc gactaacatg 960agttcttgga
caaatttgat tggacctgat gagatgatcc aacccgagga tatagcaaag
1020ctcgttcgtg cagcaatgga acggccaaac cgtgcttttg tccccaagaa
tgaggtgcta 1080tgcatgaagg aatctacccg ttgatgtcca acagtctcag
ggttaatgtc tatgtatctt 1140aaataatgtt gtcggtattt tgtaatctca
tatagatttt cactgtgcga cgcaaaaata 1200ttaaataaat attattatta
tctacgtttt gattgagata tctagatctc gaggtgtgga 1260agatatgaat
ttttttgaga aactagataa gattaatgaa tatcggtgtt ttggtttttt
1320cttgtggccg tctttgttta tattgagatt tttcaaatca gtgcgcaaga
cgtgacgtaa 1380gtatccgagt cagtttttat ttttctacta atttggtcgt
ttatttcggc gtgtaggaca 1440tggcaaccgg gcctgaattt cgcgggtatt
ctgtttctat tccaactttt tcttgatccg 1500cagccattaa cgacttttga
atagatacgt ctagggtcga ggggggatcc gtcgaggggg 1560tccaccaaaa
acgtaagcgc ttacgtacat ggtcgagggg gtccaccaaa aacgtaagcg
1620cttacgtaca tggtcgaggg ggtccaccaa aaacgtaagc gcttacgtac
atggtcgagg 1680gggtccacca aaaacgtaag cgcttacgta catggtcgac
tagagcgtga cgctcgcggt 1740gacgccattt cgccttttca gaaatggata
aatagccttg cttcctatta tatcttccca 1800aattaccaat acattacact
agcatctgaa tttcataacc aatctcgata caccaaatcg 1860cggatccgtc
gacctgcagg tcgacatgtc tccggagagg agaccagttg agattaggcc
1920agctacagca gctgatatgg ccgcggtttg tgatatcgtt aaccattaca
ttgagacgtc 1980tacagtgaac tttaggacag agccacaaac accacaagag
tggattgatg atctagagag 2040gttgcaagat agataccctt ggttggttgc
tgaggttgag ggtgttgtgg ctggtattgc 2100ttacgctggg ccctggaagg
ctaggaacgc ttacgattgg acagttgaga gtactgttta 2160cgtgtcacat
aggcatcaaa ggttgggcct aggatccaca ttgtacacac atttgcttaa
2220gtctatggag gcgcaaggtt ttaagtctgt ggttgctgtt ataggccttc
caaacgatcc 2280atctgttagg ttgcatgagg ctttgggata cacagcccgg
ggtacattgc gcgcagctgg 2340atacaagcat ggtggatggc atgatgttgg
tttttggcaa agggattttg agttgccagc 2400tcctccaagg ccagttaggc
cagttaccca gatctgagtc gacggatccc cgacatatgc 2460cccggtttcg
ttgcgactaa catgagttct tggacaaatt tgattggacc tgatgagatg
2520atccaacccg aggatatagc aaagctcgtt cgtgcagcaa tggaacggcc
aaaccgtgct 2580tttgtcccca agaatgaggt gctatgcatg aaggaatcta
cccgttgatg tccaacagtc 2640tcagggttaa tgtctatgta tcttaaataa
tgttgtcggt attttgtaat ctcatataga 2700ttttcactgt gcgacgcaaa
aatattaaat aaatattatt attatctacg ttttgattga 2760gatatcatca
atattataat aaaaatatcc attaaacacg atttgataca aatgacagtc
2820aataatctga tttgaatatt tattaattgt aacgaattac ataaagatcg
aatagaaaat 2880actgcactgc aaatgaaaat taacacatac taataaatgc
gtcaaatatc tttgccaaga 2940tcaagcggag tgagggcctc atatccggtc
tcagttacaa gcacggtatc cccgaagcgc 3000gctccaccaa tgccctcgac
atagatgccg ggctcgacgc tgaggacatt gcctaccttg 3060agcatggtct
cagcgccggc tttaagctca atcccatccc aatctgaata tcctatcccg
3120cgcccagtcc ggtgtaagaa cgggtctgtc catccacctc tgttgggaat
tctgatcttg 3180gcgcgcatgc ggatcctcat tcctccatca gaagtaactc
cacgctatca acaatgaatg 3240ttccttccgt ttctccaatc tcaatccaaa
ccttgtcggt ttctggaaag tactctaact 3300ctttggtgac atagccggct
ggtaacggtg tgtagtcccc atagcctctg ttagattcgc 3360aaggattgtc
cctacgtcca tcggtgtaag ccttctcctc ataggctgat gcatagtcag
3420cgggtacaga agagttgctc tcataggctc catcgtatcc tcgattgcga
gaagtgtaag 3480taccctcata ctcctcttga gtcgcagtgt agtcattgca
agttacggtg ttgtttgggt 3540agacttcctc ctcgacgcag ttgctgaact
tcagctcgtc ggtgttgttc tcaatctcgt 3600gtatggtgac gcaaccttct
ccgtatcctt ctttgtacgc ggtaacacga agaatgtagc 3660cacgaccagg
acagacacga acttcttgtg aaacttctgc ttcccactca ggaacaacaa
3720ggacagagcg gtgattgttc tgttcttcta catctacgtg ccctttcaca
ttccagcagg 3780ataggccatt gttgaagtca ccattcttga tgacattcct
cgcatcatac aaggagaatg 3840cagtgaagat gcgcccttct aactcttcaa
agatagcagc attgacaccc ggaatcacgc 3900taagttcagg aaggtaagct
tcccgaatgc tatgaacgcg tttgtctgca gcatgaatca 3960tagctatgtt
ggtatcagct tggagcctat catactgaga gttcacaaac agagcgtcaa
4020cgctttcttt ggcttctttg tacacaatgt ttgtttccca ttccaacttc
tctctcttgt 4080ccctccactt cttctcagcc ctcttcactc tagcgagggc
ttctccaaca agtggtttct 4140cttctagaaa ctccagattg cctagcctgg
catggccatc ttgagtcttg atcttgaaga 4200tcacccacac accgaggtct
tcgttcaggt cggtacagcc aacgtctatg tccaaggaga 4260agtggtgtga
gtgatgggca cacttgccga tgggacttgg ggctgaaagt ggccagagtg
4320aacccgtccc aggcacattg actgtctcat gtttggcgtt gtatctgatg
aggtagatct 4380caaggtcttg actgtcctcg atgtaacctc tcaactggta
tcttgtgtag gctttgagtt 4440tcgattcatc tatcttctgg tacaggtatg
ttggatagca ctcatcaaag gtacccaaga 4500gcgtaacata gttctccttg
aacacatcat cacctccttg aatggtgatg tccgtacttc 4560ccctccatcc
acgatctagt tgcctgttga tcccgcgaaa gttgggatct tgaagcaagt
4620tccgctcatc actaagtcgc ttagcatgtt tgaccttctc ggacaactcc
ttcttctcat 4680ccaaacagaa ctcatcagag aggcactcaa caaggttgga
aacgcgatcg atgtgatagt 4740cagtcacatc tgtcttgagc ccaatctgat
tggacgaagt gaacagagca ttcaccgcct 4800tctgtgctct ttccaagtca
gactctgcct cgagcgttgc agtaacggga atgaattcga 4860agcggtcgat
tatcactccg gcggttccgg agaaatttct aacacctact atgttaccta
4920gggaagaggt
gaaggcattg gcactttcga agtaaccgaa atcgctagat tggagattat
4980ccaaggatgt agctgtcgct ggtactgtat tggaaaagat ggaggaatta
ccccaattga 5040cgttgaggtg aataggggta acagaggcat accttacacg
aacacgatat ctggtagatg 5100tcgatgggaa gtgaatgggc acttcaatat
accctctatt ctggatgttg ttgccggaag 5160aattcagcct aaccaagtcg
cctccagtga atcctggtcc tgaaatgaca gaaccattaa 5220agagaaagtt
ccccttgaca gctgggatct gagtaatgct atcggatgca attatgttgt
5280taaactcagc actacgatgt atccaagaga acatcggagc tctgatgata
ctaacgctgc 5340tattactaaa gcctgaacgg aacatggaca catggctaag
gcgatggcta aacccttgcc 5400taggtggaac gttgttgttc tgtggaggga
tctcatccaa gctatcaact gttccgctct 5460ttctgtagac agcggatggc
agatttgagg aggttccata ggcaaattct gtcccgtcaa 5520gcacagacaa
ttgttgattg ttgatgccga tgttgaaagg tctcctatat agagtgctgg
5580acaaggttct atacacgccc tgaccgagtt gagcaacaat acgttgttgt
ggagctgcat 5640tgcccatagt cccgtaaagt gggaaagtga attctggtcc
agagaaccca acgggtgatg 5700ccatgatctg atgccctgac cagtagtaat
aaccgcggtg cgcatcggtg tagatcgtga 5760tactgttcaa tatgtccatc
aggtgtggag acctgatgct tctctctatg ccctgagccg 5820agcctcgaaa
gctaccgtcg aagttctcga ggactgggtt tgtgtagatt tcccgggtca
5880attgtgacac agtacggatt gggtagcgcc tagagtcgta gttgggaaag
agagcgacaa 5940tgtctaggac agttagtgtc aactctcgcc tgaactggtt
gtacctgacc caatctctag 6000aatccggtcc ccagacacgt tcgagacccg
tgttgtacca gcgaacagca taatcggtat 6060agttgccaat aagcctagtc
agatcattat aacgactatt gatagttgcg gcatcaaagc 6120cccaccgttg
tccgaacacg gagacatcgc ggagcaccga caagtgcagg ttggcagcct
6180gcacgtacac ggataaaaga ggaacttggt aattctgaac ggcgaagagc
ggaattgcgg 6240tcgtcagcgc gctgttcatg tcattgaatt gaatgcgcat
ctcctctctt aaggcaggat 6300tggtcgggtc tgcttcccac tctcgaaaag
attctgcgta aatctggtaa aggttgctga 6360ggccttctaa ccttgagatg
gcttggttcc tagcgaattc ttctattctt tggttaatta 6420actgctctat
ctgtacaaga aaggcgtccc attgagaggg accaaagatt ccccaaatga
6480tatcgacaag tccaagcacg aatccagcac cgggcacgaa ctctgacaaa
aggaattggg 6540taagtgacaa cgagatgtcg ataggtgtgt aaccagtctc
aatccgttct ccacccagca 6600cctcaacctc agggttgctc aggcagttgt
aaggaatgca ctcgttgatg ttgggattgt 6660tgtccattgt tggatcctct
agagtcgacc tgcagaagta acaccaaaca acagggtgag 6720catcgacaaa
agaaacagta ccaagcaaat aaatagcgta tgaaggcagg gctaaaaaaa
6780tccacatata gctgctgcat atgccatcat ccaagtatat caagatcaaa
ataattataa 6840aacatacttg tttattataa tagataggta ctcaaggtta
gagcatatga atagatgctg 6900catatgccat catgtatatg catcagtaaa
acccacatca acatgtatac ctatcctaga 6960tcgatatttc catccatctt
aaactcgtaa ctatgaagat gtatgacaca cacatacagt 7020tccaaaatta
ataaatacac caggtagttt gaaacagtat tctactccga tctagaacga
7080atgaacgacc gcccaaccac accacatcat cacaaccaag cgaacaaaaa
gcatctctgt 7140atatgcatca gtaaaacccg catcaacatg tatacctatc
ctagatcgat atttccatcc 7200atcatcttca attcgtaact atgaatatgt
atggcacaca catacagatc caaaattaat 7260aaatccacca ggtagtttga
aacagaattc tactccgatc tagaacgacc gcccaaccag 7320accacatcat
cacaaccaag acaaaaaaaa gcatgaaaag atgacccgac aaacaagtgc
7380acggcatata ttgaaataaa ggaaaagggc aaaccaaacc ctatgcaacg
aaacaaaaaa 7440aatcatgaaa tcgatcccgt ctgcggaacg gctagagcca
tcccaggatt ccccaaagag 7500aaacactggc aagttagcaa tcagaacgtg
tctgacgtac aggtcgcatc cgtgtacgaa 7560cgctagcagc acggatctaa
cacaaacacg gatctaacac aaacatgaac agaagtagaa 7620ctaccgggcc
ctaaccatgg accggaacgc cgatctagag aaggtagaga gggggggggg
7680gggaggacga gcggcgtacc ttgaagcgga ggtgccgacg ggtggatttg
ggggagatct 7740ggttgtgtgt gtgtgcgctc cgaacaacac gaggttgggg
aaagagggtg tggagggggt 7800gtctatttat tacggcgggc gaggaaggga
aagcgaagga gcggtgggaa aggaatcccc 7860cgtagctgcc ggtgccgtga
gaggaggagg aggccgcctg ccgtgccggc tcacgtctgc 7920cgctccgcca
cgcaatttct ggatgccgac agcggagcaa gtccaacggt ggagcggaac
7980tctcgagagg ggtccagagg cagcgacaga gatgccgtgc cgtctgcttc
gcttggcccg 8040acgcgacgct gctggttcgc tggttggtgt ccgttagact
cgtcgacggc gtttaacagg 8100ctggcattat ctactcgaaa caagaaaaat
gtttccttag tttttttaat ttcttaaagg 8160gtatttgttt aatttttagt
cactttattt tattctattt tatatctaaa ttattaaata 8220aaaaaactaa
aatagagttt tagttttctt aatttagagg ctaaaataga ataaaataga
8280tgtactaaaa aaattagtct ataaaaacca ttaaccctaa accctaaatg
gatgtactaa 8340taaaatggat gaagtattat ataggtgaag ctatttgcaa
aaaaaaagga gaacacatgc 8400acactaaaaa gataaaactg tagagtcctg
ttgtcaaaat actcaattgt cctttagacc 8460atgtctaact gttcatttat
atgattctct aaaacactga tattattgta gtactataga 8520ttatattatt
cgtagagtaa agtttaaata tatgtataaa gatagataaa ctgcacttca
8580aacaagtgtg acaaaaaaaa tatgtggtaa ttttttataa cttcgacatg
caatgctcat 8640tatctctaga gaggggcacg accgggtcac gctgcactgc
aggcatgcgc gccttaatta 8700aggaattcct cgagtttaaa cggatccctg
aaagcgacgt tggatgttaa catctacaaa 8760ttgccttttc ttatcgacca
tgtacgtaag cgcttacgtt tttggtggac ccttgaggaa 8820actggtagct
gttgtgggcc tgtggtctca agatggatca ttaatttcca ccttcaccta
8880cgatgggggg catcgcaccg gtgagtaata ttgtacggct aagagcgaat
ttggcctgta 8940gacctcaatt gcgagctttc taatttcaaa ctattcgggc
ctaacttttg gtgtgatgat 9000gctgactgaa taattacacg gccttcttgt
gagacctcgc caatctgctc ggtgtaccct 9060agcatctcca tatcctcatg
tagaagattc aaggaagttg gccttgcatt cagtaccgca 9120agaccattga
acatgttgaa tagatttaca ggttcatcag ccacgctagt gcactcagat
9180ctggccttcc ggcagtaaga aggatcccag ttccgcctat gcaacattaa
aacaaaccta 9240ggatctccga cctgcagctg agaatatttg gctggtcggg
ggactttaaa actcacgata 9300tgaagatcac aaggtaaggg agtagttaat
ggagaaaagg gcccccagtg tgtctgcaca 9360ttaatgtctt gtggcttctt
ggcaatgaat gtgtgtaagg gatgattcaa gtgagcacca 9420acacaatgcg
agagaagaga tggattcaga ggctgtgggt cagaaacggg atttgaagtc
9480gaagaaatgt tggattcgat caagatatgg aaaacaacat tcataacacg
attgtccacc 9540acaccttgtc caagaccacg accatcgtct tttgccaaac
gacggtcaag cataatctct 9600agccacccct gtttacagct tgctgcaccc
agtgactgcc gagaatggac agaataccta 9660tggccattgg acccctgcat
gaaagcgaga gaaggcatgg ggtagtaatt cccctgcaac 9720ggaatcttat
gatatgtttc tctccgactc atctgaaagc cattcaagtc ggtgtagaaa
9780attcttttgt tgtcaatatc tgtcttatat ctaacaatta attctttgtc
aataaaactg 9840ttgccgagaa gctcaacatg atattccttc tcaatcagaa
actcctggat cgtatttcct 9900ccattataaa tacgagtact atgggagatc
ggagtattct cccatgcagt cttcggataa 9960gagtacacct cctgcatcaa
gggaccctca gagataacca gatgcccacc agattgactt 10020ataggcacag
cttcaccatg gggaacaaat aagtatgcac ctccggtact tgagtaaagg
10080cttatctctt cagccacggt attttgcaga ccatttttct ggattacctt
ttgtaacaaa 10140ccatgcttga caccaaaggt gagagtttga tgctgattct
caatttcaac cacatctcct 10200tctacttctg agcaatcata cagtgtaggg
cactgtattg aactcaaatt tgagaagagc 10260ttgagttttg ccggtttagc
tttttcacat ccgacaaagc cattggcaat ataatatgtc 10320tgcaaaccca
tggcaggaac agaagctt 10348421DNAArtificial SequenceForward primer
281-14 4tgtcggctga aggtagggag g 21520DNAArtificial SequenceReverse
primer 281-15 5ccggacatga agccatttac 206603DNAArtificial
Sequence603 bp sequence of the amplicon produced using the primers
of SEQ ID NO4 and SEQ ID NO5 6tgtcggctga aggtagggag gaattaatca
atcacagttg cttgtttctg agtctataga 60atcatgaatt ttaaatttat ggaatgcatt
ttttcgaaga tattgtatgc attaagtgta 120attttagttt caatatgaaa
tttgagattt atatatatac ttacataaaa ccctccttta 180ctgaattagt
gccatggata aaagaccaat taagcaatcc ttccaacacg tgcatgcact
240ggattttcat cgcctcgtcc attgttaaat tgataggtta ataagaacaa
ttagttggct 300actgattata tggattctgg gttaaaagta tttaggttta
ctgttacata catggaggat 360ctacatctat tttcactttt gtttaattaa
tttaagttag ttttgatgag tttaaggatt 420gtactagcca atagtagtac
ataaaggaga tagagtacca aaacaaagaa aaagccgaaa 480ggtgttaatg
ctaaattgta aaagaaagtt aaaataagag actcgaatta taatatgatt
540ctctggcgca ctaattaagc tactatatat tgtcaatagt attgtaaatg
gcttcatgtc 600cgg 603721DNAArtificial SequenceForward primer 281-9
7tctctagaga ggggcacgac c 21821DNAArtificial SequenceReverse primer
281-10 8cgagctggag agaccggtga c 219562DNAArtificial Sequence562 bp
sequence of the amplicon produced using the primers of SEQ ID NO7
and SEQ ID NO8 9tctctagaga ggggcacgac cgggtcacgc tgcactgcag
ccaagtggcc cccatttgga 60cgtgaatgta gacacgtcga aataaagatt tccgaattag
aataatttgc ttattgcttt 120cgcctataaa tacgacggat cgtaatttgt
cgctttatca gaatgtactt tcattttata 180ataacgctgc ggacatctac
atttttgaat tgaaaaaaaa ttggtaatta ctctttcttt 240ttctccatat
tgaccatcat actcattgct gatccatgta gatttccctt acttgtctcc
300ctctaatctg actttattaa cccaaagcaa ttgcttattt gttccccacg
cccacaaagc 360ccagcattgt ccctaaggta ttaatttgtt gttcgattct
tgttcttgaa cccatttgga 420gaatgcaaca agggttttca tgtcagcacg
gtaatggttc tgtgtaaatt ccagtagtgc 480tgcccaagta aagtctgggt
atttcctcga atttgcggca ttaactaagc tagctgctgg 540tgtcaccggt
ctctccagct cg 5621025DNAArtificial SequenceForward primer 3006-20
10ttccaacctt taactattat cctgc 251120DNAArtificial SequenceReverse
primer 3006-22 11gctgcggaca tctacatttt 2012614DNAArtificial
Sequence614 bp sequence of the amplicon produced using the primers
of SEQ ID NO10 and SEQ ID NO11 12ttccaacctt taactattat cctgccttaa
aattcgaata catttattat ctataaacta 60tccgaatatt attatctaaa tcctaattaa
atactatttt ttatcgagta ttcgtatccg 120ccaaggaaat ccatctccaa
attttcaatt atttttcaga tatctaaatc tgtaaaattt 180caaattcaag
tacgttacaa ttctttataa ataatccaaa ttataaatat tttataacta
240ttaattcata aattaaaatt tattattcaa atattcgaat aatctatttt
taagacgtaa 300agtattacat cgaagggtta ctttcaaagg gtagtgtatt
tccatttcaa ttattcagaa 360cgttgtcgtt ttgttccggt catagaaaag
ggctctggaa gagaagaaaa tgacttgact 420tttcaatttc atgctcatcc
actcgtttca attactgttt actaaaaaaa taataaaata 480aaatattaac
aatgcattga gtatgatgtc cgggaaatct acatggatca gcaatgagta
540tgatggtcaa tatggagaaa aagaaagagt aattaccaat tttttttcaa
ttcaaaaatg 600tagatgtccg cagc 6141324DNAArtificial SequenceForward
primer 3006-9 13gacatgcaat gctcattatc tcta 241422DNAArtificial
SequenceReverse primer 3600-12 14aagtctctgc cttctaccct gg
2215662DNAArtificial Sequence662 bp sequence of the amplicon
produced using the primers of SEQ ID NO13 and SEQ ID NO14
15gacatgcaat gctcattatc tctagagagg ggcacgaccg ggtcacgctg cactgcaggc
60atgcgcgcct taattaagga attcctcgag tttaaacgga tccctgaaag cgacgttgga
120tgttaacatc tacaaattgc cttttcttat cgaccatgta cgtaagcgct
tacgtttttg 180gtggaccctt gaggaaactg gtagctgttg tgggcctgtg
gtctcaagat ggatcattaa 240tttccacctt cacctacgat ggggggcatc
gcaccggtga gtaatattgt acggctaaga 300gcgaatttgg cctgtagacc
tcaattgcga gctttctaat ttcaaactat tcgggcctaa 360cttttggtgt
gatgatgctg actggcttac gtgtggaaaa aatttgcaat ctatgtagtc
420tttaactaat gtttttttct ttaaaaaaaa agtcattatt tttggtttga
ttaatatatt 480tggtttaaat taaataaaat attaaaaagt ttagttaaat
catctattta aacgatttgt 540actgatttgt gatctattaa ttttttaact
taatctagac cagggtacta gttggtccga 600tcccatcttg aaaacactat
ctttagcttg ctggtaggtt ccagggtaga aggcagagac 660tt
6621620DNAArtificial SequenceForward primer 3006-23 16gaatcaatct
tttacgggaa 201720DNAArtificial SequenceReverse primer 3006-25
17catgtagatt tcccggacat 2018660DNAArtificial Sequence660 bp
sequence of the amplicon produced using the primers of SEQ ID NO16
and SEQ ID NO17 18gaatcaatct tttacgggaa aactcaattg gaatgaaact
tgtttccagt aaaagtatta 60aaaaatgtat aaactagtcc atgtatgata gtgtaaagag
tatattggag ttaaaaattt 120catcccatgt tactctatat tacattataa
aggaagctat aggttggctc actgcaccac 180ccaatcaaga tactcatatg
tattacttaa tatataatat taaaagatat catttctttt 240tgttatcaga
ttaaagagaa ctaagatatt acaaacttgc aaaacaatag ttaggccaac
300ccatatttta aatattttct tcttttttct actcaaacct attttgtttc
caaatattga 360agagttctgt tctgattgga tatactctcg gacaagttta
actctaaatt ttccaaatat 420tgaaatatgg atttgctcac atggaaaaac
ggttcttgaa caataaatca aaatagcata 480ttctatcaat ggaatcaagg
aattccatat tcatttcaaa gttggagcca agcatgcatg 540ataatgattt
atagcaattt tgtcaaaagg aaaaaataag gtgattggat gacaagaaac
600aaagggaaaa tgaattaaca gcctgcattt aatatcattc atgtccggga
aatctacatg 6601924DNAArtificial SequenceForward primer 3006-9
19gacatgcaat gctcattatc tcta 242024DNAArtificial SequenceReverse
primer 3006-11C 20gaacctgtaa atctattcaa catg 2421531DNAArtificial
Sequence531 bp sequence of the amplicon produced using the primers
of SEQ ID NO19 and SEQ ID NO20 21gacatgcaat gctcattatc tctagagagg
ggcacgaccg ggtcacgctg cactgcaggc 60atgcgcgcct taattaagga attcctcgag
tttaaacgga tccctgaaag cgacgttgga 120tgttaacatc tacaaattgc
cttttcttat cgaccatgta cgtaagcgct tacgtttttg 180gtggaccctt
gaggaaactg gtagctgttg tgggcctgtg gtctcaagat ggatcattaa
240tttccacctt cacctacgat ggggggcatc gcaccggtga gtaatattgt
acggctaaga 300gcgaatttgg cctgtagacc tcaattgcga gctttctaat
ttcaaactat tcgggcctaa 360cttttggtgt gatgatgctg actgaataat
tacacggcct tcttgtgaga cctcgccaat 420ctgctcggtg taccctagca
tctccatatc ctcatgtaga agattcaagg aagttggcct 480tgcattcagt
accgcaagac cattgaacat gttgaataga tttacaggtt c 531
* * * * *