U.S. patent application number 14/373442 was filed with the patent office on 2015-03-26 for down-regulation of gene expression using artificial micrornas for silencing fatty acid biosynthetic genes.
The applicant listed for this patent is E I DU PONT NEMOURS AND COMPANY. Invention is credited to Howard Glenn Damude, Brian McGonigle.
Application Number | 20150089689 14/373442 |
Document ID | / |
Family ID | 47710321 |
Filed Date | 2015-03-26 |
United States Patent
Application |
20150089689 |
Kind Code |
A1 |
Damude; Howard Glenn ; et
al. |
March 26, 2015 |
DOWN-REGULATION OF GENE EXPRESSION USING ARTIFICIAL MICRORNAS FOR
SILENCING FATTY ACID BIOSYNTHETIC GENES
Abstract
Isolated nucleic acid fragments comprising precursor miRNAs, and
artificial miRNAs and their use in down-regulating gene expression
of fatty acid biosynthetic genes are described.
Inventors: |
Damude; Howard Glenn;
(Hockessin, DE) ; McGonigle; Brian; (Wilmington,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
E I DU PONT NEMOURS AND COMPANY |
Wilmington |
DE |
US |
|
|
Family ID: |
47710321 |
Appl. No.: |
14/373442 |
Filed: |
January 23, 2013 |
PCT Filed: |
January 23, 2013 |
PCT NO: |
PCT/US13/22654 |
371 Date: |
July 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61589682 |
Jan 23, 2012 |
|
|
|
Current U.S.
Class: |
800/298 ;
435/320.1; 435/419; 435/468 |
Current CPC
Class: |
C12N 15/1137 20130101;
C12N 15/8247 20130101; C12N 15/8218 20130101 |
Class at
Publication: |
800/298 ;
435/320.1; 435/419; 435/468 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. An isolated nucleic acid sequence comprising a sequence encoding
at least one artificial microRNA precursor, operably linked to at
least one regulatory sequence, wherein said sequence encoding an
artificial microRNA precursor is transcribed so that a transcript
comprising said artificial miRNA precursor is produced, and wherein
said transcript is processed so that a mature miRNA about 21 to 22
nts in length is excised from said artificial miRNA precursor, and
expression of at least one plant fatty acid biosynthetic gene(s)
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited.
2. The isolated nucleic acid sequence of claim 1, wherein said
artificial microRNA precursor is at least one selected form the
group consisting of SEQ ID NO: 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, 156, 158,
160, 162, 164, 166, and 168.
3. A recombinant construct comprising the isolated nucleic acid
sequence of claim 1 or 2 operably linked to at least one regulatory
sequence.
4. A plant cell comprising the recombinant construct of claim
2.
5. The plant cell of claim 3 wherein the plant cell is a dicot
plant cell.
6. A method for reducing expression at least one plant fatty acid
biosynthetic gene, said method comprising: (a) transforming a plant
cell with at least one isolated nucleic acid sequence comprising a
sequence encoding at least one artificial microRNA precursor,
operably linked to at least one regulatory sequence; (b) said at
least one sequence encoding an artificial microRNA precursor is
transcribed so that a transcript comprising said at least one
artificial miRNA precursor is produced; (c) said transcript is
processed so that at least one mature miRNA about 21 to 22 nts in
length is excised from said artificial miRNA precursor, and (d)
expression of at least one plant fatty acid biosynthetic gene
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is reduced.
7. The method of claim 6 wherein the plant cell is a dicot plant
cell.
8. An isolated nucleic acid sequence comprising a sequence encoding
two or more artificial microRNA precursors, operably linked to at
least one regulatory sequence, wherein said two or more artificial
microRNA precursor can be on the same or separate transcriptional
units, further wherein said sequence encoding artificial microRNA
precursors is transcribed so that a transcript comprising said
artificial miRNA precursors is produced, and wherein said
transcript is processed so that mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
expression of two or more plant fatty acid biosynthetic gene(s)
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited.
9. A recombinant construct comprising the isolated nucleic acid
sequence of claim 8 operably linked to at least one regulatory
sequence.
10. A plant cell comprising the recombinant construct of claim
8.
11. The plant cell of claim 9 wherein the plant cell is a dicot
plant cell.
12. A method for reducing expression two or more plant fatty acid
biosynthetic gene, said method comprising: (a) transforming a plant
cell with at least one isolated nucleic acid sequence comprising a
sequence encoding two or more artificial microRNA precursors,
wherein said two or more artificial microRNA precursors can be on
the same or separate transcriptional units, operably linked to at
least one regulatory sequence; (b) said at least one sequence
encoding two or more artificial microRNA precursors is transcribed
so that at least one transcript comprising said two or more
artificial miRNA precursors is produced; (c) said at least one
transcript is processed so that at least two or more mature miRNAs
about 21 to 22 nts in length are excised from said artificial miRNA
precursors, and (d) expression of two or more plant fatty acid
biosynthetic genes selected from the group consisting of: fad2-1,
fad2-2, fad3, fatB, sad, and fael are reduced.
13. The method of claim 6 wherein the plant cell is a dicot plant
cell.
14. An isolated nucleic acid sequence comprising a sequence
encoding at least one artificial microRNA precursor, operably
linked to at least one regulatory sequence, wherein said sequence
encoding at least one artificial microRNA precursor is capable of
forming a double-stranded RNA or hairpin, wherein the at least one
amiRNA precursor comprises at least one modified miRNA precursor in
which the miRNA sequence and its complementary sequence are
replaced by at least one amiRNA sequence and at least one STAR
sequence, wherein expression of at least one plant fatty acid
biosynthetic gene(s) selected from the group consisting of: fad2-1,
fad2-2, fad3, fatB, sad, and fael is inhibited.
15. An isolated nucleic acid sequence comprising a sequence
encoding two or more artificial microRNA precursors, operably
linked to at least one regulatory sequence, wherein said two or
more artificial microRNA precursor can be on the same or separate
transcriptional units, further wherein said sequences encoding two
or more artificial microRNA precursors are capable of forming a
double-stranded RNA or hairpin, wherein the two or more amiRNA
precursors comprise a modified miRNA precursor(s) in which the
miRNA sequence(s) and its complementary sequence(s) are replaced by
two or more amiRNA sequences and two or more STAR sequences, and
further wherein expression of at least two or more plant fatty acid
biosynthetic genes selected from the group consisting of: fad2-1,
fad2-2, fad3, fatB, sad, and fael, are inhibited.
16. The isolated nucleic acid sequence of claim 14 or 15, wherein
amiRNA sequence(s) comprise(s) at least one selected form the group
consisting of: SEQ ID NO: 21, 22, 23, 24, 25, 80, 83, 86, 89, 92,
95, 98, and 101.
17. A transgenic plant or seed comprising the nucleic acid sequence
of claim 1, 14 or 15.
18. The method according to claim 6 or 12, wherein the amiRNA
sequence(s) comprise(s) at least one selected form the group
consisting of: SEQ ID NO: 21, 22, 23, 24, 25, 80, 83, 86, 89, 92,
95, 98, and 101.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/589,682, filed Jan. 23, 2012 which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The field of the present invention relates, generally, to
plant molecular biology. In particular, it relates to constructs
and methods to down-regulate expression of fatty acid biosynthetic
genes.
BACKGROUND
[0003] MicroRNAs (miRNAs) were first identified only a few years
ago, but already it is clear that they play an important role in
regulating gene activity. These 20-22 nucleotide noncoding RNAs
have the ability to hybridize via base-pairing with specific target
mRNAs and downregulate the expression of these transcripts, by
mediating either RNA cleavage or translational repression.
[0004] Recent studies have indicated that miRNAs have important
functions during development. In plants, they have been shown to
control a variety of developmental processes including flowering
time, leaf morphology, organ polarity, floral morphology, and root
development (reviewed by Mallory and Vaucheret (2006) Nat Genet 38:
S31-36). Given the established regulatory role of miRNAs, it is
likely that they are also involved in the control of some of the
major crop traits such drought tolerance and disease
resistance.
[0005] miRNAs are transcribed by RNA polymerase II as
polyadenylated and capped messages known as pri-miRNAs. These
pri-miRNAs are processed into smaller transcripts known as
pre-miRNAs and these precursors have the ability to form stable
hairpin structures (reviewed by Bartel (2004) Cell 116: 281-297;
Jones-Rhoades M W, Bartel D P, Bartel B. MicroRNAS and their
regulatory roles in plants. Annu Rev Plant Biol. 2006; 57:19-53.)
While pri-miRNAs are processed to pre-miRNAs by Drosha in the
nucleus and Dicer cleaves pre-miRNAs in the cytoplasm in metazoans,
miRNA maturation in plants differs from the pathway in animals
because plants lack a Drosha homolog. Instead, the RNase III enzyme
DICER-LIKE 1 (DCL1), which is homologous to animal Dicer, may
possess Drosha function in addition to its known function in
hairpin processing (Kurihara and Watanabe (2004) Proc Natl Acad Sci
101: 12753-12758).
[0006] Artificial microRNAs (amiRNAs) have recently been described
in Arabidopsis targeting viral mRNA sequences (Niu et al. (2006)
Nature Biotechnology 24:1420-1428) or endogenous genes (Schwab et
al. (2006) Plant Cell 18:1121-1133). The amiRNA construct can be
expressed under different promoters in order to change the spatial
pattern of silencing (Schwab et al. (2006) Plant Cell
18:1121-1133). Artificial miRNAs replace the microRNA and its
complementary star sequence in a precursor miRNA and substitute
sequences that target an mRNA to be silenced. Silencing by
endogenous miRNAs can be found in a variety of spatial, temporal,
and developmental expression patterns (Parizotto et al. (2007)
Genes Dev 18:2237-2242; Alvarez et al. (2006) Plant Cell
18:1134-51). Artificial miRNA can be constructed to both capture
and extend the diversity and specificity in the patterns of
silencing. Previously, solutions for down-regulating specific fatty
acid biosynthetic genes have been to use classic RNAi approaches,
such as co-suppression or hairpin structures. These approaches
included poor frequency of silencing (particularly with
co-suppression strategies) and non-specific silencing of other
similar genes. amiRNA technology can be designed to be very
specific to the gene of interest and silencing frequencies are on
par with RNAi (hairpin) structures.
[0007] WO 2004/009779 published Jan. 29, 2004 describes
compositions and methods for modulating gene expression in
plants.
[0008] Applicant's Assignee's US Patent Application Publication
2005/0138689 published on Jun. 23, 2005 describes miRNas and their
use in silencing a target sequence.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0009] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing, which form a part of this application.
[0010] The sequence descriptions summarize the Sequences Listing
attached hereto. The Sequence Listing contains one letter codes for
nucleotide sequence characters and the single and three letter
codes for amino acids as defined in the IUPAC-IUB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219(2):345-373 (1984).
[0011] SEQ ID NO:1 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma10g42470 targeted for
silencing.
[0012] SEQ ID NO:2 corresponds to the amino acid sequence encoded
by SEQ ID NO:1.
[0013] SEQ ID NO:3 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma20g24530 targeted for
silencing.
[0014] SEQ ID NO:4 corresponds to the amino acid sequence encoded
by SEQ ID NO:3.
[0015] SEQ ID NO:5 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma19g32940 targeted for
silencing.
[0016] SEQ ID NO:6 corresponds to the amino acid sequence encoded
by SEQ ID NO:5.
[0017] SEQ ID NO:7 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma02g15600 targeted for
silencing.
[0018] SEQ ID NO:8 corresponds to the amino acid sequence encoded
by SEQ ID NO:7.
[0019] SEQ ID NO:9 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma07g32850 targeted for
silencing.
[0020] SEQ ID NO:10 corresponds to the amino acid sequence encoded
by SEQ ID NO:9.
[0021] SEQ ID NO: 11 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma14g37350 targeted for
silencing.
[0022] SEQ ID NO:12 corresponds to the amino acid sequence encoded
by SEQ ID NO:11.
[0023] SEQ ID NO:13 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma02g39230 targeted for
silencing.
[0024] SEQ ID NO:14 corresponds to the amino acid sequence encoded
by SEQ ID NO:13.
[0025] SEQ ID NO:15 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma18g06950 targeted for
silencing.
[0026] SEQ ID NO:16 corresponds to the amino acid sequence encoded
by SEQ ID NO:15.
[0027] SEQ ID NO:17 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma05g08060 targeted for
silencing.
[0028] SEQ ID NO:18 corresponds to the amino acid sequence encoded
by SEQ ID NO:17.
[0029] SEQ ID NO:19 corresponds to the nucleotide sequence of soy
fatty acid biosynthetic gene Glyma17g12940 targeted for
silencing.
[0030] SEQ ID NO:20 corresponds to the amino acid sequence encoded
by SEQ ID NO:19.
[0031] SEQ ID NOs:21-25 corresponds to the artificial miRNA
(amiRNA) sequence used to silence the soybean fad 2-1b, fad2-2,
sad3, fad3, and fatB, respectively.
[0032] SEQ ID NOs:26-31 correspond to "star sequences" contained
within amiRNA precursors for 159fad2-1b, 396b-fad2-1b, 159-fad2-2,
396b-sad3, 159-fad3c, 159-fatbf, respectively. Star sequences are
the largely complementary sequences within the miRNA precursor that
form a duplex with the miRNA.
[0033] SEQ ID NO:32 corresponds to the nucleotide sequence of
vector PHP32511.
[0034] SEQ ID NO:33 corresponds to the nucleotide sequence of
vector PHP32510.
[0035] SEQ ID NO:34 corresponds to the nucleotide sequence of
vector PHP32843.
[0036] SEQ ID NO:35 corresponds to the nucleotide sequence of
vector PHP33705.
[0037] SEQ ID NO:36 corresponds to the nucleotide sequence of
vector PHP38557.
[0038] SEQ ID NO:37 corresponds to the nucleotide sequence of
vector PHP41103.
[0039] SEQ ID NO:38 corresponds to the nucleotide sequence of
vector pKR1756.
[0040] SEQ ID NO:39 corresponds to the nucleotide sequence of
vector pKR1757.
[0041] SEQ ID NO:40 corresponds to the nucleotide sequence of
vector pKR1766.
[0042] SEQ ID NO:41 corresponds to the nucleotide sequence of
vector pKR1771.
[0043] SEQ ID NO:42 corresponds to the nucleotide sequence of
vector PHP41784.
[0044] SEQ ID NO:43 corresponds to the nucleotide sequence to the
nucleotide sequence of vector pKR1776.
[0045] SEQ ID NO:44 corresponds to the nucleotide sequence of
Arabidopsis fatty acid biosynthetic gene fad2, At3g12120.
[0046] SEQ ID NO:45 corresponds to the amino acid sequence encoded
by SEQ ID NO:44.
[0047] SEQ ID NO:46 corresponds to the nucleotide sequence of
Arabidopsis fatty acid biosynthetic gene fad3, At2g29980.
[0048] SEQ ID NO:47 corresponds to the amino acid sequence encoded
by SEQ ID NO:46.
[0049] SEQ ID NO:48 corresponds to the nucleotide sequence of
Arabidopsis fatty acid biosynthetic gene fael, At4g34520.
[0050] SEQ ID NO:49 corresponds to the amino acid sequence encoded
by SEQ ID NO:48.
[0051] SEQ ID NO:50 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene FJ907397.
[0052] SEQ ID NO:51 corresponds to the amino acid sequence encoded
by SEQ ID NO:50.
[0053] SEQ ID NO:52 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene FJ907398.
[0054] SEQ ID NO:53 corresponds to the amino acid sequence encoded
by SEQ ID NO:52.
[0055] SEQ ID NO:54 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene FJ907399.
[0056] SEQ ID NO:55 corresponds to the amino acid sequence encoded
by SEQ ID NO:54.
[0057] SEQ ID NO:56 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene FJ907400.
[0058] SEQ ID NO:57 corresponds to the amino acid sequence encoded
by SEQ ID NO:56.
[0059] SEQ ID NO:58 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene FJ907401.
[0060] SEQ ID NO:59 corresponds to the amino acid sequence encoded
by SEQ ID NO:58.
[0061] SEQ ID NO:60 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene L01418.
[0062] SEQ ID NO:61 corresponds to the amino acid sequence encoded
by SEQ ID NO:60.
[0063] SEQ ID NO:62 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AY599884.
[0064] SEQ ID NO:63 corresponds to the amino acid sequence encoded
by SEQ ID NO:62.
[0065] SEQ ID NO:64 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene L22962.
[0066] SEQ ID NO:65 corresponds to the amino acid sequence encoded
by SEQ ID NO:64.
[0067] SEQ ID NO:66 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AF274750.
[0068] SEQ ID NO:67 corresponds to the amino acid sequence encoded
by SEQ ID NO:66.
[0069] SEQ ID NO:68 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AY888043.
[0070] SEQ ID NO:69 corresponds to the amino acid sequence encoded
by SEQ ID NO:68.
[0071] SEQ ID NO:70 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AF009563.
[0072] SEQ ID NO:71 corresponds to the amino acid sequence encoded
by SEQ ID NO:70.
[0073] SEQ ID NO:72 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene GU325719.
[0074] SEQ ID NO:73 corresponds to the amino acid sequence encoded
by SEQ ID NO:72.
[0075] SEQ ID NO:74 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AF490462.
[0076] SEQ ID NO:75 corresponds to the amino acid sequence encoded
by SEQ ID NO:74.
[0077] SEQ ID NO:76 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene AF490459.
[0078] SEQ ID NO:77 corresponds to the amino acid sequence encoded
by SEQ ID NO:76.
[0079] SEQ ID NO:78 corresponds to the nucleotide sequence of
Brassica napus fatty acid biosynthetic gene BNU50771.
[0080] SEQ ID NO:79 corresponds to the amino acid sequence encoded
by SEQ ID NO:78.
[0081] SEQ ID NO:80 corresponds to the fad2a amiRNA.
[0082] SEQ ID NO:81 corresponds to the fad2a 159 Star sequence.
[0083] SEQ ID NO:82 corresponds to the fad2a 396b Star
sequence.
[0084] SEQ ID NO:83 corresponds to the fad2b 159 amiRNA.
[0085] SEQ ID NO:84 corresponds to the fad2b 159 Star sequence.
[0086] SEQ ID NO:85 corresponds to the fad2b 396b Star
sequence.
[0087] SEQ ID NO:86 corresponds to the fad2c amiRNA.
[0088] SEQ ID NO:87 corresponds to the fad2c 159 Star sequence.
[0089] SEQ ID NO:88 corresponds to the fad2c 396b Star
sequence.
[0090] SEQ ID NO:89 corresponds to the fad3a amiRNA.
[0091] SEQ ID NO:90 corresponds to the fad3a 159 Star sequence.
[0092] SEQ ID NO:91 corresponds to the fad3a 396b Star
sequence.
[0093] SEQ ID NO:92 corresponds to the fad3b amiRNA.
[0094] SEQ ID NO:93 corresponds to the fad3b 159 Star sequence.
[0095] SEQ ID NO:94 corresponds to the fad3b 396b Star
sequence.
[0096] SEQ ID NO:95 corresponds to the faela amiRNA.
[0097] SEQ ID NO:96 corresponds to the faela 159 Star sequence.
[0098] SEQ ID NO:97 corresponds to the faela 396b Star
sequence.
[0099] SEQ ID NO:98 corresponds tot the faelb amiRNA.
[0100] SEQ ID NO:99 corresponds to the faelb 159 Star sequence.
[0101] SEQ ID NO:100 corresponds to the faelb 396b Star
sequence.
[0102] SEQ ID NO:101 corresponds to the faelc amiRNA.
[0103] SEQ ID NO:102 corresponds to the faelc 159 Star
sequence.
[0104] SEQ ID NO:103 corresponds to the faelc 396b Star
sequence.
[0105] SEQ ID NO:104 corresponds to the nucleotide sequence of
plasmid GM-159-KS332.
[0106] SEQ ID NO:105 corresponds to the nucleotide sequence of
plasmid GM-396b-KS332.
[0107] SEQ ID NO:106 corresponds to the nucleotide sequence of
plasmid pKR2007.
[0108] SEQ ID NO:107 corresponds to the nucleotide sequence of
plasmid pKR2009.
[0109] SEQ ID NO:108 corresponds to the nucleotide sequence of
plasmid pKR2032. SEQ ID NO:109 corresponds to the nucleotide
sequence of plasmid pKR2033.
[0110] SEQ ID NO: 110 corresponds to the nucleotide sequence of
plasmid pKR2034.
[0111] SEQ ID NO: 111 corresponds to the nucleotide sequence of
plasmid pKR2037
[0112] SEQ ID NO: 112 corresponds to the nucleotide sequence of
plasmid pKR2038.
[0113] SEQ ID NO:113 corresponds to the nucleotide sequence of
plasmid pKR2039.
[0114] SEQ ID NO:114 corresponds to the nucleotide sequence of
plasmid pKR2035.
[0115] SEQ ID NO:115 corresponds to the nucleotide sequence of
plasmid pKR2036.
[0116] SEQ ID NO:116 corresponds to the nucleotide sequence of
plasmid pKR2040.
[0117] SEQ ID NO:117 corresponds to the nucleotide sequence of
plasmid pKR2041.
[0118] SEQ ID NO:118 corresponds to the nucleotide sequence of
plasmid pKR2076.
[0119] SEQ ID NO:119 corresponds to the nucleotide sequence of
plasmid pKR2077.
[0120] SEQ ID NO:120 corresponds to the nucleotide sequence of
plasmid pKR2078.
[0121] SEQ ID NO:121 corresponds to the nucleotide sequence of
plasmid pKR2079.
[0122] SEQ ID NO:122 corresponds to the nucleotide sequence of
plasmid pKR2081.
[0123] SEQ ID NO:123 corresponds to the nucleotide sequence of
plasmid pKR2080.
[0124] SEQ ID NO:124 corresponds to the nucleotide sequence of
amiRNA precursor 159-fad2-1b.
[0125] SEQ ID NO:125--corresponds to the nucleotide sequence of
amiRNA precursor 396b-fad2-1b.
[0126] SEQ ID NO:126--corresponds to the nucleotide sequence of
amiRNA precursor 159-fad2-2.
[0127] SEQ ID NO:127--corresponds to the nucleotide sequence of
amiRNA precursor 396b-fad2-1b & 159-fad2-2.
[0128] SEQ ID NO:128--corresponds to the nucleotide sequence of
amiRNA precursor 396b-sad3.
[0129] SEQ ID NO:129--corresponds to the nucleotide sequence of
amiRNA precursor 396b-fad2-1b/396b-sad3.
[0130] SEQ ID NO:130--corresponds to the nucleotide sequence of
amiRNA precursor 159-fad3c.
[0131] SEQ ID NO:131--corresponds to the nucleotide sequence of
amiRNA precursor 159-fatBF.
[0132] SEQ ID NO:132--corresponds to the nucleotide sequence of
amiRNA precursor 159-fad3c/159-fatBF.
[0133] SEQ ID NO:133--corresponds to the nucleotide sequence of
amiRNA precursor 159-fatBF/159-fad3c.
[0134] SEQ ID NO:134--corresponds to the nucleotide sequence of
amiRNA precursor 159-fad2-1b/159-fatBF/159-fad3c.
[0135] SEQ ID NO:135--corresponds to the nucleotide sequence of
amiRNA precursor 396b-fad2-1b/159-fatB/159-fatB/159-fad3c.
[0136] SEQ ID NO:136--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfad2a.
[0137] SEQ ID NO:137--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfad2b.
[0138] SEQ ID NO:138 corresponds to the nucleotide sequence of
corresponds to the nucleotide sequence of amiRNA precursor
159-Atfad2c.
[0139] SEQ ID NO:139--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfad2a.
[0140] SEQ ID NO:140--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfad2b.
[0141] SEQ ID NO:141--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfad2c.
[0142] SEQ ID NO:142--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfad3a.
[0143] SEQ ID NO:143--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfad3b
[0144] SEQ ID NO:144--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfad3a.
[0145] SEQ ID NO:145--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfad3b.
[0146] SEQ ID NO:146--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfaela.
[0147] SEQ ID NO:147--corresponds to the nucleotide sequence of
amiRNA precursor 59-Atfaelb.
[0148] SEQ ID NO:148--corresponds to the nucleotide sequence of
amiRNA precursor 159-Atfaelc.
[0149] SEQ ID NO:149--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfaela.
[0150] SEQ ID NO:150--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfaelb.
[0151] SEQ ID NO:151--corresponds to the nucleotide sequence of
amiRNA precursor 396b-Atfaelc.
[0152] SEQ ID NO:152--corresponds to the nucleotide sequence of soy
genomic miRNA precursor 159.
[0153] SEQ ID NO:153--corresponds to the nucleotide sequence of soy
genomic miRNA precursor 396b.
[0154] SEQ ID NO:154--corresponds to the nucleotide sequence of the
159-fad2a/396b-fad3b amiRNA.
[0155] SEQ ID NO:155--corresponds to the nucleotide sequence of
plasmid pKR2232.
[0156] SEQ ID NO:156--corresponds to the nucleotide sequence of the
159-fad2b/396b-fad3b amiRNA.
[0157] SEQ ID NO:157--corresponds to the nucleotide sequence of
plasmid pKR2233
[0158] SEQ ID NO:158--corresponds to the nucleotide sequence of the
396b-fad3b/159-fad2a amiRNA.
[0159] SEQ ID NO:159--corresponds to the nucleotide sequence of
plasmid pKR2234.
[0160] SEQ ID NO:160--corresponds to the nucleotide sequence of the
396b-fad3b/159-fad2b amiRNA
[0161] SEQ ID NO:161--corresponds to the nucleotide sequence of
plasmid pKR2235.
[0162] SEQ ID NO:162--corresponds to the nucleotide sequence of the
159-fad2a/396b-fad3b/159-fae1a amiRNA.
[0163] SEQ ID NO:163--corresponds to the nucleotide sequence of
plasmid pKR2248.
[0164] SEQ ID NO:164--corresponds to the nucleotide sequence of the
396b-fad3b/159-fad2a/159-fae1a amiRNA.
[0165] SEQ ID NO:165--corresponds to the nucleotide sequence of
plasmid 396b-fad3b/159-fad2a/159-fae1a amiRNA.
[0166] SEQ ID NO:166--corresponds to the nucleotide sequence of the
159-fad2b/396b-fad3b/159-fae1a amiRNA.
[0167] SEQ ID NO:167--corresponds to the nucleotide sequence of
plasmid pKR2250.
[0168] SEQ ID NO:168--corresponds to the nucleotide sequence of
396b-fad3b/159-fad2b/159-fae1a amiRNA.
[0169] SEQ ID NO:169--corresponds to plasmid pKR2251.
[0170] SEQ ID NO:170--corresponds to plasmid pKR2333.
[0171] SEQ ID NO:171--corresponds to plasmid pKR2334.
[0172] SEQ ID NO:172--corresponds to plasmid pKR2335.
[0173] SEQ ID NO:173--corresponds to plasmid pKR2336.
[0174] SEQ ID NO:174--corresponds to the fad 2 nucleotide sequence
of Brassica carinata.
[0175] SEQ ID NO:175--corresponds to the amino acid sequence
encoded by SEQ ID NO:174.
SUMMARY OF THE INVENTION
[0176] The present invention concerns an isolated nucleic acid
fragment comprising a sequence encoding an artificial microRNA
precursor, operably linked to at least one regulatory sequence,
wherein said sequence encoding an artificial microRNA precursor is
transcribed so that a transcript comprising said artificial miRNA
precursor is produced, and wherein said transcript is processed so
that a mature miRNA about 21 to 22 nts in length is excised from
said artificial miRNA precursor, and expression of a plant fatty
acid biosynthetic gene selected from the group consisting of:
fad2-1, fad2-2, fad3, fatB, sad, and fael is inhibited. The plant
fatty acid biosynthetic genes include without limitation any of the
nucleotide sequences of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, and 174, which encode amino acid sequences of SEQ ID NOs:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and 175.
[0177] Another embodiment of the present invention concerns an
isolated nucleic acid fragment comprising a sequence encoding an
artificial microRNA precursor, wherein said artificial microRNA
precursor is at least one selected form the group consisting of SEQ
ID NO: 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, 156, 158, 160, 162, 164, 166, and
168.
[0178] An additional embodiment of the present invention concerns
recombinant constructs comprising the isolated nucleic acid
sequence of the invention operably linked to at least one
regulatory sequence.
[0179] Additional embodiments of the present inventions comprise an
isolated nucleic acid sequence comprising a sequence encoding two
or more artificial microRNA precursors, operably linked to at least
one regulatory sequence, wherein said two or more artificial
microRNA precursor can be on the same or separate transcriptional
units, further wherein said sequence encoding artificial microRNA
precursors is transcribed so that a transcript comprising said
artificial miRNA precursors is produced, and wherein said
transcript is processed so that mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
expression of two or more plant fatty acid biosynthetic gene(s)
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited.
[0180] Another embodiment of the instant invention includes a
method for reducing expression two or more plant fatty acid
biosynthetic gene, said method comprising: [0181] (a) transforming
a plant cell with at least one isolated nucleic acid sequence
comprising a sequence encoding two or more artificial microRNA
precursors, wherein said two or more artificial microRNA precursors
can be on the same or separate transcriptional units, operably
linked to at least one regulatory sequence; [0182] (b) said at
least one sequence encoding two or more artificial microRNA
precursors is transcribed so that at least one transcript
comprising said two or more artificial miRNA precursors is
produced; [0183] (c) said at least one transcript is processed so
that at least two or more mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
[0184] (d) expression of two or more plant fatty acid biosynthetic
genes selected from the group consisting of: fad2-1, fad2-2, fad3,
fatB, sad, and fael are reduced. Recombinant constructs and plant
cells comprising the recombinant construct of the invention are
also included. Dicot plant cells comprising the recombinant
constructs of the invention are further embodiment. In another
aspect, this invention concerns a method for reducing expression of
at least one plant fatty acid biosynthetic gene in a plant cell,
said method comprising: [0185] (a) transforming a plant cell with
at least one isolated nucleic acid sequence comprising a sequence
encoding an artificial microRNA precursor, operably linked to at
least one regulatory sequence; [0186] (b) said at least one
sequence encoding an artificial microRNA precursor is transcribed
so that a transcript comprising said at least one artificial miRNA
precursor is produced; [0187] (c) said transcript is processed so
that at least one mature miRNA about 21 to 22 nts in length is
excised from said artificial miRNA precursor, and [0188] (d)
expression of at least one plant fatty acid biosynthetic gene
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is reduced. [0189] A further embodiment of the
invention includes an isolated nucleic acid sequence comprising a
sequence encoding at least one artificial microRNA precursor,
operably linked to at least one regulatory sequence, wherein said
sequence encoding at least one artificial microRNA precursor is
capable of forming a double-stranded RNA or hairpin, wherein the at
least one amiRNA precursor comprises at least one modified miRNA
precursor in which the miRNA sequence and its complementary
sequence are replaced by at least one amiRNA sequence and at least
one STAR sequence, wherein expression of at least one plant fatty
acid biosynthetic gene(s) selected from the group consisting of:
fad2-1, fad2-2, fad3, fatB, sad, and fael is inhibited. [0190] A
further embodiment of the invention comprises an isolated nucleic
acid sequence comprising a sequence encoding two or more artificial
microRNA precursors, operably linked to at least one regulatory
sequence, wherein said two or more artificial microRNA precursor
can be on the same or separate transcriptional units, further
wherein said sequences encoding two or more artificial microRNA
precursors are capable of forming a double-stranded RNA or hairpin,
wherein the two or more amiRNA precursors comprise a modified miRNA
precursor(s) in which the miRNA sequence(s) and its complementary
sequence(s) are replaced by two or more amiRNA sequences and two or
more STAR sequences, and further wherein expression of at least two
or more plant fatty acid biosynthetic genes selected from the group
consisting of: fad2-1, fad2-2, fad3, fatB, sad, and fael, are
inhibited. [0191] In yet another embodiment the instant invention
included any of the isolated nucleic acid sequences of the instant
invention, wherein the amiRNA sequence(s) comprise(s) at least one
selected form the group consisting of: SEQ ID NO: 21, 22, 23, 24,
25, 80, 83, 86, 89, 92, 95, 98, and 101. [0192] Transgenic plants
or seed comprising these amiRNAs are also part of the invention and
methods according to the invention that use these amiRNAs are also
comprised by the instant invention.
DETAILED DESCRIPTION
[0193] Information pertinent to this application can be found in
U.S. patent application Ser. Nos. 10/963,238 and 10/963,394, filed
Oct. 12, 2004. The entire contents of the above applications are
herein incorporated by reference.
[0194] Applicant's Assignee's US Patent Application Publication
2009/0155910 A1 published on Jun. 18, 2009 describes the
down-regulation of gene expression using artificial miRNAs. The
entire contents of the above applications are herein incorporated
by reference.
[0195] Other references that may be useful in understanding the
invention include U.S. patent application Ser. No. 10/883,374,
filed Jul. 1, 2004; U.S. patent application Ser. No. 10/913,288,
filed Aug. 6, 2004; and U.S. patent application Ser. No.
11/334,776, filed Jan. 6, 2006.
[0196] The disclosure of each reference set forth herein is hereby
incorporated by reference in its entirety.
[0197] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
"a plant" includes a plurality of such plants, reference to "a
cell" includes one or more cells and equivalents thereof known to
those skilled in the art, and so forth.
[0198] In the context of this disclosure, a number of terms and
abbreviations are used. The following definitions are provided.
[0199] "microRNA or miRNA" refers to oligoribonucleic acid, which
regulates expression of a polynucleotide comprising the target
sequence. microRNAs (miRNAs) are noncoding RNAs of about 19 to
about 24 nucleotides (nt) in length that have been identified in
both animals and plants (Lagos-Quintana et al., Science 294:853-858
2001, Lagos-Quintana et al., Curr. Biol. 12:735-739 2002; Lau et
al., Science 294:858-862 2001; Lee and Ambros, Science 294:862-864
2001; Llave et al., Plant Cell 14:1605-1619 2002; Mourelatos et
al., Genes. Dev. 16:720-728 2002; Park et al., Curr. Biol.
12:1484-1495 2002; Reinhart et al., Genes. Dev. 16:1616-1626 2002)
which regulates expression of a polynucleotide comprising the
target sequence. They are processed from longer precursor
transcripts that range in size from approximately 70 to 2000 nt or
longer, and these precursor transcripts have the ability to form
stable hairpin structures. In animals, the enzyme involved in
processing miRNA precursors is called Dicer, an RNAse III-like
protein (Grishok et al., Cell 106:23-34 2001; Hutvagner et al.,
Science 293:834-838 2001; Ketting et al., Genes. Dev. 15:2654-2659
2001). Plants also have a Dicer-like enzyme, DCL1 (previously named
CARPEL FACTORY/SHORT INTEGUMENTS1/SUSPENSOR1), and recent evidence
indicates that it, like Dicer, is involved in processing the
hairpin precursors to generate mature miRNAs (Park et al., Curr.
Biol. 12:1484-1495 2002; Reinhart et al., Genes. Dev. 16:1616-1626
2002). Furthermore, it is becoming clear from recent work that at
least some miRNA hairpin precursors originate as longer
polyadenylated transcripts, and several different miRNAs and
associated hairpins can be present in a single transcript
(Lagos-Quintana et al., Science 294:853-858 2001; Lee et al., EMBO
J 21:4663-4670 2002). Recent work has also examined the selection
of the miRNA strand from the dsRNA product arising from processing
of the hairpin by DICER (Schwartz et al., 2003, Cell 115:199-208).
It appears that the stability (i.e. G:C vs. A:U content, and/or
mismatches) of the two ends of the processed dsRNA affects the
strand selection, with the low stability end being easier to unwind
by a helicase activity. The 5' end strand at the low stability end
is incorporated into the RISC complex, while the other strand is
degraded.
[0200] "pri-miRNAs" or "primary miRNAs" are long, polyadenylated
RNAs transcribed by RNA polymerase II that encode miRNAs.
"pre-miRNAs" are primary miRNAs that have been processed to form a
shorter sequence that has the capacity to form a stable hairpin and
is further processed to release a miRNA. In plants both processing
steps are carried out by dicerlike and it is therefore difficult to
functionally differentiate between "pri-miRNAs" and "pre-miRNAs".
Therefore, a precursor miRNA, or a primary miRNA, is functionally
defined herein as a nucleotide sequence that is capable of
producing a miRNA. Given this functional definition, and as will be
clear from the Examples and discussion herein, a precursor miRNA,
primary miRNA and/or a miRNA of the invention can be represented as
a ribonucleic acid or, alternatively, in a deoxyribonucleic acid
form that "corresponds substantially" to the precursor miRNA,
primary miRNA and/or miRNA. It is understood that the DNA in its
double-stranded form will comprise a strand capable of being
transcribed into the miRNA precursor described. Expression
constructs, recombinant DNA constructs, and transgenic organisms
incorporating the miRNA encoding DNA that results in the expression
of the described miRNA precursors are described.
[0201] A "variable nucleotide subsequence" refers to a portion of a
nucleotide sequence that replaces a portion of a pre-miRNA sequence
provided that this subsequence is different from the sequence that
is being replaced, i.e, it cannot be the same sequence.
[0202] A "target gene" refers to a gene that encodes a target RNA,
ie., a gene from which a target RNA is transcribed. The gene may
encode mRNA, tRNA, small RNA, etc.
[0203] A "target sequence" refers to an RNA whose expression is to
be modulated, e.g., down-regulated. The target sequence may be a
portion of an open reading frame, 5' or 3' untranslated region,
exon(s), intron(s), flanking region, etc.
[0204] A "star sequence" is the complementary sequence within a
miRNA precursor that forms a duplex with the miRNA. The
complementarity of the star sequence does not need to be perfect.
Non-helix disrupting substitutions (i.e. G:T base pairs etc.) are
sometimes found, as well as 1-3 mismatches.
[0205] The term "genome" refers to the following: (1) the entire
complement of genetic material (genes and non-coding sequences)
present in each cell of an organism, or virus or organelle; (2) a
complete set of chromosomes inherited as a (haploid) unit from one
parent.
[0206] "Progeny" comprises any subsequent generation of a plant.
Progeny will inherit, and stably segregate, genes and transgenes
from its parent plant(s).
[0207] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxyl orientation,
respectively. Numeric ranges recited within the specification are
inclusive of the numbers defining the range and include each
integer within the defined range. Amino acids may be referred to
herein by either commonly known three letter symbols or by the
one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to
by their commonly accepted single-letter codes. Unless otherwise
provided for, software, electrical, and electronics terms as used
herein are as defined in The New IEEE Standard Dictionary of
Electrical and Electronics Terms (5.sup.th edition, 1993). The
terms defined below are more fully defined by reference to the
specification as a whole.
[0208] The terms "recombinant construct", "expression construct",
"chimeric construct", "construct", and "recombinant DNA construct"
are used interchangeably herein. A recombinant construct comprises
an artificial combination of nucleic acid fragments, e.g.,
regulatory and coding sequences that are not found together in
nature. For example, a chimeric construct may comprise regulatory
sequences and coding sequences that are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source, but arranged in a manner different than that found
in nature. Such a construct may be used by itself or may be used in
conjunction with a vector. If a vector is used, then the choice of
vector is dependent upon the method that will be used to transform
host cells as is well known to those skilled in the art. For
example, a plasmid vector can be used. The skilled artisan is well
aware of the genetic elements that must be present on the vector in
order to successfully transform, select and propagate host cells
comprising any of the isolated nucleic acid fragments of the
invention. The skilled artisan will also recognize that different
independent transformation events will result in different levels
and patterns of expression (Jones et al., EMBO J. 4:2411-2418
(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)),
and thus that multiple events must be screened in order to obtain
lines displaying the desired expression level and pattern. Such
screening may be accomplished by Southern analysis of DNA, Northern
analysis of mRNA expression, immunoblotting analysis of protein
expression, or phenotypic analysis, among others.
[0209] This construct may comprise any combination of
deoxyribonucleotides, ribonucleotides, and/or modified nucleotides.
The construct may be transcribed to form an RNA, wherein the RNA
may be capable of forming a double-stranded RNA and/or hairpin
structure. This construct may be expressed in the cell, or isolated
or synthetically produced. The construct may further comprise a
promoter, or other sequences which facilitate manipulation or
expression of the construct.
[0210] As used here "suppression" or "silencing" or "inhibition"
are used interchangeably to denote the down-regulation of the
expression of a product of a target sequence relative to its normal
expression level in a wild type organism. Suppression includes
expression that is decreased by about 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
relative to the wild type expression level.
[0211] As used herein, "encodes" or "encoding" refers to a DNA
sequence which can be processed to generate an RNA and/or
polypeptide.
[0212] As used herein, "expression" or "expressing" refers to
production of a functional product, such as, the generation of an
RNA transcript from an introduced construct, an endogenous DNA
sequence, or a stably incorporated heterologous DNA sequence. The
term may also refer to a polypeptide produced from an mRNA
generated from any of the above DNA precursors. Thus, expression of
a nucleic acid fragment may refer to transcription of the nucleic
acid fragment (e.g., transcription resulting in mRNA or other
functional RNA) and/or translation of RNA into a precursor or
mature protein (polypeptide).
[0213] As used herein, "heterologous" with respect to a sequence
means a sequence that originates from a foreign species, or, if
from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, with respect to a nucleic acid, it can
be a nucleic acid that originates from a foreign species, or is
synthetically designed, or, if from the same species, is
substantially modified from its native form in composition and/or
genomic locus by deliberate human intervention. A heterologous
protein may originate from a foreign species or, if from the same
species, is substantially modified from its original form by
deliberate human intervention.
[0214] The term "host cell" refers to a cell which contains or into
which is introduced a nucleic acid construct and supports the
replication and/or expression of the construct. Host cells may be
prokaryotic cells such as E. coli, or eukaryotic cells such as
fungi, yeast, insect, amphibian, nematode, or mammalian cells.
Alternatively, the host cells are monocotyledonous or
dicotyledonous plant cells. An example of a monocotyledonous host
cell is a maize host cell.
[0215] "Plant" includes reference to whole plants, plant organs,
plant tissues, seeds and plant cells and progeny of same. Plant
cells include, without limitation, cells from seeds, suspension
cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen, and
microspores.
[0216] The term "plant parts" includes differentiated and
undifferentiated tissues including, but not limited to the
following: roots, stems, shoots, leaves, pollen, seeds, tumor
tissue and various forms of cells and culture (e.g., single cells,
protoplasts, embryos and callus tissue). The plant tissue may be in
plant or in a plant organ, tissue or cell culture.
[0217] The term "plant organ" refers to plant tissue or group of
tissues that constitute a morphologically and functionally distinct
part of a plant.
[0218] The term "introduced" means providing a nucleic acid (e.g.,
expression construct) or protein into a cell. Introduced includes
reference to the incorporation of a nucleic acid into a eukaryotic
or prokaryotic cell where the nucleic acid may be incorporated into
the genome of the cell, and includes reference to the transient
provision of a nucleic acid or protein to the cell. Introduced
includes reference to stable or transient transformation methods,
as well as sexually crossing. Thus, "introduced" in the context of
inserting a nucleic acid fragment (e.g., a recombinant DNA
construct/expression construct) into ac ell, means "transfection"
or "transformation" or "transduction" and includes reference to the
incorporation of a nucleic acid fragment into a eukaryotic or
prokaryotic cell where the nucleic acid fragment may be
incorporated into the genome of the cell (e.g., chromosome,
plasmid, plastid or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0219] The term "genome" as it applies to a plant cells encompasses
not only chromosomal DNA found within the nucleus, but organelle
DNA found within subcellular components (e.g., mitochondrial,
plastid) of the cell.
[0220] The term "isolated" refers to material, such as a nucleic
acid or a protein, which is: (1) substantially or essentially free
from components which normally accompany or interact with the
material as found in its naturally occurring environment or (2) if
the material is in its natural environment, the material has been
altered by deliberate human intervention to a composition and/or
placed at a locus in the cell other than the locus native to the
material.
[0221] As used herein, "domain" or "functional domain" refer to
nucleic acid sequence(s) that are capable of eliciting a biological
response in plants. The present invention concerns miRNAs composed
of at least 21 nucleotide sequences acting either individually, or
in concert with other miRNA sequences, therefore a domain could
refer to either individual miRNAs or groups of miRNAs. Also, miRNA
sequences associated with their backbone sequences could be
considered domains useful for processing the miRNA into its active
form. As used herein, "subdomains" or "functional subdomains" refer
to subsequences of domains that are capable of eliciting a
biological response in plants. A miRNA could be considered a
subdomain of a backbone sequence. "Contiguous" sequences or domains
refer to sequences that are sequentially linked without added
nucleotides intervening between the domains. An example of a
contiguous domain string is found in SEQ ID NO:7957 which
represents SEQ ID NOs: 1-2652 as a continuous string that can be
thought of as 2652 miRNA sequences linked together in a sequential
concatenation.
[0222] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al., Nature 391:806 1998). The
corresponding process in plants is commonly referred to as
post-transcriptional gene silencing (PTGS) or RNA silencing and is
also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes and is commonly shared by diverse
flora and phyla (Fire et al., Trends Genet. 15:358 1999). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or from the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA of viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response through a mechanism that has yet to be fully
characterized.
[0223] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as "dicer". Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs) (Berstein et al., Nature
409:363 2001) and/or pre miRNAs into miRNAs. Short interfering RNAs
derived from dicer activity are typically about 21 to about 23
nucleotides in length and comprise about 19 base pair duplexes
(Elbashir et al., Genes Dev. 15:188 2001). Dicer has also been
implicated in the excision of 21- and 22-nucleotide small temporal
RNAs (stRNAs) from precursor RNA of conserved structure that are
implicated in translational control (Hutvagner et al., 2001,
Science 293:834). The RNAi response also features an endonuclease
complex, commonly referred to as an RNA-induced silencing complex
(RISC), which mediates cleavage of single-stranded RNA having
sequence complementarity to the antisense strand of the siRNA
duplex. Cleavage of the target RNA takes place in the middle of the
region complementary to the antisense strand of the siRNA duplex
(Elbashir et al., Genes Dev. 15:188 2001). In addition, RNA
interference can also involve small RNA (e.g., microRNA, or miRNA)
mediated gene silencing, presumably through cellular mechanisms
that regulate chromatin structure and thereby prevent transcription
of target gene sequences (see, e.g., Allshire, Science
297:1818-1819 2002; Volpe et al., Science 297:1833-1837 2002;
Jenuwein, Science 297:2215-2218 2002; and Hall et al., Science
297:2232-2237 2002). As such, miRNA molecules of the invention can
be used to mediate gene silencing via interaction with RNA
transcripts or alternately by interaction with particular gene
sequences, wherein such interaction results in gene silencing
either at the transcriptional or post-transcriptional level.
[0224] RNAi has been studied in a variety of systems. Fire et al.
(Nature 391:806 1998) were the first to observe RNAi in C. elegans.
Wianny and Goetz (Nature Cell Biol. 2:70 1999) describe RNAi
mediated by dsRNA in mouse embryos. Hammond et al. (Nature 404:293
2000) describe RNAi in Drosophila cells transfected with dsRNA.
Elbashir et al., (Nature 411:494 2001) describe RNAi induced by
introduction of duplexes of synthetic 21-nucleotide RNAs in
cultured mammalian cells including human embryonic kidney and HeLa
cells.
[0225] Small RNAs play an important role in controlling gene
expression. Regulation of many developmental processes, including
flowering, is controlled by small RNAs. It is now possible to
engineer changes in gene expression of plant genes by using
transgenic constructs which produce small RNAs in the plant.
[0226] Small RNAs appear to function by base-pairing to
complementary RNA or DNA target sequences. When bound to RNA, small
RNAs trigger either RNA cleavage or translational inhibition of the
target sequence. When bound to DNA target sequences, it is thought
that small RNAs can mediate DNA methylation of the target sequence.
The consequence of these events, regardless of the specific
mechanism, is that gene expression is inhibited.
[0227] MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about
24 nucleotides (nt) in length that have been identified in both
animals and plants (Lagos-Quintana et al., Science 294:853-858
2001, Lagos-Quintana et al., Curr. Biol. 12:735-739 2002; Lau et
al., Science 294:858-862 2001; Lee and Ambros, Science 294:862-864
2001; Llave et al., Plant Cell 14:1605-1619 2002; Mourelatos et
al., Genes. Dev. 16:720-728 2002; Park et al., Curr. Biol.
12:1484-1495 2002; Reinhart et al., Genes. Dev. 16:1616-1626 2002).
They are processed from longer precursor transcripts that range in
size from approximately 70 to 200 nt, and these precursor
transcripts have the ability to form stable hairpin structures. In
animals, the enzyme involved in processing miRNA precursors is
called Dicer, an RNAse III-like protein (Grishok et al., Cell
106:23-34 2001; Hutvagner et al., Science 293:834-838 2001; Ketting
et al., Genes. Dev. 15:2654-2659 2001). Plants also have a
Dicer-like enzyme, DCL1 (previously named CARPEL FACTORY/SHORT
INTEGUMENTS1/SUSPENSOR1), and recent evidence indicates that it,
like Dicer, is involved in processing the hairpin precursors to
generate mature miRNAs (Park et al., Curr. Biol. 12:1484-1495 2002;
Reinhart et al., Genes. Dev. 16:1616-1626 2002). Furthermore, it is
becoming clear from recent work that at least some miRNA hairpin
precursors originate as longer polyadenylated transcripts, and
several different miRNAs and associated hairpins can be present in
a single transcript (Lagos-Quintana et al., Science 294:853-858
2001; Lee et al., EMBO J 21:4663-4670 2002). Recent work has also
examined the selection of the miRNA strand from the dsRNA product
arising from processing of the hairpin by DICER (Schwartz et al.,
2003, Cell 115:199-208). It appears that the stability (i.e. G:C
vs. A:U content, and/or mismatches) of the two ends of the
processed dsRNA affects the strand selection, with the low
stability end being easier to unwind by a helicase activity. The 5'
end strand at the low stability end is incorporated into the RISC
complex, while the other strand is degraded.
[0228] In animals, there is direct evidence indicating a role for
specific miRNAs in development. The lin-4 and let-7 miRNAs in C.
elegans have been found to control temporal development, based on
the phenotypes generated when the genes producing the lin-4 and
let-7 miRNAs are mutated (Lee et al., Cell 75:843-854 1993;
Reinhart et al., Nature 403-901-906 2000). In addition, both miRNAs
display a temporal expression pattern consistent with their roles
in developmental timing. Other animal miRNAs display
developmentally regulated patterns of expression, both temporal and
tissue-specific (Lagos-Quintana et al., Science 294:853-853 2001,
Lagos-Quintana et al., Curr. Biol. 12:735-739 2002; Lau et al.,
Science 294:858-862 2001; Lee and Ambros, Science 294:862-864
2001), leading to the hypothesis that miRNAs may, in many cases, be
involved in the regulation of important developmental processes.
Likewise, in plants, the differential expression patterns of many
miRNAs suggests a role in development (Llave et al., Plant Cell
14:1605-1619 2002; Park et al., Curr. Biol. 12:1484-1495 2002;
Reinhart et al., Genes. Dev. 16:1616-1626 2002). However, a
developmental role for miRNAs has not been directly proven in
plants, because to date there has been no report of a developmental
phenotype associated with a specific plant miRNA.
[0229] MicroRNAs appear to regulate target genes by binding to
complementary sequences located in the transcripts produced by
these genes. In the case of lin-4 and let-7, the target sites are
located in the 3' UTRs of the target mRNAs (Lee et al., Cell
75:843-854 1993; Wightman et al., Cell 75:855-862 1993; Reinhart et
al., Nature 403:901-906 2000; Slack et al., Mol. Cell 5:659-669
2000), and there are several mismatches between the lin-4 and let-7
miRNAs and their target sites. Binding of the lin-4 or let-7 miRNA
appears to cause downregulation of steady-state levels of the
protein encoded by the target mRNA without affecting the transcript
itself (Olsen and Ambros, Dev. Biol. 216:671-680 1999). On the
other hand, recent evidence suggests that miRNAs can, in some
cases, cause specific RNA cleavage of the target transcript within
the target site (Hutvagner and Zamore, Science 297:2056-2060 2002;
Llave et al., Plant Cell 14:1605-1619 2002). It seems likely that
miRNAs can enter at least two pathways of target gene regulation:
Protein downregulation and RNA cleavage. MicroRNAs entering the RNA
cleavage pathway incorporated into an RNA-induced silencing complex
(RISC) that is similar or identical to that seen for RNAi.
[0230] The present invention concerns an isolated nucleic acid
fragment comprising a sequence encoding an artificial microRNA
precursor, operably linked to at least one regulatory sequence,
wherein said sequence encoding an artificial microRNA precursor is
transcribed so that a transcript comprising said artificial miRNA
precursor is produced, and wherein said transcript is processed so
that a mature miRNA about 21 to 22 nts in length is excised from
said artificial miRNA precursor, and expression of a plant fatty
acid biosynthetic gene selected from the group consisting of:
fad2-1, fad2-2, fad3, fatB, sad, and fael is inhibited.
[0231] Another embodiment of the present invention concerns an
isolated nucleic acid fragment comprising a sequence encoding an
artificial microRNA precursor, wherein said artificial microRNA
precursor is at least one selected form the group consisting of SEQ
ID NO: 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, 156, 158, 160, 162, 164, 166, and
168.
[0232] Yet another embodiment of the present invention comprises an
isolated nucleic acid sequence comprising a sequence encoding two
or more artificial microRNA precursors, operably linked to at least
one regulatory sequence, wherein said two or more artificial
microRNA precursor can be on the same or separate transcriptional
units, further wherein said sequence encoding artificial microRNA
precursors is transcribed so that a transcript comprising said
artificial miRNA precursors is produced, and wherein said
transcript is processed so that mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
expression of two or more plant fatty acid biosynthetic gene(s)
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited. The plant fatty acid biosynthetic genes
include without limitation any of the nucleotide sequences of SEQ
ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 174, which
encode amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79, and 175.
[0233] Another embodiment comprises a method for reducing
expression two or more plant fatty acid biosynthetic gene, said
method comprising: [0234] (a) transforming a plant cell with at
least one isolated nucleic acid sequence comprising a sequence
encoding two or more artificial microRNA precursors, wherein said
two or more artificial microRNA precursors can be on the same or
separate transcriptional units, operably linked to at least one
regulatory sequence; [0235] (b) said at least one sequence encoding
two or more artificial microRNA precursors is transcribed so that
at least one transcript comprising said two or more artificial
miRNA precursors is produced; [0236] (c) said at least one
transcript is processed so that at least two or more mature miRNAs
about 21 to 22 nts in length are excised from said artificial miRNA
precursors, and [0237] (d) expression of two or more plant fatty
acid biosynthetic genes selected from the group consisting of:
fad2-1, fad2-2, fad3, fatB, sad, and fael are reduced.
[0238] An additional embodiment of the present invention concerns
recombinant constructs comprising the isolated nucleic acid
sequence of the invention operably linked to at least one
regulatory sequence.
[0239] Plant cells comprising the recombinant construct of the
invention are also included. Dicot plant cells comprising the
recombinant constructs of the invention are further embodiment. In
another aspect, this invention concerns a method for reducing
expression of at least one plant fatty acid biosynthetic gene in a
plant cell, said method comprising: [0240] (a) transforming a plant
cell with at least one isolated nucleic acid sequence comprising a
sequence encoding an artificial microRNA precursor, operably linked
to at least one regulatory sequence; [0241] (b) said at least one
sequence encoding an artificial microRNA precursor is transcribed
so that a transcript comprising said at least one artificial miRNA
precursor is produced; [0242] (c) said transcript is processed so
that at least one mature miRNA about 21 to 22 nts in length is
excised from said artificial miRNA precursor, and [0243] (d)
expression of at least one plant fatty acid biosynthetic gene
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is reduced.
[0244] Any of these isolated nucleic acid fragments can be used to
make a recombinant construct comprising these isolated nucleic acid
fragments operably linked to at least one regulatory sequence.
These constructs can be transformed into plant cells so that the
transformed plant cell comprises the recombinant construct in its
genome. Preferably, the plant cell can be a dicot plant cell.
Examples of dicot plant cells include, but are not limited to,
soybean, rapeseed (e.g. Brassica napus), sunflower, flax, cotton,
alfalfa, barley, bean, pea, tobacco, and Arabidopsis.
[0245] The most preferred dicot plant cell is soybean.
[0246] The present invention concerns an isolated nucleic acid
fragment comprising a sequence encoding an artificial microRNA
precursor, operably linked to at lea.
[0247] st one regulatory sequence, wherein said sequence encoding
an artificial microRNA precursor is transcribed so that a
transcript comprising said artificial miRNA precursor is produced,
and wherein said transcript is processed so that a mature miRNA
about 21 to 22 nts in length is excised from said artificial miRNA
precursor, and expression of a plant fatty acid biosynthetic gene
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited.
[0248] Another embodiment of the present invention concerns an
isolated nucleic acid fragment comprising a sequence encoding an
artificial microRNA precursor, wherein said artificial microRNA
precursor is at least one selected form the group consisting of SEQ
ID NO: 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, 156, 158, 160, 162, 164, 166, and
168.
[0249] An additional embodiment of the present invention concerns
recombinant constructs comprising the isolated nucleic acid
sequence of the invention operably linked to at least one
regulatory sequence.
[0250] Additional embodiments of the present inventions comprise an
isolated nucleic acid sequence comprising a sequence encoding two
or more artificial microRNA precursors, operably linked to at least
one regulatory sequence, wherein said two or more artificial
microRNA precursor can be on the same or separate transcriptional
units, further wherein said sequence encoding artificial microRNA
precursors is transcribed so that a transcript comprising said
artificial miRNA precursors is produced, and wherein said
transcript is processed so that mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
expression of two or more plant fatty acid biosynthetic gene(s)
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is inhibited.
[0251] Another embodiment of the instant invention includes a
method for reducing expression two or more plant fatty acid
biosynthetic gene, said method comprising: [0252] (a) transforming
a plant cell with at least one isolated nucleic acid sequence
comprising a sequence encoding two or more artificial microRNA
precursors, wherein said two or more artificial microRNA precursors
can be on the same or separate transcriptional units, operably
linked to at least one regulatory sequence; [0253] (b) said at
least one sequence encoding two or more artificial microRNA
precursors is transcribed so that at least one transcript
comprising said two or more artificial miRNA precursors is
produced; [0254] (c) said at least one transcript is processed so
that at least two or more mature miRNAs about 21 to 22 nts in
length are excised from said artificial miRNA precursors, and
[0255] (d) expression of two or more plant fatty acid biosynthetic
genes selected from the group consisting of: fad2-1, fad2-2, fad3,
fatB, sad, and fael are reduced. Recombinant constructs and plant
cells comprising the recombinant construct of the invention are
also included. Dicot plant cells comprising the recombinant
constructs of the invention are further embodiment. In another
aspect, this invention concerns a method for reducing expression of
at least one plant fatty acid biosynthetic gene in a plant cell,
said method comprising: [0256] (a) transforming a plant cell with
at least one isolated nucleic acid sequence comprising a sequence
encoding an artificial microRNA precursor, operably linked to at
least one regulatory sequence; [0257] (b) said at least one
sequence encoding an artificial microRNA precursor is transcribed
so that a transcript comprising said at least one artificial miRNA
precursor is produced; [0258] (c) said transcript is processed so
that at least one mature miRNA about 21 to 22 nts in length is
excised from said artificial miRNA precursor, and [0259] (d)
expression of at least one plant fatty acid biosynthetic gene
selected from the group consisting of: fad2-1, fad2-2, fad3, fatB,
sad, and fael is reduced. [0260] A further embodiment of the
invention includes an isolated nucleic acid sequence comprising a
sequence encoding at least one artificial microRNA precursor,
operably linked to at least one regulatory sequence, wherein said
sequence encoding at least one artificial microRNA precursor is
capable of forming a double-stranded RNA or hairpin, wherein the at
least one amiRNA precursor comprises at least one modified miRNA
precursor in which the miRNA sequence and its complementary
sequence are replaced by at least one amiRNA sequence and at least
one STAR sequence, wherein expression of at least one plant fatty
acid biosynthetic gene(s) selected from the group consisting of:
fad2-1, fad2-2, fad3, fatB, sad, and fael is inhibited. [0261] A
further embodiment of the invention comprises an isolated nucleic
acid sequence comprising a sequence encoding two or more artificial
microRNA precursors, operably linked to at least one regulatory
sequence, wherein said two or more artificial microRNA precursor
can be on the same or separate transcriptional units, further
wherein said sequences encoding two or more artificial microRNA
precursors are capable of forming a double-stranded RNA or hairpin,
wherein the two or more amiRNA precursors comprise a modified miRNA
precursor(s) in which the miRNA sequence(s) and its complementary
sequence(s) are replaced by two or more amiRNA sequences and two or
more STAR sequences, and further wherein expression of at least two
or more plant fatty acid biosynthetic genes selected from the group
consisting of: fad2-1, fad2-2, fad3, fatB, sad, and fael, are
inhibited. [0262] In yet another embodiment the instant invention
included any of the isolated nucleic acid sequences of the instant
invention, wherein the amiRNA sequence(s) comprise(s) at least one
selected form the group consisting of: SEQ ID NO: 21, 22, 23, 24,
25, 80, 83, 86, 89, 92, 95, 98, and 101. [0263] Transgenic plants
or seed comprising these amiRNAs are also part of the invention and
methods according to the invention that use these amiRNAs are also
comprised by the instant invention.
[0264] Bioinformatic approaches have been successfully used to
predict targets for plant miRNAs (Llave et al., Plant Cell
14:1605-1619 2002; Park et al., Curr. Biol. 12:1484-1495 2002;
Rhoades et al., Cell 110:513-520 2002), and thus it appears that
plant miRNAs have higher overall complementarity with their
putative targets than do animal miRNAs. Most of these predicted
target transcripts of plant miRNAs encode members of transcription
factor families implicated in plant developmental patterning or
cell differentiation.
[0265] General categories of sequences of interest include, for
example, those genes involved in regulation or information, such as
zinc fingers, transcription factors, homeotic genes, or cell cycle
and cell death modulators, those involved in communication, such as
kinases, and those involved in housekeeping, such as heat shock
proteins.
[0266] Target sequences may include coding regions and non-coding
regions such as promoters, enhancers, terminators, introns and the
like.
[0267] The target sequence may be an endogenous sequence, or may be
an introduced heterologous sequence, or transgene. For example, the
methods may be used to alter the regulation or expression of a
transgene, or to remove a transgene or other introduced sequence
such as an introduced site-specific recombination site. The target
sequence may also be a sequence from a pathogen, for example, the
target sequence may be from a plant pathogen such as a virus, a
mold or fungus, an insect, or a nematode. A miRNA could be
expressed in a plant which, upon infection or infestation, would
target the pathogen and confer some degree of resistance to the
plant.
[0268] In plants, other categories of target sequences include
genes affecting agronomic traits, insect resistance, disease
resistance, herbicide resistance, sterility, grain characteristics,
and commercial products. Genes of interest also included those
involved in oil, starch, carbohydrate, or nutrient metabolism as
well as those affecting, for example, kernel size, sucrose loading,
and the like. The quality of grain is reflected in traits such as
levels and types of oils, saturated and unsaturated, quality and
quantity of essential amino acids, and levels of cellulose. For
example, genes of the phytic acid biosynthetic pathway could be
suppressed to generate a high available phosphorous phenotype. See,
for example, phytic acid biosynthetic enzymes including inositol
polyphosphate kinase-2 polynucleotides, disclosed in WO 02/059324,
inositol 1,3,4-trisphosphate 5/6-kinase polynucleotides, disclosed
in WO 03/027243, and myo-inositol 1-phosphate synthase and other
phytate biosynthetic polynucleotides, disclosed in WO 99/05298, all
of which are herein incorporated by reference. Genes in the
lignification pathway could be suppressed to enhance digestibility
or energy availability. Genes affecting cell cycle or cell death
could be suppressed to affect growth or stress response. Genes
affecting DNA repair and/or recombination could be suppressed to
increase genetic variability. Genes affecting flowering time could
be suppressed, as well as genes affecting fertility. Any target
sequence could be suppressed in order to evaluate or confirm its
role in a particular trait or phenotype, or to dissect a molecular,
regulatory, biochemical, or proteomic pathway or network.
[0269] A number of promoters can be used. These promoters can be
selected based on the desired outcome. It is recognized that
different applications will be enhanced by the use of different
promoters in plant expression cassettes to modulate the timing,
location and/or level of expression of the miRNA. Such plant
expression cassettes may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible, constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0270] Constitutive, tissue-preferred or inducible promoters can be
employed. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),
the Nos promoter, the pEmu promoter, the rubisco promoter, the
GRP1-8 promoter and other transcription initiation regions from
various plant genes known to those of skill. If low level
expression is desired, weak promoter(s) may be used. Weak
constitutive promoters include, for example, the core promoter of
the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the
core 35S CaMV promoter, and the like. Other constitutive promoters
include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and
5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated
by reference.
[0271] Examples of inducible promoters are the Adhi promoter which
is inducible by hypoxia or cold stress, the Hsp70 promoter which is
inducible by heat stress, the PPDK promoter and the pepcarboxylase
promoter which are both inducible by light. Also useful are
promoters which are chemically inducible, such as the In2-2
promoter which is safener induced (U.S. Pat. No. 5,364,780), the
ERE promoter which is estrogen induced, and the Axig1 promoter
which is auxin induced and tapetum specific but also active in
callus (PCT US01/22169).
[0272] Examples of promoters under developmental control include
promoters that initiate transcription preferentially in certain
tissues, such as leaves, roots, fruit, seeds, or flowers. An
exemplary promoter is the anther specific promoter 5126 (U.S. Pat.
Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters
include, but are not limited to, 27 kD gamma zein promoter and waxy
promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M.
et al. Nucl. Acids Res. 18(21):6426; and Kloesgen, R. B. et al.
(1986) Mol. Gen. Genet. 203:237-244. Promoters that express in the
embryo, pericarp, and endosperm are disclosed in U.S. Pat. No.
6,225,529 and PCT publication WO 00/12733. The disclosures each of
these are incorporated herein by reference in their entirety.
[0273] In some embodiments it will be beneficial to express the
gene from an inducible promoter, particularly from a
pathogen-inducible promoter. Such promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)
Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.
4:111-116. See also WO 99/43819, herein incorporated by
reference.
[0274] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant--Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200).
[0275] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the polynucleotides. Such wound-inducible
promoters include potato proteinase inhibitor (pin II) gene (Ryan
(1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature
Biotech. 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1
and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208);
systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1
(Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et
al. (1993) FEBS Lett. 323:73-76); MPI gene (Corderok et al. (1994)
Plant J. 6(2):141-150); and the like, herein incorporated by
reference.
[0276] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0277] Tissue-preferred promoters can be utilized to target
enhanced expression of a sequence of interest within a particular
plant tissue. Tissue-preferred promoters include Yamamoto et al.
(1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell
Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.
254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;
Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et
al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996)
Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.
20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590;
and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such
promoters can be modified, if necessary, for weak expression.
[0278] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. In
addition, the promoters of cab and rubisco can also be used. See,
for example, Simpson et al. (1958) EMBO J 4:2723-2729 and Timko et
al. (1988) Nature 318:57-58.
[0279] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus comiculatus,
and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed rolC and rolD root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene, fused to nptII (neomycin phosphotransferase II) showed
similar characteristics. Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179. The phaseolin gene (Murai et al. (1983) Science
23:476-482 and Sengopta-Gopalen et al. (1988) PNAS
82:3320-3324.
[0280] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing the DNA
construct include microinjection (Crossway et al. (1986)
Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543), sexual
crossing, electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606), Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055; and U.S. Pat. No.
5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.
3:2717-2722), and ballistic particle acceleration (see, for
example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.,
U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244;
Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) "Direct
DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and
McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger
et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)
Particulate Science and Technology 5:27-37 (onion); Christou et al.
(1988) Plant Physiol. 87:671-674 (soybean); Finer and McMullen
(1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al.
(1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al.
(1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc.
Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)
Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855;
Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Klein et
al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.
(1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.
5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Aced. Sci.
USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The
Experimental Manipulation of Ovule Tissues, ed. Chapman et al.
(Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)
Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor.
Appl. Genet. 84:560-566 (whisker-mediated transformation);
D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation);
Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and
Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al.
(1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium
tumefaciens); and U.S. Pat. No. 5,736,369 (meristem
transformation), all of which are herein incorporated by
reference.
[0281] The nucleotide constructs may be introduced into plants by
contacting plants with a virus or viral nucleic acids. Generally,
such methods involve incorporating a nucleotide construct of the
invention within a viral DNA or RNA molecule. Further, it is
recognized that useful promoters encompass promoters utilized for
transcription by viral RNA polymerases. Methods for introducing
nucleotide constructs into plants and expressing a protein encoded
therein, involving viral DNA or RNA molecules, are known in the
art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367 and 5,316,931; herein incorporated by
reference.
[0282] In some embodiments, transient expression may be desired. In
those cases, standard transient transformation techniques may be
used. Such methods include, but are not limited to viral
transformation methods, and microinjection of DNA or RNA, as well
other methods well known in the art.
[0283] The cells from the plants that have stably incorporated the
nucleotide sequence may be grown into plants in accordance with
conventional ways. See, for example, McCormick et al. (1986) Plant
Cell Reports 5:81-84. These plants may then be grown, and either
pollinated with the same transformed strain or different strains,
and the resulting hybrid having constitutive expression of the
desired phenotypic characteristic imparted by the nucleotide
sequence of interest and/or the genetic markers contained within
the target site or transfer cassette. Two or more generations may
be grown to ensure that expression of the desired phenotypic
characteristic is stably maintained and inherited and then seeds
harvested to ensure expression of the desired phenotypic
characteristic has been achieved.
[0284] Fad2-1 and fad2-2 are fatty acid desaturases [Developmental
and growth temperature regulation of two different microsomal
.omega.-6 desaturase genes in soybeans. Heppard, Elmer P.; Kinney,
Anthony J.; Stecca, Kevin L.; Miao, Guo-Hua. Agric. Products Dep.,
E. I. du Pont de Nemours Co., Wilmington, Del., USA. Plant
Physiology (1996), 110(1), 311-19.] gene families, also known as
delta-12 desaturase or omega-6 desaturase (U.S. Pat. No.
6,872,872B1, U.S. Pat. No. 6,919,466B2, U.S. Pat. No. 7,105,721B2).
FatB is a thioesterase encoding a palmitoyl-thioesterase (Kinney,
A. J. (1997) Genetic engineering of oilseeds for desired traits.
In: Genetic Engineering, Vol. 19, (Setlow J. K. Plenum Press, New
York, N.Y., pp. 149-166.). Sad is a stearic acid desaturase and
belongs to the Sad gene family, also known as delta-9 stearoyl-ACP
desaturase (U.S. Pat. No. 7,498,427B2, U.S. Pat. No. 6,949,698B2).
Fad3 belongs to the fatty acid desaturase 3 (fad3) gene family
(U.S. Pat. No. 5,952,544A). Fael is a fatty acid elongase
(publication number US 2007/0204370 A1, filed Nov. 24, 2004).
EXAMPLES
Example 1
Artificial MicroRNA (amiRNA) Constructs for Silencing Soybean Fatty
Acid Biosynthetic Genes
Soy Fatty Acid Biosynthetic Genes Targeted for Silencing
[0285] Key gene family sequences targeted for silencing in soybean
are the stearic acid desaturase (Sad) gene family, also known as
delta-9 stearoyl-ACP desaturase (U.S. Pat. No. 7,498,427B2, U.S.
Pat. No. 6,949,698B2), the fatty acid desaturase 2-1 (Fad2-1) or
2-2 (fad2-2) [Developmental and growth temperature regulation of
two different microsomal .omega.-6 desaturase genes in soybeans.
Heppard, Elmer P.; Kinney, Anthony J.; Stecca, Kevin L.; Miao,
Guo-Hua. Agric. Products Dep., E. I. du Pont de Nemours Co.,
Wilmington, Del., USA. Plant Physiology (1996), 110(1), 311-19.]
gene families, also known as delta-12 desaturase or omega-6
desaturase (U.S. Pat. No. 6,872,872B1, U.S. Pat. No. 6,919,466B2,
U.S. Pat. No. 7,105,721B2), the fatty acid desaturase 3 (fad3) gene
family (U.S. Pat. No. 5,952,544A) and the fatty acid thioesterase B
(fatB) gene family, also known as palmitoyl-ACP thioesterase (U.S.
Pat. No. 5,955,650A, U.S. RE37317E1). A list of fatty acid
biosynthetic genes targeted for silencing by amiRNAs, along with
corresponding soy genome sequence (Glyma) gene identifier, nt SEQ
ID NO and aa SEQ ID NO are shown in Table 1.
TABLE-US-00001 TABLE 1 Soy Fatty Acid Biosynthetic Gene Sequences
Targeted for Silencing Gene Family Glyma Genes nt SEQ ID NO aa SEQ
ID NO GmFad2-1 Glyma10g42470 1 2 Glyma20g24530 3 4 GmFad2-2
Glyma19g32940 5 6 GmSad Glyma02g15600 7 8 Glyma07g32850 9 10 GmFad3
Glyma14g37350 11 12 Glyma02g39230 13 14 Glyma18g06950 15 16 GmFatB
Glyma05g08060 17 18 Glyma17g12940 19 20
Design of Artificial MicroRNA Sequences
[0286] Artificial microRNAs (amiRNAs) that would have the ability
to silence the desired target genes were designed largely according
to rules described in Schwab R, et al. (2005) Dev Cell 8: 517-27.
To summarize, microRNA sequences are 21 nucleotides in length,
start at their 5'-end with a "U", display 5' instability relative
to their star sequence which is achieved by including a C or G at
position 19, and their 10th nucleotide is either an "A" or an "U".
An additional requirement for artificial microRNA design was that
the amiRNA have a high free delta-G as calculated using the ZipFold
algorithm (Markham, N. R. & Zuker, M. (2005) Nucleic Acids Res.
33: W577-W581.).
[0287] Design and synthesis of amiRNA sequences for targeting the
soy fad2-1 and fad2-2 genes was previously described in
US20090155910A1 (WO 2009/079532) (the contents of which are
incorporated by reference) and are shown in Table 2. New amiRNA
sequences targeting soy fad3, fatB and sad genes were similarly
designed and are also shown in Table 2.
TABLE-US-00002 TABLE 2 amiRNAs For Soy Fatty Acid Biosynthetic Gene
Sequences Targeted for Silencing Gene Family amiRNA nt SEQ ID NO
GmFad2-1 GM-MFAD2-1B 21 GmFad2-2 GM-MFAD2-2 22 GmSad GM-MSAD3 23
GmFad3 GM-MFAD3C 24 FatB GM-MFATBF 25
Design of an Artificial Star Sequences
[0288] "Star sequences" are those that base pair with the amiRNA
sequences, in the precursor RNA, to form imperfect stem structures.
To form a perfect stem structure the star sequence would be the
exact reverse complement of the amiRNA. The soybean precursor
sequence as described in "Novel and nodulation-regulated microRNAs
in soybean roots" Subramanian S, Fu Y, Sunkar R, Barbazuk W B, Zhu
J K, Yu O BMC Genomics. 9:160(2008) and accessed on mirBase
(Conservation and divergence of microRNA families in plants"
Dezulian T, Palatnik J F, Huson D H, Weigel D (2005) Genome Biology
6:P13) was folded using mfold (M. Zuker (2003) Nucleic Acids Res.
31: 3406-15; and D. H. Mathews, J. et al. (1999) J. Mol. Biol. 288:
911-940). The miRNA sequence was then replaced with the amiRNA
sequence and the endogenous star sequence was replaced with the
exact reverse complement of the amiRNA. Changes in the artificial
star sequence were introduced so that the structure of the stem
would remain the same as the endogenous structure. The altered
sequence was then folded with mfold and the original and altered
structures were compared by eye. If necessary, further alterations
to the artificial star sequence were introduced to maintain the
original structure.
[0289] Design and synthesis of STAR sequences that pair with
amiRNAs for targeting the soy fad2-1 and fad2-2 genes was
previously described in US20090155910A1 (WO 2009/079532) and are
shown in Table 3. New STAR sequences that pair with amiRNAs for
targeting the soy fad3, fatB and sad genes were similarly designed
and are also shown in Table 3. STAR sequences differ for a given
amiRNA with which they pair depending on the amiRNA precursor
sequence used and this is also indicated in Table 3.
TABLE-US-00003 TABLE 3 Star Sequences For Soy Fatty Acid
Biosynthetic Gene Sequences Targeted for Silencing Gene Family STAR
Sequence nt SEQ ID NO GmFad2-1 159-GM-MFAD2-1B 26 396b-GM-MFAD2-1B
27 GmFad2-2 159-GM-MFAD2-2 28 GmSad 396b-GM-MSAD3 29 GmFad3
159-GM-MFAD3C 30 FatB 159-GM-FATBF 31
Example 2
Prophetic
Conversion of Genomic MicroRNA Precursors to Artificial MicroRNA
Precursors
[0290] Genomic miRNA precursor genes ("backbones"), such as those
described in US20090155909A1 (WO 2009/079548) and in
US20090155910A1 (WO 2009/079532), can be converted to amiRNAs using
overlapping PCR, and the resulting DNAs can be completely sequenced
and then cloned downstream of an appropriate promoter in a vector
capable of transformation.
[0291] Alternatively, amiRNAs can be synthesized commercially, for
example, by Codon Devices (Cambridge, Mass.), DNA 2.0 (Menlo Park,
Calif.) or Genescript (Piscataway, N.J.). The synthesized DNA is
then cloned downstream of an appropriate promoter in a vector
capable of soybean transformation.
[0292] Artificial miRNAs can also be constructed using
In-Fusion.TM. technology (Clontech, Mountain View, Calif.).
Example 3
Generation of amiRNA Precursors to Silence Soy Fatty Acid
Biosynthetic Genes
[0293] The identification of the genomic miRNA precursor sequences
159 and 396b was described previously in US20090155910A1 (WO
2009/079532) and their sequences are set forth in SEQ ID NO: 152
and SEQ ID NO: 153, respectively.
[0294] Genomic miRNA precursor genes were converted to amiRNA
precursors 159-fad2-1b, 396b-fad2-1b and 159-fad2-2 using
overlapping PCR as previously described in US20090155910A1 (WO
2009/079532). Precursor amiRNA 159-fad2-1b and 396b-fad2-1b were
then individually cloned downstream of the beta-conglycinin
promoter in plasmid PHP27753 (also known as plasmid KS332,
described in U.S. patent application Ser. No. 13/295,345,
applicant's designation BB-1623 USCNT), to form expression
constructs PHP32511 and PHP32510, respectively, as described in
US20090155910A1. US20090155910A1 further describes the cloning of
amiRNA precursor 159-fad2-2 downstream of either 396b-fad2-1b or
159-fad2-1b to produce PHP32843 and PHP32869, respectively. The SEQ
ID NOs of sequences for precursor amiRNAs 159-fad2-1b,
396b-fad2-1b, 159-fad2-2 and 396b-fad2-1b/159-fad2-2, as well as
plasmids PHP32511, PHP32510 and PHP32843 are shown in Table 4.
[0295] Genomic miRNA precursor gene 396b was converted to amiRNA
precursor 396b-sad3 using overlapping PCR by the method previously
described in US20090155910A1 (WO 2009/079532). amiRNA precursor
396b-sad3 was cloned 3' (downstream) of 396b-fad2-1b in expression
vector PHP27753 to produce construct PHP33705. The SEQ ID NO of the
sequence of precursor amiRNA 396b-sad3 and 396b-sad3/396b-fad2-1b,
as well as plasmid PHP33705 is shown in Table 4.
[0296] Genomic miRNA precursor gene 159 was converted to amiRNA
precursor 159-fad3c using overlapping PCR by the method previously
described in US20090155910A1 (WO 2009/079532). amiRNA precursor
159-fad3c was cloned into expression vector PHP27753 to produce
construct PHP38557. The SEQ ID NO of the sequence of precursor
amiRNA 159-fad3c, as well as plasmid PHP38557 is shown in Table
4.
[0297] Genomic miRNA precursor gene 159 was converted to amiRNA
precursor 159-fatBF using overlapping PCR by the method previously
described in US20090155910A1 (WO 2009/079532). amiRNA precursor
159-fatBF was cloned into expression vector PHP27753 to produce
construct PHP41103. The SEQ ID NO of the sequence of precursor
amiRNA 159-fatb, as well as plasmid PHP41103 is shown in Table
4.
[0298] The NotI fragment of PHP38557, containing the amiRNA
precursor 159-fad3c, was cloned into the NotI site of vector PKR72,
described in U.S. Pat. No. 8,049,062, to produce construct pKR1756.
The SEQ ID NO of the sequence of precursor amiRNA 159-fad3c, as
well as plasmid pKR1756 is shown in Table 4.
[0299] The NotI fragment of PHP41103, containing the amiRNA
precursor 159-fatBF, was cloned into the NotI site of vector PKR72,
described in U.S. Pat. No. 8,049,062, to produce construct pKR1757.
The SEQ ID NO of the sequence of precursor amiRNA 159-fatb, as well
as plasmid pKR1757 is shown in Table 4.
[0300] amiRNA precursors 159-fad3c and 159-fatBF were generated as
described above and cloned together (in that order) downstream of
the beta-conglycinin promoter into expression vector PKR72,
described in U.S. Pat. No. 8,049,062, to produce construct pKR1766.
The SEQ ID NO of the sequence of precursor amiRNA
159-fad3c/159-fatBF, as well as plasmid pKR1766 is shown in Table
4.
[0301] amiRNA precursors 159-fatBF and 159-fad3c were generated as
described above and cloned together (in that order) downstream of
the beta-conglycinin promoter into expression vector PKR72,
described in U.S. Pat. No. 8,049,062, to produce construct pKR1771.
The SEQ ID NO of the sequence of precursor amiRNA
159-fatBF/159-fad3c, as well as plasmid pKR1771 is shown in Table
4.
[0302] amiRNA precursors 159-fad2-1b, 159-fatBF and 159-fad3c were
generated as described above and cloned together (in that order)
into expression vector PHP27753 to produce construct PHP41784. The
SEQ ID NO of the sequence of precursor amiRNA
159-fad2-1b/159-fatBF/159-fad3c, as well as plasmid PHP41784 is
shown in Table 4.
[0303] amiRNA precursors 396b-fad2-1b, 159-fatBF and 159-fad3c were
generated as described above and cloned together (in that order)
downstream of the beta-conglycinin promoter into expression vector
PKR72, described in U.S. Pat. No. 8,049,062, to produce construct
pKR1776. The SEQ ID NO of the sequence of precursor amiRNA
396b-fad2-1b/159-fatBF/159-fad3c, as well as plasmid pKR1776 is
shown in Table 4.
TABLE-US-00004 TABLE 4 Precursor amiRNAs and amiRNA Expression
Constructs For Soy Fatty Acid Biosynthetic Gene Sequences Targeted
for Silencing amiRNA amiRNA Precursor Plasmid Plasmid Gene Family
Precursor SEQ ID NO Name SEQ ID NO GmFad2-1 159-fad2-1b 124
PHP32511 32 GmFad2-1 396b-fad2-1b 125 PHP32510 33 GmFad2-2
159-fad2-2 126 -- -- GmFad2-1 & 396b-fad2- 127 PHP32843 34
GmFad2-2 1b/159-fad2-2 GmSad 396b-sad3 128 -- -- GmFad2-1 &
396b-fad2- 129 PHP33705 35 GmSad 1b/396b-sad3 GmFad3 159-fad3c 130
PHP38557 36 GmFatB 159-fatBF 131 PHP41103 37 GmFad3 159-fad3c 130
pKR1756 38 GmFatB 159-fatBF 131 pKR1757 39 GmFad3 &
159-fad3c/159- 132 pKR1766 40 GmFatB fatBF GmFatB &
159-fatBF/159- 133 pKR1771 41 GmFad3 fad3c GmFad2-1 &
159-fad2-1b/159- 134 PHP41784 42 GmFatB & fatBF/159-fad3c
GmFad3 GmFad2-1 & 396b-fad2- 135 pKR1776 43 GmFatB &
1b/159-fatB/159- GmFad3 fad3c
[0304] From Table 4, the amiRNA precursor 159-fad2-1b (SEQ ID NO:
124) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
GM-MFAD2-1B amiRNA (SEQ ID NO: 21) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-GM-MFAD2-1B Star Sequence
(SEQ ID NO: 26).
[0305] From Table 4, the amiRNA precursor 396-fad2-1b (SEQ ID NO:
124) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 152 are replaced by
GM-MFAD2-1B amiRNA (SEQ ID NO: 21) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-GM-MFAD2-1B Star
Sequence (SEQ ID NO: 26).
[0306] From Table 4, the amiRNA precursor 159-fad2-2 (SEQ ID NO:
126) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by GM-MFAD2-2
amiRNA (SEQ ID NO: 22) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-GM-MFAD2-2 Star Sequence (SEQ ID NO:
28).
[0307] From Table 4, the amiRNA precursor 396b-fad2-1b/159-fad2-2
(SEQ ID NO: 127), which combines amiRNA precursors 396b-fad2-1b
(SEQ ID NO: 126) and 159-fad2-2 (SEQ ID NO: 127) into one
transcriptional unit, is 1568 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 1 to 574 of 396b-fad2-1b/159-fad2-2) wherein
nucleotides 196 to 216 of SEQ ID NO: 152 are replaced by
GM-MFAD2-1B amiRNA (SEQ ID NO: 21) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-GM-MFAD2-1B Star
Sequence (SEQ ID NO: 26). The amiRNA precursor
396b-fad2-1b/159-fad2-2 (SEQ ID NO: 127) is also, substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 611 to 1568 of 396b-fad2-1b/159-fad2-2) wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by GM-MFAD2-2
amiRNA (SEQ ID NO: 22) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-GM-MFAD2-2 Star Sequence (SEQ ID NO:
28). In amiRNA precursor 396b-fad2-1b/159-fad2-2, nt 575 to 610 are
derived from cloning.
[0308] From Table 4, the amiRNA precursor 396b-sad3 (SEQ ID NO:
128) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 152 are replaced by GM-MSAD3
amiRNA (SEQ ID NO: 23) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by GM-MSAD3 amiRNA Star Sequence (SEQ ID NO:
29).
[0309] From Table 4, the amiRNA precursor 396b-fad2-1b/396b-sad3
(SEQ ID NO: 129), which combines amiRNA precursors 396b-fad2-1b
(SEQ ID NO: 126) and 396b-sad3 (SEQ ID NO: 128) into one
transcriptional unit, is 1184 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 1 to 574 of 396b-fad2-1b/396b-sad3) wherein
nucleotides 196 to 216 of SEQ ID NO: 152 are replaced by
GM-MFAD2-1B amiRNA (SEQ ID NO: 21) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-GM-MFAD2-1B Star
Sequence (SEQ ID NO: 26). The amiRNA precursor
396b-fad2-1b/396b-sad3 (SEQ ID NO: 129) is also, substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 611 to 1184 of 396b-fad2-1b/396b-sad) wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by GM-MSAD3
amiRNA (SEQ ID NO: 23) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by GM-MSAD3 Star Sequence (SEQ ID NO: 29). In
amiRNA precursor 396b-fad2-1b/396b-sad3, nt 575 to 610 are derived
from cloning.
[0310] From Table 4, the amiRNA precursor 159-fad3c (SEQ ID NO:
130) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by GM-MFAD3C
amiRNA (SEQ ID NO: 24) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by GM-MFAD3C Star Sequence (SEQ ID NO:
30).
[0311] From Table 4, the amiRNA precursor 159-fatBF (SEQ ID NO:
131) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by GM-MFATBF
amiRNA (SEQ ID NO: 25) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by GM-MFATBF Star Sequence (SEQ ID NO:
31).
[0312] From Table 4, the amiRNA precursor 159-fad3c/159-fatBF (SEQ
ID NO: 132), which combines amiRNA precursors 159-fad3c (SEQ ID NO:
130) and 159-fatBF (SEQ ID NO: 131) into one transcriptional unit,
is 1924 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 (from nt 1
to 958 of 159-fad3c/159-fatBF) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFAD3C amiRNA (SEQ ID NO: 24) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFAD3C Star Sequence (SEQ ID NO: 30). The amiRNA precursor
159-fad3c/159-fatBF (SEQ ID NO: 132) is also, substantially similar
to the deoxyribonucleotide sequence set forth in SEQ ID NO: 152
(from nt 967 to 1924 of 159-fad3c/159-fatBF) wherein nucleotides
276 to 296 of SEQ ID NO: 152 are replaced by GM-MFATBF amiRNA (SEQ
ID NO: 25) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-GM-MFATBF Star Sequence (SEQ ID NO: 31). In amiRNA
precursor 159-fad3c/159-fatBF, nt 959 to 966 are derived from
cloning.
[0313] From Table 4, the amiRNA precursor 159-fatBF/159-fad3c (SEQ
ID NO: 133), which combines amiRNA precursors 159-fatBF (SEQ ID NO:
131) and 159-fad3c (SEQ ID NO: 130) into one transcriptional unit,
is 1924 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 (from nt 1
to 958 of 159-fatBF/159-fad3c) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFATBF amiRNA (SEQ ID NO: 25) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFATBF Star Sequence (SEQ ID NO: 31). The amiRNA precursor
159-fatBF/159-fad3c (SEQ ID NO: 133) is also, substantially similar
to the deoxyribonucleotide sequence set forth in SEQ ID NO: 152
(from nt 967 to 1924 of 159-fatBF/159-fad3c) wherein nucleotides
276 to 296 of SEQ ID NO: 152 are replaced by GM-MFAD3C amiRNA (SEQ
ID NO: 24) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by GM-MFAD3C Star Sequence (SEQ ID NO: 30). In amiRNA
precursor 159-fatBF/159-fad3c, nt 959 to 966 are derived from
cloning.
[0314] From Table 4, the amiRNA precursor
159-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 134), which combines
amiRNA precursors 159-fad2-1b (SEQ ID NO: 124), 159-fatBF (SEQ ID
NO: 131) and 159-fad3c (SEQ ID NO: 130) into one transcriptional
unit, is 2886 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 (from nt 1
to 958 of 159-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 276
to 296 of SEQ ID NO: 152 are replaced by GM-MFAD2-1B amiRNA (SEQ ID
NO: 21) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-GM-MFAD2-1B Star Sequence (SEQ ID NO: 26). The
amiRNA precursor 159-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 134)
is also, substantially similar to the deoxyribonucleotide sequence
set forth in SEQ ID NO: 152 (from nt 965 to 1922 of
159-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFATBF amiRNA (SEQ ID NO: 25) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFATBF Star Sequence (SEQ ID NO: 31). The amiRNA precursor
159-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 134) is also,
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 152 (from nt 1929 to 2886 of
159-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFAD3C amiRNA (SEQ ID NO: 24) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFAD3C Star Sequence (SEQ ID NO: 30). In amiRNA precursor
159-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 134), nt 959 to 964 and
1923 to 1928 are derived from cloning.
[0315] From Table 4, the amiRNA precursor
396b-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 135), which combines
amiRNA precursors 396b-fad2-1b (SEQ ID NO: 125), 159-fatBF (SEQ ID
NO: 131) and 159-fad3c (SEQ ID NO: 130) into one transcriptional
unit, is 2543 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 (from nt 1
to 574 of 396b-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 196
to 216 of SEQ ID NO: 152 are replaced by GM-MFAD2-1B amiRNA (SEQ ID
NO: 21) and wherein nucleotides 262 to 282 of SEQ ID NO: 153 are
replaced by 396b-GM-MFAD2-1B Star Sequence (SEQ ID NO: 26). The
amiRNA precursor 396b-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 135)
is also, substantially similar to the deoxyribonucleotide sequence
set forth in SEQ ID NO: 152 (from nt 620 to 1577 of
396b-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFATBF amiRNA (SEQ ID NO: 25) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFATBF Star Sequence (SEQ ID NO: 31). The amiRNA precursor
396b-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 135) is also,
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 152 (from nt 1586 to 2543 of
396b-fad2-1b/159-fatBF/159-fad3c) wherein nucleotides 276 to 296 of
SEQ ID NO: 152 are replaced by GM-MFAD3C amiRNA (SEQ ID NO: 24) and
wherein nucleotides 121 to 141 of SEQ ID NO: 152 are replaced by
GM-MFAD3C Star Sequence (SEQ ID NO: 30). In amiRNA precursor
396b-fad2-1b/159-fatBF/159-fad3c (SEQ ID NO: 135), nt 575 to 619
and 1578 to 1585 are derived from cloning.
Example 4
Expression of amiRNAs for Silencing Soy Fatty Acid Biosynthetic
Genes in Soybean Somatic Embryos
[0316] Plasmids pKR1756 (SEQ ID NO: 38), pKR1757 (SEQ ID NO: 39),
pKR1766 (SEQ ID NO: 40), pKR1771 (SEQ ID NO: 41) and pKR1776 (SEQ
ID NO: 43) were transformed into soybean embryogenic suspension
cultures (cv. Jack or 93B86) as described below.
Soybean Embryogenic Suspension Culture Initiation:
[0317] Soybean cultures were initiated twice each month with 5-7
days between each initiation. Pods with immature seeds from
available soybean plants 45-55 days after planting were picked,
removed from their shells and placed into a sterilized magenta box.
The soybean seeds were sterilized by shaking them for 15 min in a
5% Clorox solution with 1 drop of ivory soap (i.e., 95 mL of
autoclaved distilled water plus 5 mL Clorox and 1 drop of soap,
mixed well). Seeds were rinsed using 2 1-liter bottles of sterile
distilled water and those less than 4 mm were placed on individual
microscope slides. The small end of the seed was cut and the
cotyledons pressed out of the seed coat. Cotyledons were
transferred to plates containing SB199 medium (25-30 cotyledons per
plate) for 2 weeks, then transferred to SB1 for 2-4 weeks. Plates
were wrapped with fiber tape. After this time, secondary embryos
were cut and placed into SB196 liquid media for 7 days.
Culture Conditions
[0318] Soybean embryogenic suspension cultures (cv. Jack) were
maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker,
150 rpm, 26.degree. C. with cool white fluorescent lights on 16:8 h
day/night photoperiod at light intensity of 60-85 .mu.E/m2/s.
Cultures were subcultured every 7 days to two weeks by inoculating
approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the
preferred subculture interval is every 7 days).
Preparation of DNA for Bombardment:
[0319] Plasmids pKR1756 (SEQ ID NO: 38), pKR1757 (SEQ ID NO: 39),
pKR1766 (SEQ ID NO: 40), pKR1771 (SEQ ID NO: 41) and pKR1776 (SEQ
ID NO: 43) were prepared for transformation in the following
way.
[0320] A 50 .mu.L aliquot of sterile distilled water containing 1
mg of gold particles was added to 5 .mu.L of a 1 .mu.g/.mu.L DNA
solution, 50 .mu.L 2.5M CaCl.sub.2 and 20 .mu.L of 0.1 M
spermidine. The mixture was pulsed 5 times on level 4 of a vortex
shaker and spun for 5 sec in a bench microfuge. After a wash with
150 .mu.L of 100% ethanol, the pellet was suspended by sonication
in 85 .mu.L of 100% ethanol. Five .mu.L of DNA suspension was
dispensed to each flying disk of the Biolistic PDS1000/HE
instrument disk. Each 5 .mu.L aliquot contained approximately 0.058
mg gold particles per bombardment (i.e., per disk).
Tissue Preparation and Bombardment with DNA:
[0321] Soybean embryogenic suspension cultures were transformed by
the method of particle gun bombardment (Klein et al., Nature 327:70
(1987)) using a DuPont Biolistic PDS1000/HE instrument (helium
retrofit) for all transformations.
[0322] Approximately 100-150 mg of 7 day old embryonic suspension
cultures were placed in an empty, sterile 60.times.15 mm petri dish
and the dish was placed inside of an empty 150.times.25 mm Petri
dish. Tissue was bombarded 1 shot per plate with membrane rupture
pressure set at 650 PSI and the chamber was evacuated to a vacuum
of 27-28 inches of mercury. Tissue was placed approximately 2.5
inches from the retaining/stopping screen.
Selection of Transformed Embryos:
[0323] Transformed embryos were selected using hygromycin as the
selectable marker. Specifically, following bombardment, the tissue
was placed into fresh SB196 media and cultured as described above.
Six to eight days post-bombardment, the SB196 is exchanged with
fresh SB196 containing 30 mg/L hygromycin. The selection media was
refreshed weekly. Four to six weeks post-selection, green,
transformed tissue was observed growing from untransformed,
necrotic embryogenic clusters. Isolated, green tissue was removed
and inoculated into multi-well plates to generate new, clonally
propagated, transformed embryogenic suspension cultures.
Embryo Maturation:
[0324] Transformed embryogenic clusters were cultured for one-three
weeks at 26.degree. C. in SB196 under cool white fluorescent
(Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40
Agro) bulbs (40 watt) on a 16:8 hrphotoperiod with light intensity
of 90-120 .mu.E/m.sup.2s. Embryo clusters were removed to SB228
(SHaM) liquid media, 35 mL in 250 mL Erlenmeyer flask, for 2-3
weeks. Tissue cultured in SB228 was maintained on a rotary shaker,
130 rpm, 26.degree. C. with cool white fluorescent lights on 16:8 h
day/night photoperiod at light intensity of 60-85 .mu.E/m2/s.
During this period, individual embryos were removed from the
clusters and screened for alterations in their fatty acid
compositions as described supra. Media Recipes:
SB196--FN Lite Liquid Proliferation Medium (Per Liter)
TABLE-US-00005 [0325] MS FeEDTA - 100x Stock 1 10 mL MS Sulfate -
100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P,
B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10
mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm
(NH.sub.4).sub.2SO.sub.4 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10
gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00006 [0326] Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x
Stock Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g
1.392 g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g
MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g
0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides
100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x
Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve
in dark bottle while stirring
SB1 Solid Medium (Per Liter)
[0327] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0328] 1 mL B5 vitamins 1000.times. stock
[0329] 31.5 g Glucose
[0330] 2 mL 2,4-D (20 mg/L final concentration)
[0331] pH 5.7
[0332] 8 g TC agar
SB199 Solid Medium (Per Liter)
[0333] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0334] 1 mL B5 vitamins 1000.times. stock
[0335] 30 g Sucrose
[0336] 4 ml 2,4-D (40 mg/L final concentration)
[0337] pH 7.0
[0338] 2 gm Gelrite
SB 166 Solid Medium (Per Liter)
[0339] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0340] 1 mL B5 vitamins 1000.times. stock
[0341] 60 g maltose
[0342] 750 mg MgCl.sub.2 hexahydrate
[0343] 5 g Activated charcoal
[0344] pH 5.7
[0345] 2 g Gelrite
SB 103 Solid Medium (Per Liter)
[0346] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0347] 1 mL B5 vitamins 1000.times. stock
[0348] 60 g maltose
[0349] 750 mg MgCl2 hexahydrate
[0350] pH 5.7
[0351] 2 g Gelrite
SB 71-4 Solid Medium (Per Liter)
[0352] 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL--Cat. No.
21153-036)
[0353] pH 5.7
[0354] 5 g TC agar
2,4-D Stock
[0355] Obtain premade from Phytotech Cat. No. D 295--concentration
1 mg/mL
B5 Vitamins Stock (Per 100 mL)
[0356] Store aliquots at -20.degree. C.
[0357] 10 g Myo-inositol
[0358] 100 mg Nicotinic acid
[0359] 100 mg Pyridoxine HCl
[0360] 1 g Thiamine
[0361] If the solution does not dissolve quickly enough, apply a
low level of heat via the hot stir plate.
SB 228--Soybean Histodifferentiation & Maturation (SHaM) (Per
Liter)
TABLE-US-00007 [0362] DDI H2O 600 ml FN-Lite Macro Salts for SHaM
10X 100 ml MS Micro Salts 1000x 1 ml MS FeEDTA 100x 10 ml CaCl 100x
6.82 ml B5 Vitamins 1000x 1 ml L-Methionine 0.149 g Sucrose 30 g
Sorbitol 30 g Adjust volume to 900 mL pH 5.8 Autoclave Add to
cooled media (.ltoreq.30 C.): *Glutamine (Final conc. 30 mM) 4% 110
mL *Note: Final volume will be 1010 mL after glutamine
addition.
[0363] Because glutamine degrades relatively rapidly, it may be
preferable to add immediately prior to using media. Expiration 2
weeks after glutamine is added; base media can be kept longer w/o
glutamine.
FN-Lite Macro for SHAM 10.times.--Stock #1 (Per Liter
TABLE-US-00008 [0364] (NH.sub.4)2SO.sub.4 (Ammonium Sulfate) 4.63 g
KNO.sub.3 (Potassium Nitrate) 28.3 g MgSO.sub.4*7H.sub.20
(Magnesium Sulfate Heptahydrate) 3.7 g KH.sub.2PO.sub.4 (Potassium
Phosphate, Monobasic) 1.85 g Bring to volume Autoclave
MS Micro 1000.times.--Stock #2 (Per 1 Liter)
TABLE-US-00009 [0365] H.sub.3BO.sub.3 (Boric Acid) 6.2 g
MnSO.sub.4*H.sub.2O (Manganese Sulfate Monohydrate) 16.9 g
ZnSO4*7H20 (Zinc Sulfate Heptahydrate) 8.6 g Na.sub.2MoO.sub.4*2H20
(Sodium Molybdate Dihydrate) 0.25 g CuSO.sub.4*5H.sub.20 (Copper
Sulfate Pentahydrate) 0.025 g CoCl.sub.2*6H.sub.20 (Cobalt Chloride
Hexahydrate) 0.025 g KI (Potassium Iodide) 0.8300 g Bring to volume
Autoclave
FeEDTA 100.times.--Stock #3 (Per Liter)
TABLE-US-00010 [0366] Na.sub.2EDTA* (Sodium EDTA) 3.73 g
FeSO.sub.4*7H.sub.20 (Iron Sulfate Heptahydrate) 2.78 g *EDTA must
be completely dissolved before adding iron. Bring to Volume
Solution is photosensitive. Bottle(s) should be wrapped in foil to
omit light. Autoclave
Ca 100.times.--Stock #4 (Per Liter)
TABLE-US-00011 [0367] CaCl.sub.2*2H.sub.20 (Calcium Chloride
Dihydrate) 44 g Bring to Volume Autoclave
B5 Vitamin 1000.times.--Stock #5 (Per Liter)
TABLE-US-00012 [0368] Thiamine*HCl 10 g Nicotinic Acid 1 g
Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store
frozen
4% Glutamine--Stock #6 (Per Liter)
TABLE-US-00013 [0369] DDI water heated to 30.degree. C. 900 ml
L-Glutamine 40 g Gradually add while stirring and applying low
heat. Do not exceed 35.degree. C. Bring to Volume Filter Sterilize
Store frozen * * Note: Warm thawed stock in 31.degree. C. bath to
fully dissolve crystals.
Functional Analysis in Somatic Soybean Embryos
[0370] Mature somatic soybean embryos are a good model for zygotic
embryos. While in the globular embryo state in liquid culture,
somatic soybean embryos contain very low amounts of triacylglycerol
(TAG) or storage proteins typical of maturing, zygotic soybean
embryos. At this developmental stage, the ratio of total
triacylglyceride to total polar lipid (phospholipids and
glycolipid) is about 1:4, as is typical of zygotic soybean embryos
at the developmental stage from which the somatic embryo culture
was initiated. At the globular stage as well, the mRNAs for the
prominent seed proteins, .alpha.'-subunit of .beta.-conglycinin,
kunitz trypsin inhibitor 3, and seed lectin are essentially absent.
Upon transfer to hormone-free media to allow differentiation to the
maturing somatic embryo state, TAG becomes the most abundant lipid
class. As well, mRNAs for .alpha.'-subunit of .beta.-conglycinin,
kunitz trypsin inhibitor 3 and seed lectin become very abundant
messages in the total mRNA population. On this basis, the somatic
soybean embryo system behaves very similarly to maturing zygotic
soybean embryos in vivo, and is thus a good and rapid model system
for analyzing the phenotypic effects of modifying the expression of
genes in the fatty acid biosynthesis pathway (see PCT Publication
No. WO 2002/00904). The model system is also predictive of the
fatty acid composition of seeds from plants derived from transgenic
embryos.
Fatty Acid Analysis:
[0371] Somatic embryos were harvested after two weeks of culture in
the liquid maturation medium SB228 (SHaM) liquid media.
Approximately 30 events were created in transformations with
plasmids pKR1756 (SEQ ID NO: 38), pKR1757 (SEQ ID NO: 39), pKR1766
(SEQ ID NO: 40), pKR1771 (SEQ ID NO: 41) and pKR1776 (SEQ ID NO:
43), having experiment names MSE2887, MSE2888, MSE2889, MSE2890 and
MSE2723, respectively.
[0372] All embryos generated for a given event were harvested in
bulk and processed as follows. Approximately 10-20 embryos were
frozen by incubation in a -80.degree. C. freezer for 24 h followed
by lyophilization for 48 h.
[0373] Dried embryos were ground to a fine powder using a
genogrinder vial (1/2''.times.2'' polycarbonate) and a steel ball
(SPEX Centriprep (Metuchen, N.J., U.S.A.). Grinding time was 30 sec
at 1450 oscillations per min.
[0374] Lipids from approximately 20-50 mg of dried embryo powder
were transesterified to fatty acid methyl esters (FAME) and
analyzed by GC as described in PCT Publication No. WO
2008/147935.
[0375] Briefly, 50 .mu.L of trimethylsulfonium hydroxide (TMSH)
reagent and 0.5 mL of hexane were added to the dried embryo powder
in glass GC vials and incubated for 30 min at room temperature
while shaking. Fatty acid methyl esters (1 .mu.L injected from
hexane layer) were separated and quantified using a Hewlett-Packard
6890 Gas Chromatograph fitted with an Omegawax 320 fused silica
capillary column (Catalog #24152, Supelco Inc.). The oven
temperature was programmed to hold at 220.degree. C. for 2.6 min,
increase to 240.degree. C. at 20.degree. C./min and then hold for
an additional 2.4 min. Carrier gas was supplied by a Whatman
hydrogen generator. Retention times were compared to those for
methyl esters of standards commercially available (Nu-Chek Prep,
Inc.).
[0376] The resulting fatty acid profiles for events from MSE2887,
MSE2888, MSE2889, MSE2890 and MSE2723 are summarized in TABLE 5. In
TABLE 5, fatty acids are identified as 16:0 (palmitate), 18:0
(stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid) and 18:3
(alpha-linolenic acid) and are expressed as a weight percent (wt.
%) of total fatty acids. For MSE2887 and MSE2889 results are sorted
for 18:3 in ascending order. For MSE2888, MSE2890 and MSE2723,
results are sorted for 16:0 in ascending order. The average fatty
acid profile for all events for each experiment is shown as
avg.
TABLE-US-00014 TABLE 5 Fatty Acid Profiles For MSE2887, MSE2888,
MSE2889, MSE2890 and MSE2723 Event Construct amiRNAs 16:0 18:0 18:1
18:2 18:3 MSE2887-12 pKR1756 fad3 16.8 5.5 19.4 53.1 5.1 MSE2887-8
pKR1756 fad3 16.2 5.9 22.0 50.5 5.5 MSE2887-24 pKR1756 fad3 17.2
7.2 23.5 46.6 5.5 MSE2887-13 pKR1756 fad3 17.7 5.5 20.0 51.1 5.8
MSE2887-23 pKR1756 fad3 17.7 5.8 21.2 49.1 6.2 MSE2887-30 pKR1756
fad3 17.0 6.4 21.6 47.4 7.6 MSE2887-28 pKR1756 fad3 17.9 6.0 15.1
53.4 7.6 MSE2887-9 pKR1756 fad3 17.3 5.7 20.2 48.8 8.0 MSE2887-29
pKR1756 fad3 16.5 4.7 15.1 55.5 8.1 MSE2887-25 pKR1756 fad3 16.6
5.1 19.4 50.6 8.2 MSE2887-16 pKR1756 fad3 17.6 5.9 19.4 48.7 8.4
MSE2887-6 pKR1756 fad3 17.1 5.3 18.9 49.8 8.9 MSE2887-11 pKR1756
fad3 16.0 6.6 21.3 46.6 9.5 MSE2887-18 pKR1756 fad3 16.3 6.1 21.7
45.8 10.0 MSE2887-27 pKR1756 fad3 16.2 7.1 23.8 42.6 10.2
MSE2887-19 pKR1756 fad3 16.5 4.9 20.2 47.0 11.4 MSE2887-31 pKR1756
fad3 17.4 5.8 17.6 47.6 11.5 MSE2887-2 pKR1756 fad3 16.4 5.2 18.9
46.9 12.6 MSE2887-22 pKR1756 fad3 17.4 5.9 20.1 43.5 13.1
MSE2887-14 pKR1756 fad3 16.2 6.3 21.1 43.3 13.2 MSE2887-4 pKR1756
fad3 14.2 5.4 21.9 45.0 13.4 MSE2887-17 pKR1756 fad3 16.6 4.9 13.4
51.3 13.8 MSE2887-1 pKR1756 fad3 17.5 5.0 17.0 46.4 14.1 MSE2887-26
pKR1756 fad3 17.7 5.4 14.1 48.1 14.7 MSE2887-21 pKR1756 fad3 17.8
6.0 17.3 43.6 15.4 MSE2887-20 pKR1756 fad3 17.9 5.1 12.1 49.2 15.6
MSE2887-15 pKR1756 fad3 17.9 6.4 16.5 43.6 15.7 MSE2887-10 pKR1756
fad3 17.3 4.8 16.9 45.2 15.7 MSE2887-5 pKR1756 fad3 17.6 5.2 18.5
42.7 16.1 MSE2887-3 pKR1756 fad3 18.4 4.8 14.4 45.5 16.8 MSE2887-7
pKR1756 fad3 16.5 5.1 18.4 41.9 18.1 Avg. 17.0 5.6 18.7 47.4 11.2
MSE2888-12 pKR1757 fatBF 4.7 3.6 22.9 56.7 12.1 MSE2888-30 pKR1757
fatBF 5.3 3.7 22.0 56.9 12.2 MSE2888-27 pKR1757 fatBF 6.1 5.4 28.0
46.9 13.5 MSE2888-26 pKR1757 fatBF 6.6 4.3 22.4 55.3 11.4
MSE2888-22 pKR1757 fatBF 6.8 4.6 21.5 54.5 12.7 MSE2888-28 pKR1757
fatBF 6.9 4.5 22.7 51.4 14.4 MSE2888-13 pKR1757 fatBF 7.5 4.2 21.9
55.2 11.2 MSE2888-2 pKR1757 fatBF 7.6 5.0 26.0 46.9 14.6 MSE2888-21
pKR1757 fatBF 7.8 4.3 23.8 52.0 12.0 MSE2888-15 pKR1757 fatBF 7.9
3.7 21.4 55.3 11.6 MSE2888-31 pKR1757 fatBF 8.1 5.5 30.3 44.3 11.7
MSE2888-11 pKR1757 fatBF 8.4 5.1 24.2 48.8 13.5 MSE2888-20 pKR1757
fatBF 8.7 5.2 23.2 48.0 14.9 MSE2888-18 pKR1757 fatBF 8.8 6.6 26.6
46.1 11.9 MSE2888-14 pKR1757 fatBF 8.8 4.2 20.9 53.9 12.3
MSE2888-29 pKR1757 fatBF 9.4 6.1 28.0 44.4 12.2 MSE2888-16 pKR1757
fatBF 9.4 4.8 23.0 49.7 13.0 MSE2888-19 pKR1757 fatBF 9.6 5.3 22.4
48.5 14.3 MSE2888-23 pKR1757 fatBF 9.7 5.0 22.7 47.9 14.8 MSE2888-6
pKR1757 fatBF 11.5 5.5 21.3 50.5 11.2 MSE2888-17 pKR1757 fatBF 12.3
4.4 20.6 49.4 13.3 MSE2888-5 pKR1757 fatBF 13.3 5.3 21.4 47.7 12.3
MSE2888-10 pKR1757 fatBF 13.5 5.5 22.6 45.9 12.4 MSE2888-9 pKR1757
fatBF 14.5 6.6 28.3 37.6 13.0 MSE2888-3 pKR1757 fatBF 15.0 8.0 25.4
39.2 12.5 MSE2888-25 pKR1757 fatBF 15.1 6.1 23.8 42.1 13.0
MSE2888-8 pKR1757 fatBF 15.1 6.7 26.1 40.9 11.2 MSE2888-1 pKR1757
fatBF 15.5 6.9 24.2 41.2 12.2 MSE2888-7 pKR1757 fatBF 16.0 5.3 20.2
46.6 11.9 MSE2888-4 pKR1757 fatBF 16.4 4.9 20.1 45.5 13.2
MSE2888-24 pKR1757 fatBF 16.5 5.1 19.3 46.4 12.7 Avg. 10.4 5.2 23.4
48.2 12.7 MSE2889-7 pKR1766 fad3/fatBF 6.9 3.8 22.9 60.6 5.9
MSE2889-8 pKR1766 fad3/fatBF 7.3 3.4 22.6 60.8 5.9 MSE2889-16
pKR1766 fad3/fatBF 9.5 4.3 21.9 57.6 6.7 MSE2889-3 pKR1766
fad3/fatBF 16.4 5.2 19.2 52.4 6.8 MSE2889-15 pKR1766 fad3/fatBF
12.5 5.5 23.0 51.8 7.1 MSE2889-1 pKR1766 fad3/fatBF 11.3 4.9 22.8
53.7 7.2 MSE2889-2 pKR1766 fad3/fatBF 8.7 5.6 27.5 51.0 7.2
MSE2889-31 pKR1766 fad3/fatBF 10.4 4.2 19.2 58.8 7.3 MSE2889-18
pKR1766 fad3/fatBF 9.9 4.1 19.3 59.4 7.4 MSE2889-20 pKR1766
fad3/fatBF 9.2 4.3 21.5 57.5 7.5 MSE2889-14 pKR1766 fad3/fatBF 11.5
5.3 23.5 51.8 7.9 MSE2889-29 pKR1766 fad3/fatBF 11.1 4.4 19.8 56.6
8.1 MSE2889-9 pKR1766 fad3/fatBF 9.5 4.1 21.6 56.6 8.2 MSE2889-12
pKR1766 fad3/fatBF 10.7 4.1 20.1 56.8 8.2 MSE2889-11 pKR1766
fad3/fatBF 11.3 4.7 20.4 54.0 9.5 MSE2889-10 pKR1766 fad3/fatBF
11.5 4.9 24.3 49.7 9.6 MSE2889-19 pKR1766 fad3/fatBF 11.8 5.2 23.3
49.5 10.2 MSE2889-23 pKR1766 fad3/fatBF 13.7 6.4 23.3 46.4 10.2
MSE2889-13 pKR1766 fad3/fatBF 12.7 4.2 20.4 52.3 10.3 MSE2889-22
pKR1766 fad3/fatBF 13.9 6.0 22.2 47.5 10.3 MSE2889-28 pKR1766
fad3/fatBF 12.0 4.9 22.7 50.0 10.4 MSE2889-30 pKR1766 fad3/fatBF
16.0 4.6 18.9 49.7 10.7 MSE2889-4 pKR1766 fad3/fatBF 12.4 5.9 23.6
46.9 11.1 MSE2889-26 pKR1766 fad3/fatBF 16.3 5.0 19.5 47.7 11.5
MSE2889-6 pKR1766 fad3/fatBF 14.9 5.8 20.9 46.7 11.7 MSE2889-5
pKR1766 fad3/fatBF 15.8 5.6 21.0 45.9 11.7 MSE2889-21 pKR1766
fad3/fatBF 15.4 4.2 19.9 48.5 12.0 MSE2889-17 pKR1766 fad3/fatBF
15.7 6.7 21.9 43.6 12.0 MSE2889-25 pKR1766 fad3/fatBF 16.4 5.2 19.8
46.3 12.2 MSE2889-24 pKR1766 fad3/fatBF 15.6 6.0 22.3 43.5 12.6
MSE2889-27 pKR1766 fad3/fatBF 15.9 5.5 20.0 44.0 14.7 Avg. 12.5 5.0
21.6 51.5 9.4 MSE2890-14 pKR1771 fatBF/fad3 5.0 3.2 21.9 55.6 14.3
MSE2890-29 pKR1771 fatBF/fad3 6.7 5.3 25.7 55.5 6.8 MSE2890-17
pKR1771 fatBF/fad3 7.3 5.2 27.9 46.3 13.3 MSE2890-13 pKR1771
fatBF/fad3 7.6 5.0 24.8 50.1 12.6 MSE2890-6 pKR1771 fatBF/fad3 7.8
3.8 20.9 58.8 8.7 MSE2890-5 pKR1771 fatBF/fad3 8.3 3.7 22.7 54.7
10.7 MSE2890-10 pKR1771 fatBF/fad3 9.0 4.5 22.0 54.5 9.9 MSE2890-12
pKR1771 fatBF/fad3 9.3 5.3 23.0 52.7 9.7 MSE2890-26 pKR1771
fatBF/fad3 9.3 4.8 22.8 53.5 9.5 MSE2890-19 pKR1771 fatBF/fad3 10.2
5.5 23.2 51.8 9.4 MSE2890-1 pKR1771 fatBF/fad3 10.8 5.2 21.3 51.1
11.6 MSE2890-9 pKR1771 fatBF/fad3 10.8 5.4 22.7 47.6 13.4
MSE2890-23 pKR1771 fatBF/fad3 11.0 4.2 19.3 55.1 10.5 MSE2890-24
pKR1771 fatBF/fad3 11.4 5.9 23.4 50.4 8.8 MSE2890-30 pKR1771
fatBF/fad3 11.6 3.8 20.2 53.3 11.2 MSE2890-16 pKR1771 fatBF/fad3
11.6 4.2 18.4 53.4 12.4 MSE2890-15 pKR1771 fatBF/fad3 12.9 4.9 19.7
49.3 13.2 MSE2890-2 pKR1771 fatBF/fad3 13.0 5.6 24.6 47.2 9.6
MSE2890-21 pKR1771 fatBF/fad3 13.8 6.3 23.1 45.6 11.1 MSE2890-18
pKR1771 fatBF/fad3 14.1 4.3 20.4 50.3 10.9 MSE2890-3 pKR1771
fatBF/fad3 15.2 5.6 20.9 45.3 13.0 MSE2890-25 pKR1771 fatBF/fad3
15.6 5.2 21.0 44.8 13.4 MSE2890-4 pKR1771 fatBF/fad3 15.6 5.7 20.9
46.1 11.6 MSE2890-8 pKR1771 fatBF/fad3 15.8 5.5 19.2 42.8 16.6
MSE2890-7 pKR1771 fatBF/fad3 15.9 4.7 19.1 45.8 14.5 MSE2890-11
pKR1771 fatBF/fad3 16.0 6.0 18.8 42.5 16.7 MSE2890-22 pKR1771
fatBF/fad3 16.0 4.2 18.6 47.3 13.9 MSE2890-20 pKR1771 fatBF/fad3
16.2 5.2 19.7 47.3 11.7 MSE2890-27 pKR1771 fatBF/fad3 16.5 6.5 21.0
43.4 12.6 MSE2890-28 pKR1771 fatBF/fad3 16.7 4.4 18.1 48.1 12.6
Avg. 12.0 5.0 21.5 49.7 11.8 MSE2723-14 pKR1776 fad2/fatB/fad3 6.2
2.5 36.3 39.9 15.1 MSE2723-16 pKR1776 fad2/fatB/fad3 6.9 2.7 44.4
37.1 8.9 MSE2723-25 pKR1776 fad2/fatB/fad3 7.5 3.9 49.2 29.6 9.7
MSE2723-18 pKR1776 fad2/fatB/fad3 8.2 2.6 36.4 36.6 16.2 MSE2723-4
pKR1776 fad2/fatB/fad3 8.5 3.5 24.9 52.4 10.7 MSE2723-2 pKR1776
fad2/fatB/fad3 8.7 2.9 32.1 44.1 12.2 MSE2723-13 pKR1776
fad2/fatB/fad3 9.0 5.1 50.6 25.0 10.3 MSE2723-15 pKR1776
fad2/fatB/fad3 9.1 4.4 38.9 37.7 10.0 MSE2723-7 pKR1776
fad2/fatB/fad3 11.0 4.4 25.5 42.8 16.3 MSE2723-12 pKR1776
fad2/fatB/fad3 11.2 5.1 37.2 33.5 13.1 MSE2723-3 pKR1776
fad2/fatB/fad3 11.2 4.6 45.7 28.7 9.7 MSE2723-26 pKR1776
fad2/fatB/fad3 11.9 4.5 35.2 34.4 14.0 MSE2723-17 pKR1776
fad2/fatB/fad3 11.9 3.9 25.5 40.4 18.2 MSE2723-27 pKR1776
fad2/fatB/fad3 12.4 4.5 26.7 36.2 20.1 MSE2723-22 pKR1776
fad2/fatB/fad3 13.4 5.0 19.9 48.4 13.2 MSE2723-21 pKR1776
fad2/fatB/fad3 13.6 3.4 21.5 40.4 21.1 MSE2723-31 pKR1776
fad2/fatB/fad3 13.7 4.9 17.4 51.5 12.5 MSE2723-6 pKR1776
fad2/fatB/fad3 14.1 4.6 29.0 35.7 16.6 MSE2723-1 pKR1776
fad2/fatB/fad3 14.5 4.1 25.5 39.2 16.7 MSE2723-10 pKR1776
fad2/fatB/fad3 14.7 5.7 30.1 34.7 14.9 MSE2723-30 pKR1776
fad2/fatB/fad3 14.9 5.2 13.8 40.4 25.7 MSE2723-23 pKR1776
fad2/fatB/fad3 15.0 4.3 24.3 43.8 12.6 MSE2723-8 pKR1776
fad2/fatB/fad3 15.7 4.5 12.8 38.8 28.1 MSE2723-28 pKR1776
fad2/fatB/fad3 15.9 3.7 17.0 49.7 13.7 MSE2723-29 pKR1776
fad2/fatB/fad3 16.3 5.2 17.2 44.0 17.3 MSE2723-24 pKR1776
fad2/fatB/fad3 16.3 4.8 17.9 41.8 19.2 MSE2723-9 pKR1776
fad2/fatB/fad3 16.4 3.8 12.8 44.0 23.1 MSE2723-20 pKR1776
fad2/fatB/fad3 16.7 6.3 22.1 36.9 18.0 MSE2723-5 pKR1776
fad2/fatB/fad3 16.8 4.2 15.5 48.1 15.5 MSE2723-11 pKR1776
fad2/fatB/fad3 17.1 5.0 18.0 44.1 15.8 MSE2723-19 pKR1776
fad2/fatB/fad3 17.5 4.1 19.3 45.3 13.7 Avg. 12.8 4.3 27.2 40.2
15.6
[0377] A summary of the average fatty acid profiles for each
experiment is shown in Table 6. Also included in Table 6 is the
range of fatty acid content for 16:0, 18:1 and 18:3 for each
experiment.
TABLE-US-00015 TABLE 6 Average Fatty Acid Profiles For MSE2887,
MSE2888, MSE2889, MSE2890 and MSE2723 Avg. Avg. Avg. Avg. Avg.
Range Range Range Experiment 16:0 18:0 18:1 18:2 18:3 16:0 18:1
18:3 MSE2887 17.0 5.6 18.7 47.4 11.2 14.2-18.4 12.1-23.8 5.1-18.1
MSE2888 10.4 5.2 23.4 48.2 12.7 4.7-16.5 19.3-30.3 11.2-12.7
MSE2889 12.5 5.0 21.6 51.5 9.4 6.9-16.4 18.9-27.5 5.9-14.7 MSE2890
12.0 5.0 21.5 49.7 11.8 5.0-16.7 18.1-27.9 6.8-16.7 MSE2723 12.8
4.3 27.2 40.2 15.6 6.2-17.5 12.8-50.6 8.9-28.1
[0378] Table 6 shows that in all experiments where the fad3 amiRNA
is present, the 18:3 content is significantly lowered while in
MSE2888, 18:3 content resembles that for wild-type embryos.
Similarly, in all experiments where the fatB amiRNA is present,
16:0 content is significantly lowered while in MSE2887, 16:0
content resembles that of wild-type embryos. When the fad2 amiRNA
is added oleic acid contents increase significantly above what is
seen when the fatB amiRNA, fad3 amiRNA or both fatB and fad3
amiRNAs are present.
Example 5
Expression of amiRNAs for Silencing Soy Fatty Acid Biosynthetic
Genes in Soybean Seed
[0379] Plasmid DNA fragments from PHP32511, containing the
159-fad2-1b amiRNA (SEQ ID NO: 32), PHP32510, containing the
396b-fad2-1b amiRNA (SEQ ID NO: 33), PHP32843, containing the
369b-fad2-1b & 159-fad2-2 amiRNAs (SEQ ID NO: 34), PHP33705,
containing the 396-fad2-1b & 396b-sad3 amiRNA (SEQ ID NO: 35),
PHP38557, containing the 159-fad3c amiRNA (SEQ ID NO: 36) and
PHP41103, containing the 159-fatBF amiRNA (SEQ ID NO: 37) were
transformed into soybean embryogenic suspension cultures (cv. Jack)
as described in Example 4.
[0380] Plasmid DNA fragment from PHP41784 (SEQ ID NO: 42),
containing the 159-fad2-1b & 159-fatBF& 159-fad3c amiRNAs,
was transformed into soybean embryogenic suspension culture (cv.
93B86) also as described in Example 4.
[0381] DNA was prepared, transgenic embryos were matured and
transgenic plants were obtained as described below.
Preparation of DNA for Bombardment:
[0382] For every seventeen bombardment transformations, 85 .mu.L of
suspension is prepared containing 1 to 90 picograms (pg) of
purified fragment from plasmid DNA per base pair of each DNA
fragment. DNA fragments were co-precipitated onto gold particles as
follows. The DNAs in suspension were added to 50 .mu.L of a 20-60
mg/mL 0.6 .mu.m gold particle suspension and then combined with 50
.mu.L CaCl.sub.2 (2.5 M) and 20 .mu.L spermidine (0.1 M). The
mixture was vortexed for 5 sec, spun in a microfuge for 5 sec, and
the supernatant removed. The DNA-coated particles were then washed
once with 150 .mu.L of 100% ethanol, vortexed and spun in a
microfuge again, then resuspended in 85 .mu.L of anhydrous ethanol.
Five .mu.L of the DNA-coated gold particles were then loaded on
each macrocarrier disk.
Embryo Maturation:
[0383] Seven days after bombardment, the liquid medium was
exchanged with fresh SB196 medium supplemented with 30-50 mg/L
hygromycin. The selective medium was subsequently refreshed weekly
or biweekly. Seven weeks post-bombardment, bright green,
transformed tissue was observed growing from untransformed,
chlorotic or necrotic embryogenic clusters. Isolated green tissue
was removed and inoculated into individual wells in six-well
culture dishes to generate new, clonally-propagated, transformed
embryogenic suspension cultures. Thus, each new line was treated as
independent transformation event in an individual well. These
suspensions can then be maintained as suspensions of embryos
clustered in an immature developmental stage through subculture or
they can be regenerated into whole plants by maturation and
germination of individual somatic embryos.
[0384] After two weeks in individual cell wells, transformed
embryogenic clusters were removed from liquid culture and placed on
solidified medium (SB166) containing no hormones or antibiotics for
one week. Embryos were cultured for at 26.degree. C. with mixed
fluorescent and incandescent lights on a 16 h day/8 h night
schedule. After one week, the cultures were then transferred to
SB103 medium and maintained in the same growth conditions for 3
additional weeks.
Plant Regeneration:
[0385] Somatic embryos became suitable for germination after four
weeks. As described in Example 4, somatic embryos are a good model
zygotic embryos and for each experiment, a subset of somatic
embryos at this stage from each experiment were removed and
analyzed for changes in fatty acid profile as described in Example
4.
[0386] In order to regenerate plants, remaining somatic embryos
were then removed from the maturation medium and dried in empty
petri dishes for one to five days. The dried embryos were then
planted in SB71-4 medium where they were allowed to germinate under
the same light and temperature conditions as described above.
Germinated embryos were transferred to sterile soil and grown to
maturity for seed production.
[0387] T1 seed were screened for alterations in fatty acid
composition. For every event, approximately 10-20 T1 seed were
analyzed. Soybean seed chips were produced by cutting the seed with
a razorblade avoiding the embryonic axis. Seed chips of
approximately 2 mg were placed in a vial containing 50 .mu.L TMSH
and 0.5 mL hexane and after incubation at room temperature for 30
min., fatty acid methyl esters (FAMES) from the hexane phase were
analyzed by GC as described in Example 4. Subsequent generations of
greenhouse or field grown seed was analyzed similarly for changes
in fatty acid composition.
[0388] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP32510, containing the 369b-fad2-1b amiRNA is
shown in Table 7a.
[0389] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP32511, containing the 159-fad2-1b amiRNA is
shown in Table 7b.
[0390] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP32843, containing the 369b-fad2-1b &
159-fad2-2 amiRNAs is shown in Table 7c.
[0391] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP33705, containing the 396-fad2-1b &
396b-sad3 amiRNAs is shown in Table 17d.
[0392] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP38557, containing the 159-fad3c amiRNA, is
shown in Table 7e.
[0393] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP41103, containing the 159-fatBF amiRNA, is
shown in Table 7f.
[0394] The fatty acid profile for T1 seed analyzed from soy
transformed with PHP41784, containing the 159-fad2-1b &
159-fatBF& 159-fad3c amiRNAs is shown in Table 7g.
TABLE-US-00016 TABLE 7a Fatty acid profile for T1 seed analyzed
from soy transformed with PHP32510, containing the 369b-fad2-1b
amiRNA grown in the greenhouse. Seed T1 Seed amiRNA Construct Event
No. Planted 16:0 18:0 18:1 18:2 18:3 396b-fad2-1b PHP32510 AFS
5260.2.3 1 7.9 3.2 77.5 3.4 7.9 396b-fad2-1b PHP32510 AFS 5260.2.3
2 8GR31-2 7.6 3.2 80.2 2.3 6.7 396b-fad2-1b PHP32510 AFS 5260.2.3 3
11.5 3.9 15.4 55.7 13.4 396b-fad2-1b PHP32510 AFS 5260.2.3 4 11.3
3.9 11.4 56.4 17.0 396b-fad2-1b PHP32510 AFS 5260.2.3 5 9.4 3.1
72.7 5.3 9.5 396b-fad2-1b PHP32510 AFS 5260.2.3 6 8.3 3.1 77.6 2.9
8.2 396b-fad2-1b PHP32510 AFS 5260.2.4 1 12.1 3.7 14.5 55.6 14.1
396b-fad2-1b PHP32510 AFS 5260.2.4 2 13.1 2.7 12.5 54.8 16.9
396b-fad2-1b PHP32510 AFS 5260.2.4 3 11.8 3.5 15.8 57.1 11.7
396b-fad2-1b PHP32510 AFS 5260.2.4 4 11.7 3.5 17.4 56.1 11.3
396b-fad2-1b PHP32510 AFS 5260.2.4 5 8.5 3.6 74.6 4.5 8.8
396b-fad2-1b PHP32510 AFS 5260.2.4 6 9.1 3.9 72.0 5.9 9.1
396b-fad2-1b PHP32510 AFS 5260.3.10 1 13.1 3.4 22.6 46.7 14.3
396b-fad2-1b PHP32510 AFS 5260.3.10 2 11.7 3.4 18.0 52.2 14.7
396b-fad2-1b PHP32510 AFS 5260.3.10 3 11.2 3.1 22.2 49.9 13.6
396b-fad2-1b PHP32510 AFS 5260.3.10 4 12.6 3.0 20.3 47.4 16.6
396b-fad2-1b PHP32510 AFS 5260.3.10 5 12.4 3.4 17.5 52.6 14.1
396b-fad2-1b PHP32510 AFS 5260.3.10 6 14.2 3.1 12.6 50.5 19.6
396b-fad2-1b PHP32510 AFS 5260.3.11 1 8GR31-7 8.3 2.9 79.8 1.9 7.0
396b-fad2-1b PHP32510 AFS 5260.3.11 2 7.6 2.5 80.5 1.6 7.9
396b-fad2-1b PHP32510 AFS 5260.3.11 3 7.9 2.8 79.9 2.2 7.2
396b-fad2-1b PHP32510 AFS 5260.3.11 4 8.6 3.2 76.1 3.8 8.3
396b-fad2-1b PHP32510 AFS 5260.3.11 5 8.0 2.2 81.8 2.2 5.8
396b-fad2-1b PHP32510 AFS 5260.3.11 6 8.5 2.3 77.0 2.3 9.9
396b-fad2-1b PHP32510 AFS 5260.3.3 1 10.8 3.3 18.8 56.5 10.7
396b-fad2-1b PHP32510 AFS 5260.3.3 2 12.4 3.2 20.2 53.7 10.6
396b-fad2-1b PHP32510 AFS 5260.3.3 3 9.3 2.9 78.6 3.5 5.7
396b-fad2-1b PHP32510 AFS 5260.3.3 4 8.1 3.3 76.4 4.2 8.1
396b-fad2-1b PHP32510 AFS 5260.3.3 5 9.0 3.0 76.3 3.8 7.8
396b-fad2-1b PHP32510 AFS 5260.3.3 6 8GR31-16 9.3 3.3 74.4 5.7 7.3
396b-fad2-1b PHP32510 AFS 5260.3.6 1 8GR31-19 12.1 3.5 18.5 54.0
11.9 396b-fad2-1b PHP32510 AFS 5260.3.6 2 7.6 3.5 82.8 1.3 4.8
396b-fad2-1b PHP32510 AFS 5260.3.6 3 12.1 4.0 19.5 53.4 11.0
396b-fad2-1b PHP32510 AFS 5260.3.6 4 8.4 4.2 75.3 3.9 8.1
396b-fad2-1b PHP32510 AFS 5260.3.6 5 8.1 3.3 79.5 2.3 6.9
396b-fad2-1b PHP32510 AFS 5260.3.6 6 8.4 3.5 77.8 3.2 7.1
396b-fad2-1b PHP32510 AFS 5260.4.11 1 11.1 3.6 10.0 54.5 20.9
396b-fad2-1b PHP32510 AFS 5260.4.11 2 8.2 3.1 73.1 6.2 9.5
396b-fad2-1b PHP32510 AFS 5260.4.11 3 11.9 3.2 12.7 55.4 16.7
396b-fad2-1b PHP32510 AFS 5260.4.11 4 8.6 3.0 70.8 7.8 9.8
396b-fad2-1b PHP32510 AFS 5260.4.11 5 11.2 3.3 13.0 56.2 16.3
396b-fad2-1b PHP32510 AFS 5260.4.11 6 11.8 3.5 12.0 53.7 19.0
396b-fad2-1b PHP32510 AFS 5260.4.5 1 11.2 3.4 12.4 57.1 15.9
396b-fad2-1b PHP32510 AFS 5260.4.5 2 8.1 3.2 75.8 4.4 8.5
396b-fad2-1b PHP32510 AFS 5260.4.5 3 8GR31-24 8.4 3.1 75.8 4.8 7.9
396b-fad2-1b PHP32510 AFS 5260.4.5 4 11.4 3.2 15.1 54.2 16.2
396b-fad2-1b PHP32510 AFS 5260.4.5 5 7.8 3.4 78.7 2.9 7.2
396b-fad2-1b PHP32510 AFS 5260.4.5 6 8.2 3.4 72.2 7.0 9.2
396b-fad2-1b PHP32510 AFS 5260.4.8 1 8.0 3.2 78.4 3.2 7.2
396b-fad2-1b PHP32510 AFS 5260.4.8 2 10.9 3.6 11.8 58.0 15.7
396b-fad2-1b PHP32510 AFS 5260.4.8 3 7.7 3.4 77.2 3.6 8.1
396b-fad2-1b PHP32510 AFS 5260.4.8 4 7.9 3.1 76.1 4.2 8.7
396b-fad2-1b PHP32510 AFS 5260.4.8 5 8.6 2.9 73.7 5.9 9.0
396b-fad2-1b PHP32510 AFS 5260.4.8 6 8.3 2.9 77.6 3.5 7.7
396b-fad2-1b PHP32510 AFS 5260.4.9 1 12.6 2.5 17.2 51.3 16.4
396b-fad2-1b PHP32510 AFS 5260.4.9 2 11.9 3.1 12.7 54.3 17.9
396b-fad2-1b PHP32510 AFS 5260.4.9 3 11.5 3.6 13.0 56.3 15.6
396b-fad2-1b PHP32510 AFS 5260.4.9 4 12.2 2.9 13.5 52.5 18.9
396b-fad2-1b PHP32510 AFS 5260.4.9 5 11.5 2.7 9.9 54.0 21.9
396b-fad2-1b PHP32510 AFS 5260.4.9 6 9.7 2.6 17.0 58.8 12.0
396b-fad2-1b PHP32510 AFS 5260.3.1 1 13.0 3.6 14.1 57.1 12.2
396b-fad2-1b PHP32510 AFS 5260.3.1 2 7.9 3.9 79.5 2.7 6.0
396b-fad2-1b PHP32510 AFS 5260.3.1 3 8.5 4.1 76.1 4.3 7.1
396b-fad2-1b PHP32510 AFS 5260.3.1 4 8.5 3.8 78.1 2.9 6.7
396b-fad2-1b PHP32510 AFS 5260.3.1 5 12.0 4.3 14.3 56.1 13.3
396b-fad2-1b PHP32510 AFS 5260.3.1 6 9.0 3.8 75.9 4.0 7.3
TABLE-US-00017 TABLE 7b Fatty acid profile for T1 seed analyzed
from soy transformed with PHP32511, containing the 159-fad2-1b
amiRNA grown in the greenhouse. Seed T1 Seed amiRNA Construct Event
No. Planted 16:0 18:0 18:1 18:2 18:3 159-fad2-1b PHP32511 AFS
5292.5.8 1 9.8 3.3 70.1 7.0 9.9 159-fad2-1b PHP32511 AFS 5292.5.8 2
9.1 3.3 74.5 4.3 8.8 159-fad2-1b PHP32511 AFS 5292.5.8 3 8GR31-38
9.9 3.2 72.9 4.8 9.2 159-fad2-1b PHP32511 AFS 5292.5.8 4 12.3 3.5
17.8 54.0 12.3 159-fad2-1b PHP32511 AFS 5292.5.8 5 13.1 3.7 13.5
56.4 13.4 159-fad2-1b PHP32511 AFS 5292.5.8 6 8.6 3.5 74.4 4.7 8.8
159-fad2-1b PHP32511 AFS 5292.6.8 1 14.1 3.5 11.1 56.3 15.0
159-fad2-1b PHP32511 AFS 5292.6.8 2 10.6 3.3 69.6 6.7 9.9
159-fad2-1b PHP32511 AFS 5292.6.8 3 12.8 3.2 12.7 58.5 12.8
159-fad2-1b PHP32511 AFS 5292.6.8 4 12.5 3.5 14.9 58.1 11.1
159-fad2-1b PHP32511 AFS 5292.6.8 5 12.0 3.6 12.9 59.0 12.4
159-fad2-1b PHP32511 AFS 5292.6.8 6 13.2 3.1 10.9 59.0 13.8
159-fad2-1b PHP32511 AFS 5292.7.6 1 10.9 3.6 69.9 5.6 10.0
159-fad2-1b PHP32511 AFS 5292.7.6 2 8GR31-42 9.3 3.4 73.9 4.8 8.6
159-fad2-1b PHP32511 AFS 5292.7.6 3 10.9 3.2 68.7 6.2 11.0
159-fad2-1b PHP32511 AFS 5292.7.6 4 8GR31-44 9.5 3.4 67.1 8.9 11.0
159-fad2-1b PHP32511 AFS 5292.7.6 5 11.0 2.8 72.7 4.5 9.0
159-fad2-1b PHP32511 AFS 5292.7.6 6 8.7 3.2 74.5 5.0 8.7
159-fad2-1b PHP32511 AFS 5292.8.3 1 8.8 3.9 74.1 4.4 8.8
159-fad2-1b PHP32511 AFS 5292.8.3 2 12.0 3.9 13.5 58.6 12.1
159-fad2-1b PHP32511 AFS 5292.8.3 3 8.2 4.3 75.2 4.5 7.8
159-fad2-1b PHP32511 AFS 5292.8.3 4 9.6 3.4 65.4 10.6 11.0
159-fad2-1b PHP32511 AFS 5292.8.3 5 8.3 3.4 73.1 6.2 9.0
159-fad2-1b PHP32511 AFS 5292.8.3 6 83 3.0 73.8 6.3 8.5 159-fad2-1b
PHP32511 AFS 5292.8.8 1 11.6 3.3 19.6 52.7 12.7 159-fad2-1b
PHP32511 AFS 5292.8.8 2 9.1 3.4 74.5 4.7 8.3 159-fad2-1b PHP32511
AFS 5292.8.8 3 8.7 3.3 80.2 1.4 6.5 159-fad2-1b PHP32511 AFS
5292.8.8 4 9.2 2.9 42.2 39.9 5.8 159-fad2-1b PHP32511 AFS 5292.8.8
5 8.8 3.5 77.8 3.2 6.8 159-fad2-1b PHP32511 AFS 5292.8.8 6 8.8 3.2
77.5 2.6 7.8 159-fad2-1b PHP32511 AFS 5292.1.4 1 8.2 3.5 76.4 4.1
7.8 159-fad2-1b PHP32511 AFS 5292.1.4 2 8.0 3.2 77.1 3.7 8.0
159-fad2-1b PHP32511 AFS 5292.1.4 3 8GR31-58 8.2 3.4 78.1 2.7 7.6
159-fad2-1b PHP32511 AFS 5292.1.4 4 8GR31-59 8.2 3.2 78.6 2.6 7.3
159-fad2-1b PHP32511 AFS 5292.1.4 5 8.1 3.6 76.2 4.6 7.6
159-fad2-1b PHP32511 AFS 5292.1.4 6 8GR31-61 8.3 4.2 77.9 2.8 6.7
159-fad2-1b PHP32511 AFS 5292.5.2 1 10.1 3.9 76.2 2.6 7.3
159-fad2-1b PHP32511 AFS 5292.5.2 2 7.8 3.4 82.7 1.4 4.8
159-fad2-1b PHP32511 AFS 5292.5.2 3 8.4 3.3 81.5 1.3 5.6
159-fad2-1b PHP32511 AFS 5292.5.2 4 7.6 3.6 82.3 1.4 5.1
159-fad2-1b PHP32511 AFS 5292.5.2 5 12.8 3.7 13.0 56.4 14.2
159-fad2-1b PHP32511 AFS 5292.5.2 6 8.4 3.2 82.0 1.5 5.0
159-fad2-1b PHP32511 AFS 5292.6.5 1 13.2 3.3 19.5 54.8 9.2
159-fad2-1b PHP32511 AFS 5292.6.5 2 12.2 3.2 18.6 54.6 11.3
159-fad2-1b PHP32511 AFS 5292.6.5 3 8GR31-67 8.0 3.0 79.5 2.6 6.8
159-fad2-1b PHP32511 AFS 5292.6.5 4 8.4 2.9 80.9 1.9 5.7
159-fad2-1b PHP32511 AFS 5292.6.5 5 8.8 4.1 75.5 4.2 7.5
159-fad2-1b PHP32511 AFS 5292.6.5 6 12.8 3.5 20.6 53.1 10.0
159-fad2-1b PHP32511 AFS 5292.7.1 1 11.3 3.1 63.9 10.0 11.7
159-fad2-1b PHP32511 AFS 5292.7.1 2 7.3 3.5 82.8 1.4 5.0
159-fad2-1b PHP32511 AFS 5292.7.1 3 12.5 2.9 32.6 44.8 7.2
159-fad2-1b PHP32511 AFS 5292.7.1 4 7.6 3.6 81.0 2.2 5.6
159-fad2-1b PHP32511 AFS 5292.7.1 5 11.6 3.4 15.6 58.7 10.7
159-fad2-1b PHP32511 AFS 5292.7.1 6 11.3 3.4 18.9 56.4 10.1
159-fad2-1b PHP32511 AFS 5292.7.2 1 8GR31-73 8.6 3.2 80.4 2.1 5.7
159-fad2-1b PHP32511 AFS 5292.7.2 2 8GR31-74 8.3 3.1 81.9 1.4 5.3
159-fad2-1b PHP32511 AFS 5292.7.2 3 8GR31-75 8.7 3.2 81.5 1.6 4.9
159-fad2-1b PHP32511 AFS 5292.7.2 4 8GR31-76 8.1 3.2 80.2 2.1 6.5
159-fad2-1b PHP32511 AFS 5292.7.2 5 9.0 3.3 77.5 2.7 7.5
159-fad2-1b PHP32511 AFS 5292.7.2 6 8GR31-78 8.0 3.3 81.3 1.9 5.6
159-fad2-1b PHP32511 AFS 5292.8.2 1 13.3 3.2 16.4 54.4 12.7
159-fad2-1b PHP32511 AFS 5292.8.2 2 12.9 3.0 17.5 54.9 11.8
159-fad2-1b PHP32511 AFS 5292.8.2 3 12.5 3.1 17.0 56.1 11.4
159-fad2-1b PHP32511 AFS 5292.8.2 4 12.7 3.0 17.6 55.1 11.7
159-fad2-1b PHP32511 AFS 5292.8.2 5 12.5 3.0 19.0 54.9 10.6
159-fad2-1b PHP32511 AFS 5292.8.2 6 13.0 3.1 17.2 54.1 12.6
TABLE-US-00018 TABLE 7c Fatty acid profile for T1 seed analyzed
from soy transformed with PHP32843, containing the 369b-fad2-1b
& 159-fad2-2 amiRNAs grown in the greenhouse. Seed T1 Seed
amiRNA Construct Event No. Planted 16:0 18:0 18:1 18:2 18:3 396b-
PHP32843 AFS 1 8.2 3.0 85.8 0.4 2.7 fad2- 5396.1.11 1b/159- fad2-2
396b- PHP32843 AFS 2 7.9 3.3 85.4 0.5 2.9 fad2- 5396.1.11 1b/159-
fad2-2 396b- PHP32843 AFS 3 8.2 3.2 85.8 0.4 2.6 fad2- 5396.1.11
1b/159- fad2-2 396b- PHP32843 AFS 4 8.1 3.3 85.6 0.4 2.6 fad2-
5396.1.11 1b/159- fad2-2 396b- PHP32843 AFS 5 8.3 3.1 85.2 0.4 2.9
fad2- 5396.1.11 1b/159- fad2-2 396b- PHP32843 AFS 6 13.1 3.5 11.4
55.1 16.8 fad2- 5396.1.11 1b/159- fad2-2 396b- PHP32843 AFS 1 11.5
3.3 20.0 55.0 10.2 fad2- 5396.1.2 1b/159- fad2-2 396b- PHP32843 AFS
2 7.2 3.2 87.3 0.3 2.0 fad2- 5396.1.2 1b/159- fad2-2 396b- PHP32843
AFS 3 7.6 2.9 86.7 0.4 2.4 fad2- 5396.1.2 1b/159- fad2-2 396b-
PHP32843 AFS 4 7.6 3.3 85.9 0.5 2.7 fad2- 5396.1.2 1b/159- fad2-2
396b- PHP32843 AFS 5 7.9 3.0 86.7 0.3 2.1 fad2- 5396.1.2 1b/159-
fad2-2 396b- PHP32843 AFS 6 8.1 3.0 84.0 1.0 4.0 fad2- 5396.1.2
1b/159- fad2-2 396b- PHP32843 AFS 1 7.8 3.3 83.4 1.3 4.2 fad2-
5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 2 7.5 3.2 85.7 0.7 2.9
fad2- 5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 3 7.7 3.1 83.7 1.2
4.3 fad2- 5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 4 8.4 3.6 82.1
1.3 4.7 fad2- 5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 5 7.2 3.2
86.0 0.7 2.9 fad2- 5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 6 7.4
3.4 85.7 0.6 2.9 fad2- 5396.1.4 1b/159- fad2-2 396b- PHP32843 AFS 1
8.3 2.9 80.5 2.9 5.4 fad2- 5396.1.5 1b/159- fad2-2 396b- PHP32843
AFS 1 12.9 3.2 16.0 53.5 14.4 fad2- 5396.1.7 1b/159- fad2-2 396b-
PHP32843 AFS 2 7.5 3.1 85.2 1.0 3.3 fad2- 5396.1.7 1b/159- fad2-2
396b- PHP32843 AFS 3 7.8 2.8 84.0 1.3 4.1 fad2- 5396.1.7 1b/159-
fad2-2 396b- PHP32843 AFS 4 7.1 3.3 87.3 0.4 1.9 fad2- 5396.1.7
1b/159- fad2-2 396b- PHP32843 AFS 5 7.4 3.2 85.2 0.9 3.3 fad2-
5396.1.7 1b/159- fad2-2 396b- PHP32843 AFS 6 11.8 3.4 18.0 54.2
12.6 fad2- 5396.1.7 1b/159- fad2-2 396b- PHP32843 AFS 1 8.1 2.9
83.8 1.2 4.0 fad2- 5396.2.2 1b/159- fad2-2 396b- PHP32843 AFS 2
8GR31- 7.8 2.5 86.6 0.6 2.5 fad2- 53962.2 140 1b/159- fad2-2 396b-
PHP32843 AFS 3 13.5 2.8 14.3 56.7 12.7 fad2- 5396.2.2 1b/159-
fad2-2 396b- PHP32843 AFS 4 8.0 2.8 85.0 1.1 3.1 fad2- 5396.2.2
1b/159- fad2-2 396b- PHP32843 AFS 5 8.3 2.4 84.6 1.3 3.4 fad2-
5396.2.2 1b/159- fad2-2 396b- PHP32843 AFS 6 7.7 2.4 87.2 0.7 2.0
fad2- 5396.2.2 1b/159- fad2-2 396b- PHP32843 AFS 1 13.2 3.3 16.6
56.3 10.7 fad2- 5396.2.3 1b/159- fad2-2 396b- PHP32843 AFS 2 7.4
2.7 85.8 1.1 3.0 fad2- 5396.2.3 1b/159- fad2-2 396b- PHP32843 AFS 3
8GR31- 7.3 2.6 87.5 0.7 2.0 fad2- 5396.2.3 145 1b/159- fad2-2 396b-
PHP32843 AFS 4 13.2 3.0 13.0 60.9 10.0 fad2- 5396.2.3 1b/159-
fad2-2 396b- PHP32843 AFS 5 7.5 2.5 86.6 0.9 2.5 fad2- 5396.2.3
1b/159- fad2-2 396b- PHP32843 AFS 6 12.7 3.0 20.3 55.1 8.9 fad2-
5396.2.3 1b/159- fad2-2 396b- PHP32843 AFS 1 7.8 3.0 86.1 0.5 2.6
fad2- 5396.2.4 1b/159- fad2-2 396b- PHP32843 AFS 2 7.6 3.0 86.7 0.4
2.3 fad2- 5396.2.4 1b/159- fad2-2 396b- PHP32843 AFS 3 7.4 3.3 86.1
0.6 2.6 fad2- 5396.2.4 1b/159- fad2-2 396b- PHP32843 AFS 4 7.5 3.4
86.3 0.6 2.2 fad2- 5396.2.4 1b/159- fad2-2 396b- PHP32843 AFS 5
12.6 3.9 17.4 54.0 12.1 fad2- 5396.2.4 1b/159- fad2-2 396b-
PHP32843 AFS 6 7.6 3.1 86.4 0.7 2.2 fad2- 5396.2.4 1b/159- fad2-2
396b- PHP32843 AFS 1 7.5 3.1 86.7 0.6 2.0 fad2- 5396.2.9 1b/159-
fad2-2 396b- PHP32843 AFS 2 13.0 3.5 17.9 53.3 12.3 fad2- 5396.2.9
1b/159- fad2-2 396b- PHP32843 AFS 3 7.5 3.1 85.8 0.8 2.8 fad2- 5396
2.9 1b/159- fad2-2 396b- PHP32843 AFS 4 13.1 3.4 17.6 53.9 12.0
fad2- 5396.2.9 1b/159- fad2-2 396b- PHP32843 AFS 5 7.5 3.2 85.4 0.9
2.9 fad2- 5396.2.9 1b/159- fad2-2 396b- PHP32843 AFS 6 7.5 3.2 86.6
0.6 2.1 fad2- 5396.2.9 1b/159- fad2-2 396b- PHP32843 AFS 1 13.9 3.0
10.9 58.0 14.2 fad2- 5396.3.3 1b/159- fad2-2 396b- PHP32843 AFS 2
13.3 2.9 8.2 60.9 14.7 fad2- 5396.3.3 1b/159- fad2-2 396b- PHP32843
AFS 1 8GR31- 7.2 3.0 87.0 0.4 2.3 fad2- 5396.3.8 156 1b/159- fad2-2
396b- PHP32843 AFS 2 8GR31- 7.9 2.9 84.8 0.8 3.6 fad2- 5396.3.8 157
1b/159- fad2-2 396b- PHP32843 AFS 3 7.6 2.7 86.0 0.7 2.9 fad2-
5396.3.8 1b/159- fad2-2 396b- PHP32843 AFS 4 12.1 3.4 12.6 57.9
14.0 fad2- 5396.3.8 1b/159- fad2-2 396b- PHP32843 AFS 5 11.6 3.1
17.4 55.1 12.7 fad2- 5396.3.8 1b/159- fad2-2 396b- PHP32843 AFS 6
11.5 3.3 19.3 54.2 11.8 fad2- 5396.3.8 1b/159- fad2-2
TABLE-US-00019 TABLE 7d Fatty acid profile for T1 seed analyzed
from soy transformed with PHP33705, containing the 396-fad2-1b
& 396b-sad3 amiRNAs grown in the greenhouse. Seed T1 Seed
amiRNA Construct Event No. Planted 16:0 18:0 18:1 18:2 18:3
396-fad2- PHP33705 AFS 5489.1.1 1 12.8 2.5 22.0 54.1 8.7
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.1 2 8.8 10.3 59.8 10.4
10.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.1 3 12.8 2.7 22.4
53.0 9.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.1 4 7.8 12.7
66.1 5.3 8.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.1 5 7.9
14.9 65.9 3.9 7.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.1 6
13.4 3.4 20.6 54.7 7.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.2
1 12.6 2.8 17.7 56.5 10.4 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.1.2 2 8.5 0.0 73.2 8.0 10.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.1.2 3 8.1 13.1 65.7 5.4 7.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.1.2 4 13.3 3.5 19.2 52.9 11.2 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.1.2 5 7.7 5.5 70.6 6.2 10.0
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.1.2 6 12.2 3.1 23.4 52.3
8.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.2 1 11.3 3.3 21.2
53.5 10.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.2 2 11.3 3.3
17.0 57.8 10.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.2 3 10.6
3.6 15.2 59.2 11.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.2 4
12.1 3.5 16.2 58.1 10.1 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.2.2 5 12.6 4.0 12.9 58.2 12.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.2.2 6 11.9 3.2 18.8 56.2 9.9 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.2.5 1 12.4 3.2 15.5 57.1 11.8 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.2.5 2 11.9 3.4 15.8 56.9 12.0
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.5 3 11.9 3.0 17.8 56.7
10.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.5 4 12.9 3.2 11.5
57.2 15.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.5 5 7.6 9.4
69.8 5.1 8.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.5 6 10.9
2.2 28.6 49.9 8.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.7 1
7.4 12.6 70.4 2.9 6.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.7
2 7.9 7.6 71.1 3.8 9.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.2.7
3 6.7 12.1 72.9 2.5 5.8 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.2.7 4 7.8 11.6 65.3 5.6 9.7 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.2.7 5 7.2 14.6 68.2 1.8 8.2 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.2.7 6 7.8 8.1 72.2 3.2 8.6 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.1 1 11.5 2.8 24.0 50.3 11.5 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.1 2 6.6 13.2 64.4 4.8 11.0
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.1 3 6.3 16.4 65.0 3.4
8.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.1 4 6.5 14.1 64.2
5.1 10.0 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.1 5 6.1 17.9
64.3 3.5 8.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.1 6 10.8
3.7 58.9 11.5 15.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.3 1
6.4 8.6 75.2 3.1 6.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.3 2
7.1 7.7 73.0 3.8 8.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.3 3
7.3 8.2 72.5 4.2 7.8 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.3 4
7.8 7.4 68.7 5.8 10.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.3
5 11.5 3.2 18.1 56.0 11.3 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.3 6 12.4 3.0 16.9 56.2 11.6 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.7 1 7.4 7.6 71.9 4.4 8.7 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.7 2 11.2 3.2 17.6 58.0 10.0 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.7 3 7.3 9.1 71.3 3.8 8.4 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.7 4 6.8 8.1 72.1 4.2 8.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.7 5 6.6 6.6 74.7 4.2 7.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.7 6 6.7 8.0 73.6 4.0 7.6 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.2 1 11.5 2.4 25.8 49.4 10.9 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.4.2 2 11.3 3.4 35.6 41.2 8.5
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.2 3 6.9 9.5 71.8 3.5 8.3
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.2 4 11.1 2.8 28.7 48.6
8.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.2 5 6.3 10.9 68.7
4.8 9.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.2 6 12.1 3.2
16.0 56.2 12.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.3 1 6.5
9.3 74.3 3.2 6.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.3 2 7.1
12.3 70.6 2.7 7.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.3 3
10.6 2.7 29.9 48.3 8.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.3
4 11.6 2.2 22.7 53.6 9.9 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.4.3 5 7.1 13.0 71.7 1.4 6.9 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.3 6 6.8 13.1 70.4 2.9 6.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.7 1 11.6 3.2 19.3 54.2 11.7 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.4.7 2 11.4 3.2 20.5 54.0 10.9
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.7 3 11.8 3.1 19.3 53.1
12.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.7 4 12.3 3.1 16.4
55.8 12.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.7 5 12.6 3.1
10.3 54.9 19.0 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.7 6 12.2
3.3 19.7 54.9 9.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.8 1
6.7 14.4 68.7 3.0 7.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.8
2 13.2 2.6 14.6 52.6 17.0 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.4.8 3 6.3 14.9 67.9 3.6 7.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.8 4 7.9 8.6 68.9 5.3 9.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.8 5 6.4 12.1 69.3 4.2 8.0 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.8 6 11.7 2.7 21.8 54.7 9.1 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.2 1 8.0 8.6 63.5 9.1 10.8
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 2 6.5 13.6 62.6 7.7
9.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 3 7.4 13.9 58.7
7.1 13.0 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 4 12.8 3.5
21.3 49.5 13.0 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 5 13.1
3.4 16.1 52.1 15.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 6
7.9 8.1 65.4 7.2 11.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2
7 6.9 16.8 61.2 5.1 10.0 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.2 8 7.5 11.2 60.5 8.9 12.0 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.2 9 7.5 9.4 65.3 6.9 10.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.2 10 12.4 3.4 18.8 51.1 14.2 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.2 11 7.3 7.8 69.4 5.3 10.2
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 12 6.6 13.8 62.0 7.8
9.8 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 13 10GR13-6 6.2
14.8 63.5 5.4 10.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 14
10GR13-7 11.7 3.1 19.7 52.6 13.0 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.2 15 10GR13-8 13.5 3.1 16.7 51.0 15.6 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.2 16 7.1 12.9 63.2 6.6 10.2
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 17 7.4 9.1 67.3 5.5
10.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 18 12.2 3.4 18.6
52.4 13.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 19 7.6 7.9
64.7 7.4 12.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 20 13.2
3.2 19.1 49.4 15.2 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2 21
7.1 12.7 60.1 8.9 11.2 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.2
22 8.3 6.8 63.6 9.6 11.7 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.2 23 6.5 13.1 62.5 7.0 10.8 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.2 24 7.7 9.7 60.0 11.3 11.3 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.4 1 7.5 7.2 69.8 7.1 8.4 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.4 2 6.8 10.7 69.1 4.6 8.8 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.4 3 10GR13-14 6.8 13.3 66.2 5.1 8.6
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 4 10GR13-15 6.0 16.6
66.0 3.9 7.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 5 6.6 9.7
72.7 3.0 7.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 6 8.8 6.2
64.5 8.6 12.0 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 7
10GR13-16 6.2 11.7 71.1 3.5 7.6 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.4 8 10GR13-17 11.2 3.2 15.0 58.3 12.3 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.4 9 8.7 6.7 63.8 10.2 10.7 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.4 10 7.1 9.0 72.6 3.4 7.9
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 11 6.2 13.5 67.5 4.1
8.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 12 7.2 9.0 70.8
4.0 8.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 13 7.0 9.7
69.0 5.2 9.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 14
10GR13-19 7.7 10.6 65.0 7.7 9.1 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.4 15 10GR13-20 7.3 10.8 67.8 5.8 8.3 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.4 16 6.8 12.6 65.5 5.3 9.8 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.4 17 6.5 9.3 69.3 5.9 9.0
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 18 6.1 11.9 70.3 3.1
8.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 19 7.3 7.0 71.9
4.6 9.2 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 20 7.4 7.6
71.1 4.3 9.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.4 21
10GR13-23 6.1 13.7 69.6 3.2 7.5 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.4 22 10.9 3.4 19.0 56.4 10.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.4 23 7.2 8.4 69.6 5.2 9.5 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.4 24 6.9 9.4 69.3 5.2 9.3 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.11 1 11.4 4.1 15.8 57.5 11.1
1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.11 2 11.8 3.3 30.2 45.1 9.6
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 3 7.3 15.9 65.4 3.4
8.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 4 6.6 14.1 68.8
2.6 7.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 5 13.1 3.3
14.4 55.0 14.2 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 6 10.8
3.6 20.7 55.7 9.3 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 7
13.7 4.7 29.4 46.2 6.0 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.11 8 5.5 23.6 63.9 1.6 5.4 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.11 9 5.2 17.6 68.8 2.4 6.0 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.11 10 5.6 15.0 70.4 3.4 5.6 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.11 11 12.2 3.5 19.7 53.8 10.8
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 12 5.6 10.9 72.9 4.3
6.2 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 13 8.2 12.1 58.2
8.6 12.8 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 14 11.7 3.6
13.7 57.6 13.4 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 15 6.6
24.9 59.4 2.6 6.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 16
8.2 8.8 61.8 10.0 11.3 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.3.11 17 11.2 3.6 32.4 44.6 8.1 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.3.11 18 6.7 13.2 72.0 1.8 6.3 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.3.11 19 6.4 15.9 67.4 2.8 7.6 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.3.11 20 11.6 3.5 17.7 54.3 12.9
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 21 6.4 23.5 61.2 2.1
6.8 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 22 7.4 12.7 66.5
2.9 10.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 23 7.7 14.4
68.1 2.1 7.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.3.11 24 8.1
15.2 65.1 2.9 8.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 1
6.2 14.6 70.5 2.0 6.8 1b/396b-ad3 396-fad2- PHP33705 AFS 5489.4.1 2
5.7 14.5 71.2 3.0 5.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1
3 8.3 5.5 69.5 6.2 10.6 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.4.1 4 6.8 14.3 65.6 4.4 8.9 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.1 5 5.9 18.0 66.3 2.1 7.6 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.1 6 6.4 16.0 67.8 2.0 7.8 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.4.1 7 7.4 17.3 64.7 2.2 8.4
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 8 10GR13-48 11.8 3.1
19.3 54.7 11.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 9 10.1
3.0 29.0 49.0 8.9 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 10
5.0 20.2 66.7 2.4 5.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1
11 5.6 14.3 71.5 3.7 5.1 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.4.1 12 10.7 3.8 26.2 53.0 6.3 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.1 13 11.5 3.5 17.2 56.1 11.8 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.1 14 5.7 21.5 63.8 1.7 7.3 1b/396b-sad3
396-fad2- PHP33705 AFS 5489.4.1 15 11.6 3.5 23.5 53.2 8.2
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 16 10GR13-53 10.8 3.0
18.6 57.5 10.1 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 17
10GR13-54 5.5 16.4 69.8 1.3 7.0 1b/396b-sad3 396-fad2- PHP33705 AFS
5489.4.1 18 10.3 3.3 31.5 47.8 7.1 1b/396b-sad3 396-fad2- PHP33705
AFS 5489.4.1 19 4.4 22.2 65.4 2.5 5.5 1b/396b-sad3 396-fad2-
PHP33705 AFS 5489.4.1 20 10GR13-56 4.8 20.5 67.4 1.4 5.9
1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 21 6.5 15.6 65.5 4.0
8.5 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 22 6.3 14.8 66.0
3.3 9.6 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 23 6.5 13.4
66.1 4.4 9.7 1b/396b-sad3 396-fad2- PHP33705 AFS 5489.4.1 24 5.7
15.4 70.8 1.4 6.7 1b/396b-sad3
TABLE-US-00020 TABLE 7e Fatty acid profile for T1 seed analyzed
from soy transformed with PHP38557, containing the 159-fad3c amiRNA
grown in the greenhouse. Seed T1 Seed amiRNA Construct Event No.
Planted 16:0 18:0 18:1 18:2 18:3 159-fad3c PHP38557 AFS 6272.1.4 1
10GR13-63 13.2 3.3 12.5 67.8 3.2 159-fad3c PHP38557 AFS 6272.1.4 2
13.0 3.2 16.8 63.1 4.0 159-fad3c PHP38557 AFS 6272.1.4 3 10GR13-65
13.3 3.4 14.1 65.3 3.9 159-fad3c PHP38557 AFS 6272.1.4 4 10.1 2.5
30.5 45.4 11.5 159-fad3c PHP38557 AFS 6272.1.4 5 10GR13-66 12.5 3.4
13.5 53.4 17.2 159-fad3c PHP38557 AFS 6272.1.4 6 12.3 3.8 12.5 67.1
4.3 159-fad3c PHP38557 AFS 6272.1.4 7 12.8 3.4 15.3 65.1 3.4
159-fad3c PHP38557 AFS 6272.1.4 8 12.4 3.3 14.4 65.6 4.3 159-fad3c
PHP38557 AFS 6272.1.4 9 10GR13-70 10.7 3.2 18.9 55.0 12.2 159-fad3c
PHP38557 AFS 6272.1.4 10 13.1 3.6 15.1 64.7 3.5 159-fad3c PHP38557
AFS 6272.1.4 11 11.8 2.8 20.9 61.6 2.8 159-fad3c PHP38557 AFS
6272.1.4 12 12.3 3.5 15.8 64.9 3.5 159-fad3c PHP38557 AFS 6272.1.4
13 10GR13-73 12.0 3.7 13.8 68.3 2.2 159-fad3c PHP38557 AFS 6272.1.4
14 12.4 3.6 15.8 65.0 3.2 159-fad3c PHP38557 AFS 6272.1.4 15 12.0
3.2 15.2 65.9 3.7 159-fad3c PHP38557 AFS 6272.1.4 16 11.4 3.1 19.6
63.0 2.9 159-fad3c PHP38557 AFS 6272.1.4 17 11.2 3.2 16.2 55.0 14.4
159-fad3c PHP38557 AFS 6272.1.4 18 12.0 3.3 14.6 66.5 3.5 159-fad3c
PHP38557 AFS 6272.1.4 19 11.4 3.1 19.1 63.3 3.0 159-fad3c PHP38557
AFS 6272.2.1 1 12.7 3.1 15.0 65.8 3.5 159-fad3c PHP38557 AFS
6272.2.1 2 13.0 3.2 19.1 61.3 3.4 159-fad3c PHP38557 AFS 6272.2.1 3
10GR13-81 11.3 2.9 15.4 55.3 15.2 159-fad3c PHP38557 AFS 6272.2.1 4
10GR13-82 12.1 2.7 16.9 52.5 15.8 159-fad3c PHP38557 AFS 6272.2.1 5
10GR13-83 12.9 3.5 15.9 65.3 2.4 159-fad3c PHP38557 AFS 6272.2.1 6
12.2 3.1 16.0 53.5 15.2 159-fad3c PHP38557 AFS 6272.2.1 7 12.2 3.4
16.4 64.8 3.1 159-fad3c PHP38557 AFS 6272.2.1 8 12.5 2.9 17.6 51.9
15.1 159-fad3c PHP38557 AFS 6272.2.1 9 12.3 3.2 15.2 65.6 3.7
159-fad3c PHP38557 AFS 6272.2.1 10 13.3 3.6 13.7 65.4 4.1 159-fad3c
PHP38557 AFS 6272.2.1 11 12.2 3.6 14.2 66.3 3.6 159-fad3c PHP38557
AFS 6272.2.1 12 11.4 3.2 15.1 56.4 13.9 159-fad3c PHP38557 AFS
6272.2.1 13 10GR13-90 12.8 3.3 13.0 67.8 3.2 159-fad3c PHP38557 AFS
6272.2.1 14 10GR13-91 12.2 4.2 13.6 67.6 2.4 159-fad3c PHP38557 AFS
6272.2.1 15 11.9 3.4 14.6 66.5 3.5 159-fad3c PHP38557 AFS 6272.2.1
16 10GR13-93 12.1 3.8 14.5 66.2 3.4 159-fad3c PHP38557 AFS 6272.2.1
17 11.8 3.4 14.1 56.6 14.1 159-fad3c PHP38557 AFS 6272.2.1 18 11.1
3.6 12.5 57.8 15.0 159-fad3c PHP38557 AFS 6272.2.1 19 10GR13-94
12.3 3.2 14.4 67.2 2.9 159-fad3c PHP38557 AFS 6272.2.3 1 10GR13-95
12.9 3.0 14.4 64.7 5.0 159-fad3c PHP38557 AFS 6272.2.3 2 10GR13-96
12.7 3.4 14.1 64.7 5.1 159-fad3c PHP38557 AFS 6272.2.3 3 10GR13-97
12.7 3.5 15.5 64.9 3.4 159-fad3c PHP38557 AFS 6272.2.3 4 10GR13-98
13.5 3.6 11.8 66.2 4.8 159-fad3c PHP38557 AFS 6272.2.3 5 12.6 3.1
15.7 63.7 4.9 159-fad3c PHP38557 AFS 6272.2.3 6 12.3 3.2 15.8 63.9
4.8 159-fad3c PHP38557 AFS 6272.2.3 7 12.6 3.2 14.5 64.8 4.9
159-fad3c PHP38557 AFS 6272.2.3 8 10GR13-101 12.6 2.8 18.0 62.0 4.5
159-fad3c PHP38557 AFS 6272.2.3 9 10GR13-102 13.1 3.3 15.5 62.5 5.6
159-fad3c PHP38557 AFS 6272.2.3 10 13.0 3.4 13.7 65.6 4.4 159-fad3c
PHP38557 AFS 6272.2.3 11 10GR13-103 14.0 3.5 11.9 63.5 7.1
159-fad3c PHP38557 AFS 6272.2.3 12 10GR13-104 12.0 3.3 15.0 64.7
5.0 159-fad3c PHP38557 AFS 6272.2.3 13 12.5 3.7 13.7 65.2 4.9
159-fad3c PHP38557 AFS 6272.2.3 14 11.7 3.4 13.9 66.5 4.4 159-fad3c
PHP38557 AFS 6272.2.3 15 10GR13-106 11.7 3.6 16.1 66.0 2.7
159-fad3c PHP38557 AFS 6272.2.3 16 10GR13-107 12.2 3.5 14.2 54.2
15.9 159-fad3c PHP38557 AFS 6272.2.3 17 12.3 3.1 15.7 64.5 4.4
159-fad3c PHP38557 AFS 6272.2.3 18 10GR13-109 12.5 3.5 13.6 55.3
14.9 159-fad3c PHP38557 AFS 6272.2.3 19 12.3 3.1 15.0 64.5 5.0
TABLE-US-00021 TABLE 7f Fatty acid profile for T2 seed analyzed
from soy transformed with PHP41103, containing the 159-fatBF
amiRNA, grown in the greenhouse. Seed T1 Seed amiRNA Construct
Event No. Planted 16:0 18:0 18:1 18:2 18:3 159-fatBF PHP41103 AFS
6671.1.4 1 2.5 2.2 22.0 64.3 9.1 159-fatBF PHP41103 AFS 6671.1.4 2
10GR13-222 2.5 2.5 22.4 62.9 9.7 159-fatBF PHP41103 AFS 6671.1.4 3
2.7 2.5 21.0 63.2 10.5 159-fatBF PHP41103 AFS 6671.1.4 4 2.9 2.5
24.7 60.1 9.8 159-fatBF PHP41103 AFS 6671.1.4 5 11.8 3.0 19.7 55.9
9.5 159-fatBF PHP41103 AFS 6671.1.4 6 10GR13-226 11.1 3.1 17.4 58.3
10.1 159-fatBF PHP41103 AFS 6671.1.4 7 2.3 1.9 26.8 57.8 11.2
159-fatBF PHP41103 AFS 6671.1.4 8 2.2 1.8 22.4 61.5 12.1 159-fatBF
PHP41103 AFS 6671.1.4 9 10GR13-229 2.4 1.9 23.2 61.1 11.5 159-fatBF
PHP41103 AFS 6671.1.4 10 2.9 2.6 20.0 64.2 10.4 159-fatBF PHP41103
AFS 6671.1.4 11 2.5 2.0 20.9 62.5 12.0 159-fatBF PHP41103 AFS
6671.1.4 12 2.5 2.1 23.5 61.1 10.8 159-fatBF PHP41103 AFS 6671.1.4
13 2.8 2.3 21.3 63.1 10.5 159-fatBF PHP41103 AFS 6671.1.4 14 2.8
2.2 22.2 63.1 9.7 159-fatBF PHP41103 AFS 6671.1.4 15 2.8 2.3 19.8
62.7 12.4 159-fatBF PHP41103 AFS 6671.1.4 16 2.3 1.8 33.0 52.9 9.9
159-fatBF PHP41103 AFS 6671.1.4 17 10GR13-237 11.1 2.7 20.5 54.1
11.5 159-fatBF PHP41103 AFS 6671.1.4 18 2.3 1.8 24.5 57.3 14.2
159-fatBF PHP41103 AFS 6671.1.4 19 11.2 3.0 17.6 57.3 10.9
159-fatBF PHP41103 AFS 6671.1.4 20 2.4 1.9 23.8 59.7 12.2 159-fatBF
PHP41103 AFS 6671.1.4 21 2.1 1.7 26.0 58.3 12.0 159-fatBF PHP41103
AFS 6671.1.6 1 10GR13-242 2.1 1.8 39.8 47.1 9.1 159-fatBF PHP41103
AFS 6671.1.6 2 10GR13-243 2.2 1.8 28.5 56.8 10.8 159-fatBF PHP41103
AFS 6671.1.6 3 10GR13-244 2.2 2.0 27.9 57.8 10.1 159-fatBF PHP41103
AFS 6671.1.6 4 2.4 1.8 35.0 50.4 10.4 159-fatBF PHP41103 AFS
6671.1.6 5 10.7 2.8 31.9 45.0 9.6 159-fatBF PHP41103 AFS 6671.1.6 6
10GR13-247 1.9 1.6 27.5 54.4 14.6 159-fatBF PHP41103 AFS 6671.1.6 7
11.8 2.6 18.9 53.9 12.8 159-fatBF PHP41103 AFS 6671.1.6 8
10GR13-249 2.2 1.6 27.3 56.6 12.3 159-fatBF PHP41103 AFS 6671.1.6 9
2.5 1.8 30.1 54.6 11.0 159-fatBF PHP41103 AFS 6671.1.6 10 2.4 1.7
26.5 55.0 14.3 159-fatBF PHP41103 AFS 6671.1.6 11 10GR13-252 2.4
1.8 27.0 58.8 10.1 159-fatBF PHP41103 AFS 6671.1.6 12 10GR13-253
2.3 1.6 25.6 57.2 13.2 159-fatBF PHP41103 AFS 6671.1.6 13 2.3 1.6
37.6 47.8 10.7 159-fatBF PHP41103 AFS 6671.1.6 14 2.3 1.5 25.9 57.3
13.0 159-fatBF PHP41103 AFS 6671.1.6 15 10GR13-256 2.3 2.1 18.4
65.4 11.7 159-fatBF PHP41103 AFS 6671.1.6 16 2.2 2.0 35.0 51.1 9.7
159-fatBF PHP41103 AFS 6671.1.6 17 10GR13-258 2.3 1.7 26.0 58.6
11.3 159-fatBF PHP41103 AFS 6671.1.6 18 2.6 2.1 28.6 56.7 10.0
159-fatBF PHP41103 AFS 6671.1.6 19 10GR13-260 2.4 1.9 32.1 52.7
10.8 159-fatBF PHP41103 AFS 6671.1.6 20 10GR13-261 11.2 2.4 20.0
54.4 12.1 159-fatBF PHP41103 AFS 6671.1.6 21 10GR13-262 11.9 2.6
22.8 51.4 11.4 159-fatBF PHP41103 AFS 6671.3.1 1 2.4 2.3 20.6 64.0
10.6 159-fatBF PHP41103 AFS 6671.3.1 2 2.5 2.6 19.3 64.8 10.8
159-fatBF PHP41103 AFS 6671.3.1 3 2.9 2.8 19.4 66.4 8.4 159-fatBF
PHP41103 AFS 6671.3.1 4 3.1 2.4 19.4 65.0 10.2 159-fatBF PHP41103
AFS 6671.3.1 5 10.9 3.0 19.0 56.5 10.6 159-fatBF PHP41103 AFS
6671.3.1 6 2.5 2.4 18.5 62.8 13.9 159-fatBF PHP41103 AFS 6671.3.1 7
10GR13-269 11.1 2.7 16.6 57.0 12.6 159-fatBF PHP41103 AFS 6671.3.1
8 10GR13-270 2.7 2.6 18.1 66.0 10.6 159-fatBF PHP41103 AFS 6671.3.1
9 2.5 2.0 18.5 61.7 15.3 159-fatBF PHP41103 AFS 6671.3.1 10 2.7 2.4
18.0 64.0 13.0 159-fatBF PHP41103 AFS 6671.3.1 11 10.7 2.7 16.1
57.7 12.8 159-fatBF PHP41103 AFS 6671.3.1 12 10GR13-274 11.5 2.7
16.6 57.5 11.7 159-fatBF PHP41103 AFS 6671.3.1 13 11.2 2.7 13.3
56.8 16.0 159-fatBF PHP41103 AFS 6671.3.1 14 12.1 2.8 14.8 57.4
12.9 159-fatBF PHP41103 AFS 6671.3.1 15 10GR13-277 12.0 2.5 14.6
56.1 14.7 159-fatBF PHP41103 AFS 6671.3.1 16 10GR13-278 2.3 2.2
18.2 64.8 12.5 159-fatBF PHP41103 AFS 6671.3.1 17 2.7 2.2 14.9 66.4
13.7 159-fatBF PHP41103 AFS 6671.3.1 18 11.6 2.7 16.0 56.0 13.7
159-fatBF PHP41103 AFS 6671.3.1 19 11.5 3.2 14.6 54.5 16.1
159-fatBF PHP41103 AFS 6671.3.1 20 2.7 2.3 17.3 64.9 12.8 159-fatBF
PHP41103 AFS 6671.3.1 21 11.2 2.8 15.4 58.2 12.3 159-fatBF PHP41103
AFS 6671.3.2 1 2.7 2.1 21.9 63.0 10.4 159-fatBF PHP41103 AFS
6671.3.2 2 2.8 2.0 25.3 60.3 9.6 159-fatBF PHP41103 AFS 6671.3.2 3
3.2 2.5 24.6 60.2 9.4 159-fatBF PHP41103 AFS 6671.3.2 4 10.8 3.1
22.5 53.8 9.7 159-fatBF PHP41103 AFS 6671.3.2 5 11.6 3.0 19.4 56.0
9.9 159-fatBF PHP41103 AFS 6671.3.2 6 2.4 2.0 32.9 52.2 10.6
159-fatBF PHP41103 AFS 6671.3.2 7 11.9 2.8 19.1 54.8 11.4 159-fatBF
PHP41103 AFS 6671.3.2 8 2.7 2.1 27.9 57.8 9.5 159-fatBF PHP41103
AFS 6671.3.2 9 2.6 2.1 23.8 60.2 11.3 159-fatBF PHP41103 AFS
6671.3.2 10 2.7 2.1 18.4 64.7 12.1 159-fatBF PHP41103 AFS 6671.3.2
11 2.9 2.1 24.6 59.9 10.6 159-fatBF PHP41103 AFS 6671.3.2 12 2.5
2.4 24.0 58.6 12.6 159-fatBF PHP41103 AFS 6671.3.2 13 11.1 2.5 21.4
54.5 10.5 159-fatBF PHP41103 AFS 6671.3.2 14 2.8 2.2 21.0 61.9 12.1
159-fatBF PHP41103 AFS 6671.3.2 15 2.4 1.8 20.6 61.6 13.5 159-fatBF
PHP41103 AFS 6671.3.2 16 2.2 1.8 34.9 49.2 11.8 159-fatBF PHP41103
AFS 6671.3.2 17 2.3 1.8 25.8 58.7 11.4 159-fatBF PHP41103 AFS
6671.3.2 18 2.9 2.2 26.4 58.6 9.8 159-fatBF PHP41103 AFS 6671.3.2
19 11.7 3.0 16.7 53.1 15.5 159-fatBF PHP41103 AFS 6671.3.2 20 2.4
2.2 14.8 67.2 13.4 159-fatBF PHP41103 AFS 6671.3.2 21 2.5 2.0 23.7
60.2 11.6 159-fatBF PHP41103 AFS 6671.4.2 1 2.4 2.6 16.8 67.5 10.7
159-fatBF PHP41103 AFS 6671.4.2 2 2.5 2.1 26.6 59.0 9.7 159-fatBF
PHP41103 AFS 6671.4.2 3 3.1 3.1 17.1 65.6 11.1 159-fatBF PHP41103
AFS 6671.4.2 4 3.3 2.3 24.5 60.8 9.1 159-fatBF PHP41103 AFS
6671.4.2 5 11.4 3.0 20.4 54.2 11.0 159-fatBF PHP41103 AFS 6671.4.2
6 10.5 2.3 20.3 54.7 12.2 159-fatBF PHP41103 AFS 6671.4.2 7 3.0 2.2
19.0 63.0 12.8 159-fatBF PHP41103 AFS 6671.4.2 8 11.5 3.3 16.6 55.7
12.9 159-fatBF PHP41103 AFS 6671.4.2 9 11.4 3.0 17.6 55.8 12.1
159-fatBF PHP41103 AFS 6671.4.2 10 11.1 3.2 19.4 54.8 11.4
159-fatBF PHP41103 AFS 6671.4.2 11 11.1 2.9 15.8 58.2 12.1
159-fatBF PHP41103 AFS 6671.4.2 12 2.7 2.0 25.4 59.2 10.7 159-fatBF
PHP41103 AFS 6671.4.2 13 2.3 1.9 25.8 59.0 11.1 159-fatBF PHP41103
AFS 6671.4.2 14 3.1 2.2 21.0 62.0 11.8 159-fatBF PHP41103 AFS
6671.4.2 15 2.6 2.1 23.4 60.2 11.7 159-fatBF PHP41103 AFS 6671.4.2
16 2.8 2.7 17.1 63.6 13.8 159-fatBF PHP41103 AFS 6671.4.2 17 2.6
2.8 19.9 63.0 11.7 159-fatBF PHP41103 AFS 6671.4.2 18 11.1 3.0 17.3
57.3 11.4 159-fatBF PHP41103 AFS 6671.4.2 19 10.9 2.7 19.4 55.4
11.6 159-fatBF PHP41103 AFS 6671.4.2 20 3.0 2.3 19.0 63.3 12.4
159-fatBF PHP41103 AFS 6671.4.2 21 2.5 2.6 18.9 63.9 12.1 159-fatBF
PHP41103 AFS 6671.6.1 1 2.3 2.1 19.7 65.3 10.6 159-fatBF PHP41103
AFS 6671.6.1 2 2.6 2.1 26.8 60.8 7.7 159-fatBF PHP41103 AFS
6671.6.1 3 10GR13-286 11.4 3.5 17.0 57.4 10.7 159-fatBF PHP41103
AFS 6671.6.1 4 12.4 3.2 14.5 58.9 11.0 159-fatBF PHP41103 AFS
6671.6.1 5 10GR13-288 12.5 3.3 13.0 59.0 12.3 159-fatBF PHP41103
AFS 6671.6.1 6 10GR13-289 2.3 2.4 18.0 65.7 11.7 159-fatBF PHP41103
AFS 6671.6.1 7 2.8 2.3 16.4 66.7 11.7 159-fatBF PHP41103 AFS
6671.6.1 8 11.2 2.9 16.2 56.9 12.8 159-fatBF PHP41103 AFS 6671.6.1
9 2.5 2.2 18.7 66.1 10.4 159-fatBF PHP41103 AFS 6671.6.1 10 2.4 2.4
15.8 65.2 14.3 159-fatBF PHP41103 AFS 6671.6.1 11 2.6 2.2 15.6 67.7
12.0 159-fatBF PHP41103 AFS 6671.6.1 12 2.4 1.8 22.7 60.9 12.1
159-fatBF PHP41103 AFS 6671.6.1 13 11.1 3.3 17.1 55.7 12.8
159-fatBF PHP41103 AFS 6671.6.1 14 2.3 2.4 16.5 65.7 13.1 159-fatBF
PHP41103 AFS 6671.6.1 15 2.4 2.1 16.2 66.3 12.9 159-fatBF PHP41103
AFS 6671.6.1 16 2.5 2.5 17.0 64.3 13.7 159-fatBF PHP41103 AFS
6671.6.1 17 10.8 3.1 14.2 58.2 13.7 159-fatBF PHP41103 AFS 6671.6.1
18 2.6 2.1 20.9 63.1 11.3 159-fatBF PHP41103 AFS 6671.6.1 19 2.3
1.8 20.1 62.8 13.0 159-fatBF PHP41103 AFS 6671.6.1 20 2.3 2.3 14.1
68.2 13.0 159-fatBF PHP41103 AFS 6671.6.1 21 10GR13-304 2.4 2.5
15.5 67.8 11.8 159-fatBF PHP41103 AFS 6671.6.3 1 2.8 2.8 16.5 66.0
11.9 159-fatBF PHP41103 AFS 6671.6.3 2 4.0 2.9 19.1 64.1 9.9
159-fatBF PHP41103 AFS 6671.6.3 3 10GR13-307 4.1 2.6 17.7 65.3 10.2
159-fatBF PHP41103 AFS 6671.6.3 4 13.3 3.6 12.3 57.5 13.3 159-fatBF
PHP41103 AFS 6671.6.3 5 10GR13-309 13.3 3.2 15.3 57.2 11.0
159-fatBF PHP41103 AFS 6671.6.3 6 10GR13-310 3.4 2.6 17.9 65.3 10.9
159-fatBF PHP41103 AFS 6671.6.3 7 3.7 2.7 15.3 63.9 14.4 159-fatBF
PHP41103 AFS 6671.6.3 8 3.3 2.7 15.5 61.3 17.2 159-fatBF PHP41103
AFS 6671.6.3 9 10GR13-313 2.9 2.4 19.4 63.2 12.1 159-fatBF PHP41103
AFS 6671.6.3 10 3.9 2.9 15.0 65.4 12.8 159-fatBF PHP41103 AFS
6671.6.3 11 3.8 2.6 15.5 64.3 13.8 159-fatBF PHP41103 AFS 6671.6.3
12 2.9 2.3 14.6 65.2 15.0 159-fatBF PHP41103 AFS 6671.6.3 13 3.4
2.1 17.6 64.6 12.3 159-fatBF PHP41103 AFS 6671.6.3 14 10GR13-318
11.2 3.0 14.4 58.3 13.2 159-fatBF PHP41103 AFS 6671.6.3 15 3.0 2.1
16.0 64.3 14.7 159-fatBF PHP41103 AFS 6671.6.3 16 11.4 3.2 13.2
57.9 14.4 159-fatBF PHP41103 AFS 6671.6.3 17 10GR13-321 2.8 2.5
15.7 63.8 15.1 159-fatBF PHP41103 AFS 6671.6.3 18 3.6 2.3 19.5 61.1
13.5 159-fatBF PHP41103 AFS 6671.6.3 19 2.8 2.5 13.4 66.3 15.0
159-fatBF PHP41103 AFS 6671.6.3 20 11.4 3.2 14.1 55.7 15.5
159-fatBF PHP41103 AFS 6671.6.3 21 3.6 2.6 18.3 63.5 12.0 159-fatBF
PHP41103 AFS 6671.10.4 1 2.2 2.1 32.6 53.0 10.2 159-fatBF PHP41103
AFS 6671.10.4 2 2.3 1.9 32.8 53.1 9.8 159-fatBF PHP41103 AFS
6671.10.4 3 2.4 1.9 39.4 47.2 9.1 159-fatBF PHP41103 AFS 6671.10.4
4 2.5 2.3 17.2 66.1 11.9 159-fatBF PHP41103 AFS 6671.10.4 5 11.4
2.7 26.5 51.2 8.2 159-fatBF PHP41103 AFS 6671.10.4 6 11.3 2.6 20.4
52.3 13.4 159-fatBF PHP41103 AFS 6671.10.4 7 11.3 2.2 23.2 52.2
11.3 159-fatBF PHP41103 AFS 6671.10.4 8 11.7 3.2 15.9 54.9 14.3
159-fatBF PHP41103 AFS 6671.10.4 9 2.3 2.0 19.1 62.8 13.8 159-fatBF
PHP41103 AFS 6671.10.4 10 2.3 2.3 18.0 64.5 12.9 159-fatBF PHP41103
AFS 6671.10.4 11 11.0 2.5 24.2 50.9 11.5 159-fatBF PHP41103 AFS
6671.10.4 12 2.1 1.4 37.9 48.2 10.4 159-fatBF PHP41103 AFS
6671.10.4 13 10.9 3.1 13.5 58.8 13.8 159-fatBF PHP41103 AFS
6671.10.4 14 11.9 3.0 12.3 54.3 18.5 159-fatBF PHP41103 AFS
6671.10.4 15 2.0 2.2 14.8 64.3 16.7 159-fatBF PHP41103 AFS
6671.10.4 16 11.5 3.3 12.4 56.2 16.5 159-fatBF PHP41103 AFS
6671.10.4 17 2.5 2.1 17.4 64.7 13.2 159-fatBF PHP41103 AFS
6671.10.4 18 2.2 1.7 19.5 64.0 12.5 159-fatBF PHP41103 AFS
6671.10.4 19 11.8 2.5 13.8 55.4 16.5 159-fatBF PHP41103 AFS
6671.10.4 20 11.2 3.0 14.1 57.8 14.0 159-fatBF PHP41103 AFS
6671.10.4 21 2.5 1.5 27.0 56.0 13.0 159-fatBF PHP41103 AFS
6671.11.2 1 2.3 1.9 28.8 56.1 10.9 159-fatBF PHP41103 AFS 6671.11.2
2 2.3 2.1 37.8 47.8 9.9 159-fatBF PHP41103 AFS 6671.11.2 3
10GR13-328 11.3 2.8 21.6 52.9 11.5 159-fatBF PHP41103 AFS 6671.11.2
4 10GR13-329 11.8 2.7 21.9 53.2 10.4 159-fatBF PHP41103 AFS
6671.11.2 5 10GR13-330 12.3 3.0 19.6 54.1 11.0 159-fatBF PHP41103
AFS 6671.11.2 6 10GR13-331 2.4 2.3 24.2 58.2 13.0 159-fatBF
PHP41103 AFS 6671.11.2 7 2.6 2.3 22.7 60.5 11.9 159-fatBF PHP41103
AFS 6671.11.2 8 10GR13-333 2.5 2.1 18.4 64.5 12.6 159-fatBF
PHP41103 AFS 6671.11.2 9 10GR13-334 2.3 2.4 16.9 65.4 13.0
159-fatBF PHP41103 AFS 6671.11.2 10 3.0 2.2 21.2 61.1 12.5
159-fatBF PHP41103 AFS 6671.11.2 11 2.3 1.8 18.2 62.5 15.2
159-fatBF PHP41103 AFS 6671.11.2 12 11.5 3.0 19.3 52.8 13.4
159-fatBF PHP41103 AFS 6671.11.2 13 2.5 2.4 13.3 62.0 19.8
159-fatBF PHP41103 AFS 6671.11.2 14 2.4 2.1 19.7 63.2 12.7
159-fatBF PHP41103 AFS 6671.11.2 15 2.6 1.7 21.3 59.8 14.6
159-fatBF PHP41103 AFS 6671.11.2 16 10GR13-341 2.8 2.4 14.7 65.9
14.1 159-fatBF PHP41103 AFS 6671.11.2 17 2.5 1.7 32.3 51.1 12.5
159-fatBF PHP41103 AFS 6671.11.2 18 2.6 2.1 28.6 55.2 11.6
159-fatBF PHP41103 AFS 6671.11.2 19 3.0 2.1 18.4 65.0 11.5
159-fatBF PHP41103 AFS 6671.11.2 20 2.7 1.7 14.3 65.7 15.5
159-fatBF PHP41103 AFS 6671.11.2 21 2.5 1.8 29.7 53.2 12.9
159-fatBF PHP41103 AFS 6671.11.3 1 10GR13-347 2.2 2.2 22.6 61.7
11.4 159-fatBF PHP41103 AFS 6671.11.3 2 2.4 2.2 24.0 61.8 9.6
159-fatBF PHP41103 AFS 6671.11.3 3 2.7 2.0 21.8 62.8 10.8 159-fatBF
PHP41103 AFS 6671.11.3 4 2.7 2.2 21.4 63.6 10.1 159-fatBF PHP41103
AFS 6671.11.3 5 3.2 2.6 16.0 67.9 10.3 159-fatBF PHP41103 AFS
6671.11.3 6 3.2 2.4 18.4 64.7 11.4 159-fatBF PHP41103 AFS 6671.11.3
7 10GR13-353 11.6 2.7 16.4 56.5 12.7 159-fatBF PHP41103 AFS
6671.11.3 8 2.4 1.9 18.8 63.6 13.2 159-fatBF PHP41103 AFS 6671.11.3
9 10GR13-355 2.0 1.7 16.2 65.3 14.7 159-fatBF PHP41103 AFS
6671.11.3 10 3.1 2.4 20.4 62.6 11.5 159-fatBF PHP41103 AFS
6671.11.3 11 159-fatBF PHP41103 AFS 6671.11.3 12 12.2 2.7 11.9 53.3
19.9 159-fatBF PHP41103 AFS 6671.11.3 13 10GR13-359 12.1 2.4 12.3
57.9 15.2 159-fatBF PHP41103 AFS 6671.11.3 14 10GR13-360 3.2 2.2
20.0 63.5 11.2 159-fatBF PHP41103 AFS 6671.11.3 15 2.4 2.2 16.0
65.7 13.6 159-fatBF PHP41103 AFS 6671.11.3 16 2.2 2.2 16.4 64.2
14.9 159-fatBF PHP41103 AFS 6671.11.3 17 2.3 1.7 23.2 60.3 12.5
159-fatBF PHP41103 AFS 6671.11.3 18 2.4 1.6 27.5 57.2 11.3
159-fatBF PHP41103 AFS 6671.11.3 19 2.6 1.8 20.6 63.6 11.5
159-fatBF PHP41103 AFS 6671.11.3 20 2.7 2.5 18.0 65.6 11.2
159-fatBF PHP41103 AFS 6671.11.3 21 10GR13-367 2.3 2.3 13.7 67.3
14.5 159-fatBF PHP41103 AFS 6671.11.4 1 2.1 2.1 24.9 60.9 10.0
159-fatBF PHP41103 AFS 6671.11.4 2 2.6 2.2 33.0 54.1 8.1 159-fatBF
PHP41103 AFS 6671.11.4 3 2.6 2.0 30.0 55.6 9.8 159-fatBF PHP41103
AFS 6671.11.4 4 2.8 2.0 42.3 44.8 8.2 159-fatBF PHP41103 AFS
6671.11.4 5 11.0 2.8 33.3 44.3 8.6 159-fatBF PHP41103 AFS 6671.11.4
6 11.1 2.3 16.6 55.8 14.2 159-fatBF PHP41103 AFS 6671.11.4 7 2.4
1.8 20.4 63.7 11.7 159-fatBF PHP41103 AFS 6671.11.4 8 2.8 1.7 22.4
60.8 12.3 159-fatBF PHP41103 AFS 6671.11.4 9 10.9 2.5 24.5 50.8
11.3 159-fatBF PHP41103 AFS 6671.11.4 10 2.4 1.8 23.9 59.9 11.9
159-fatBF PHP41103 AFS 6671.11.4 11 2.2 1.9 20.8 62.5 12.6
159-fatBF PHP41103 AFS 6671.11.4 12 11.0 2.5 24.6 51.3 10.6
159-fatBF PHP41103 AFS 6671.11.4 13 11.3 2.8 18.3 54.2 13.4
159-fatBF PHP41103 AFS 6671.11.4 14 11.1 2.7 20.1 55.8 10.3
159-fatBF PHP41103 AFS 6671.11.4 15 2.3 1.5 16.2 64.4 15.7
159-fatBF PHP41103 AFS 6671.11.4 16 11.7 2.2 15.3 58.5 12.2
159-fatBF PHP41103 AFS 6671.11.4 17 2.6 1.8 23.3 60.9 11.4
159-fatBF PHP41103 AFS 6671.11.4 18 11.5 2.5 24.4 49.9 11.7
159-fatBF PHP41103 AFS 6671.11.4 19 11.0 2.6 22.6 52.3 11.5
159-fatBF PHP41103 AFS 6671.11.4 20 11.3 2.5 19.0 55.2 12.1
159-fatBF PHP41103 AFS 6671.11.4 21 2.8 1.9 27.1 57.1 11.1
159-fatBF PHP41103 AFS 6671.12.2 1 2.4 2.0 29.5 56.9 9.2 159-fatBF
PHP41103 AFS 6671.12.2 2 2.4 2.1 37.9 48.3 9.3 159-fatBF PHP41103
AFS 6671.12.2 3 11.6 3.0 23.4 53.3 8.7 159-fatBF PHP41103 AFS
6671.12.2 4 11.7 2.8 23.1 53.1 9.4 159-fatBF PHP41103 AFS 6671.12.2
5 11.8 3.1 21.3 55.0 8.8 159-fatBF PHP41103 AFS 6671.12.2 6 2.2 1.7
28.0 56.5 11.6 159-fatBF PHP41103 AFS 6671.12.2 7 11.7 2.7 14.6
57.2 13.8 159-fatBF PHP41103 AFS 6671.12.2 8 12.2 2.5 15.0 53.8
16.5 159-fatBF PHP41103 AFS 6671.12.2 9 11.4 2.3 14.6 56.0 15.8
159-fatBF PHP41103 AFS 6671.12.2 10 2.7 1.7 33.5 49.7 12.4
159-fatBF PHP41103 AFS 6671.12.2 11 10.8 2.9 40.5 34.7 11.1
159-fatBF PHP41103 AFS 6671.12.2 12 2.4 1.7 30.9 50.2 14.7
159-fatBF PHP41103 AFS 6671.12.2 13 2.4 2.4 42.6 41.7 10.9
159-fatBF PHP41103 AFS 6671.12.2 14 11.7 2.9 16.7 53.1 15.6
159-fatBF PHP41103 AFS 6671.12.2 15 2.4 1.9 18.3 64.7 12.7
159-fatBF PHP41103 AFS 6671.12.2 16 2.2 2.1 28.4 54.7 12.6
159-fatBF PHP41103 AFS 6671.12.2 17 2.4 2.1 20.0 62.8 12.7
159-fatBF PHP41103 AFS 6671.12.2 18 11.5 2.6 26.9 46.8 12.2
159-fatBF PHP41103 AFS 6671.12.2 19 2.6 2.4 41.6 43.2 10.2
159-fatBF PHP41103 AFS 6671.12.2 20 2.7 1.9 25.1 56.6 13.8
159-fatBF PHP41103 AFS 6671.12.2 21 2.5 1.7 23.1 59.7 12.9
TABLE-US-00022 TABLE 7g Fatty acid profile for T1 seed analyzed
from soy transformed with PHP41784, containing the 159-fad2-1b
& 159-fatBF& 159-fad3c amiRNAs, grown in the greenhouse.
Seed T1 Seed amiRNA Construct Event No. Planted 16:0 18:0 18:1 18:2
18:3 159-fad2-1b/159- PHP41784A AFS 6783.2.2 1a 5.6 2.3 30.5 55.9
5.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.2.2 2a 4.3
2.2 54.3 35.9 3.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.2.2 3a 12.7 2.6 20.2 55.8 8.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.2.2 4a 4.1 2.2 50.2 39.1 4.4 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.2.2 5a 12.4 2.9 18.2 58.7 7.8
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.5.1 1a 11.3 3.1
28.8 51.0 5.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.5.1 2a 2.7 1.9 84.7 8.6 2.1 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.5.1 3a 2.6 2.0 88.1 6.1 1.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.5.1 4a 2.7 2.0 84.8 8.3 2.3
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.5.1 5a 2.4 2.0
88.5 5.9 1.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.5.2 1a 12.2 3.3 33.7 44.3 6.5 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.5.2 2a 2.7 2.8 86.6 6.2 1.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.5.2 3a 2.8 1.8 81.3 11.7 2.4
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.5.2 4a 2.2 1.6
91.2 3.6 1.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.5.2 5a 4.0 2.3 79.1 12.2 2.4 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.3 1a 12.9 2.6 27.1 51.7 5.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.3 2a 6.8 2.3 40.3 47.6 3.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.3 3a 4.1 2.3
48.0 42.1 3.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.3 4a 5.4 2.4 41.5 46.7 3.9 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.3 5a 2.3 2.1 86.9 6.7 2.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 1a 2.9 1.7 89.7 4.1 1.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 2a 2.3 1.9
91.0 3.9 0.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 3a 3.5 2.1 85.8 6.6 2.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 4a 2.8 1.9 89.9 4.5 1.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 5a 2.6 2.1 88.7 5.4 1.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 1 2.1 1.8
89.3 5.2 1.5 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 2 0.0 0.0 94.9 5.1 0.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 3 2.3 2.4 89.2 6.1 0.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 4 2.2 2.3 87.1 6.7 1.8
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 5 1.9 1.7
92.8 3.6 0.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 6 2.1 2.1 89.0 5.5 1.2 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 7 2.0 1.9 90.7 4.1 1.3 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 8 2.1 2.3 88.4 5.6 1.6
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 9 2.2 2.3
89.9 4.5 1.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 10 1.9 2.3 89.3 5.2 1.3 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 11 14.0 3.8 1.5 67.0 13.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 12 1.8 1.7 90.8 4.6 1.1
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 13 1.9 1.8
88.0 6.4 1.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 14 1.6 1.3 91.2 4.2 1.7 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 15 1.8 2.0 89.6 5.0 1.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 16 2.0 1.6 90.0 4.8 1.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 17 2.3 2.1
85.6 7.6 2.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 18 2.2 1.9 90.4 4.4 1.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 19 1.9 1.5 92.3 4.3 0.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 20 1.9 1.8 90.0 4.8 1.6
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.6.1 21 2.0 1.7
89.4 4.9 2.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.6.1 22 1.8 1.7 87.9 6.7 2.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.6.1 23 1.9 1.8 90.7 4.5 1.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.6.1 24 3.1 2.2 74.8 11.1 8.8
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 1a 10GR32-1
2.5 1.9 79.2 12.3 4.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6783.8.1 2a 2.8 2.2 82.5 10.1 2.4 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 3a 2.7 2.2 82.1 10.7 23
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 4a 13.1 2.7
15.9 59.9 8.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 5a 10GR32-2 2.5 1.9 78.7 12.9 4.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 1 2.5 2.3 80.6 14.6 0.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 2 10GR32-4
10.7 3.1 15.3 59.7 11.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6783.8.1 3 2.6 2.2 73.4 17.6 4.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 4 2.3 2.1 85.5 7.3 2.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 5 2.2 1.9
90.1 5.8 0.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 6 2.5 2.1 80.1 11.7 3.5 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.1 7 2.2 2.1 83.8 8.5 3.3 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 8 2.3 2.0 82.4 9.7 3.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 9 2.6 2.1
78.8 12.1 4.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 10 10GR32-11 2.0 2.3 79.3 12.7 3.8 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 11 0.0 0.0 77.4 22.6 0.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 12 11.4 3.4
14.0 59.1 12.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 13 12.2 3.6 1.5 64.0 18.7 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.1 14 10GR32-13 10.9 3.3 14.7 58.6 12.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 15 2.5 2.1
85.8 9.6 0.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 16 2.2 1.9 79.8 12.3 3.9 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.1 17 13.6 3.9 1.7 65.6 15.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 18 10GR32-16 10.8 3.1 15.8
59.7 10.7 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1
19 2.0 2.1 85.7 10.2 0.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6783.8.1 20 2.4 2.1 75.5 15.9 4.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 21 12.6 3.7 1.5 69.3 13.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.1 22 11.6 2.7
12.8 60.9 12.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.1 23 10GR32-19 2.5 2.1 71.3 19.6 4.6 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.1 24 2.3 2.3 81.5 10.4 3.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 1a
10GR32-21 2.6 2.0 88.4 5.2 1.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 2a 12.9 3.1 14.1 58.3 11.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 3a 3.1 2.3 82.4 9.5 2.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 4a
10GR32-22 2.5 2.1 90.7 3.4 1.5 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 5a 2.6 2.0 87.5 5.7 2.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 1 12.0 3.1 21.0 55.4 8.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 2 10GR32-24
11.7 3.5 18.2 53.7 12.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6783.8.2 3 10GR32-25 2.0 2.0 86.5 6.7 2.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 4 2.4 2.3 82.3 9.5 3.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 5 14.3 3.7
1.4 66.9 13.7 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.2 6 12.3 3.0 15.0 57.4 12.3 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 7 10GR32-27 2.3 2.0 87.4 5.7 2.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 8 10GR32-28
2.1 2.4 88.3 5.1 2.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.2 9 14.1 3.2 1.8 68.4 12.4 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 10 11.0 4.2 36.5 41.4 7.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 11 4.0 2.6 73.4 15.9 4.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 12 2.6 2.8
83.1 8.6 3.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.2 13 14.6 3.8 1.7 68.3 11.6 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 14 10GR32-30 2.4 2.3 76.5 14.5 4.4
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 15 11.2 3.7
25.3 51.3 8.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.2 16 2.7 2.5 82.4 8.9 3.5 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 17 10GR32-33 2.1 2.5 85.8 7.1 2.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 18 11.5 3.3
20.2 55.8 9.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6783.8.2 19 10GR32-35 2.6 2.4 79.7 11.8 3.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 20 2.4 2.4 81.1 10.7 3.3
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 21
10GR32-37 2.2 2.1 84.4 8.3 3.1 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6783.8.2 22 2.5 2.2 86.4 6.4 2.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6783.8.2 23 2.1 2.4 87.6 5.5 2.4
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6783.8.2 24
10GR32-40 11.5 3.0 16.5 57.8 11.2 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.1.1 1a 2.5 1.9 90.3 3.5 1.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 2a 2.4 2.2 85.9 3.2 6.3
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 3a 2.8 1.7
89.5 4.0 2.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 4a 10GR32-41 2.3 2.0 90.1 3.6 2.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 5a 10GR32-42 2.2 1.9 90.4
3.2 2.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 1
13.3 3.5 1.5 69.8 12.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6784.1.1 2 10GR32-43 10.3 2.5 23.8 53.3
10.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 3 2.1
2.1 86.1 6.4 3.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 4 10GR32-45 2.2 2.4 89.5 4.1 1.8 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 5 13.5 3.3 1.6 64.2 17.3
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 6 2.3 1.9
87.6 3.1 5.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 7 10GR32-46 2.1 1.7 89.8 0.8 5.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 8 10GR32-47 1.8 2.0 90.1
4.0 2.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 9
10GR32-48 2.0 2.2 89.7 3.1 2.9 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.1.1 10 2.2 1.8 87.4 3.7 5.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 11 14.3 3.2 1.8 67.6 13.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 12 2.0 1.8
90.5 3.9 1.7 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 13 2.1 2.0 87.9 4.9 3.2 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.1.1 14 10GR32-52 1.9 2.1 89.7 3.4 2.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 15 2.4 2.4
86.0 6.7 2.5 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 16 2.0 1.7 87.9 5.2 3.3 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.1.1 17 10.4 2.5 33.1 47.2 6.8 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 18 2.3 2.2 87.2 5.3 2.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 19 10.2 2.8
24.2 57.0 5.8 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 20 10GR32-56 2.2 2.3 85.2 7.1 3.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 21 12.8 3.7 1.7 73.3 8.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.1.1 22 2.0 1.5
91.9 2.9 1.7 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.1.1 23 10GR32-59 1.9 2.2 90.8 3.5 1.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.1.1 24 2.0 1.6 92.5 2.4 1.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 1a 3.9 1.8
84.7 7.3 2.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.2.1 2a 3.9 2.1 76.0 15.6 2.4 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.2.1 3a 10GR32-61 3.6 2.3 83.2 8.5 2.4
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 4a
10GR32-62 12.1 2.8 18.8 58.4 8.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.2.1 5a 3.2 2.3 86.6 5.8 2.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 1 12.2 3.4 1.5 69.5 13.4
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 2 10GR32-64
2.9 1.9 87.9 5.7 1.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.2.1 3 10GR32-65 2.0 2.4 88.5 5.3 1.8 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 4 10GR32-66 2.9 2.2 89.3
4.3 1.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 5
15.2 3.0 2.2 65.7 13.8 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6784.2.1 6 10GR32-67 2.2 2.3 89.4 4.9 1.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 7 10GR32-68 3.1 2.0 85.2
7.5 2.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 8
11.0 3.0 16.6 59.8 9.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6784.2.1 9 5.7 2.4 83.6 6.2 2.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 10 11.5 2.8 15.9 58.0 11.8
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 11 6.1 2.9
84.0 5.7 1.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.2.1 12 10GR32-71 11.7 2.6 21.7 54.7 9.3 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 13 10GR32-72 2.8 2.0 86.1
6.6 2.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 14
10GR32-73 3.4 1.9 83.6 8.4 2.6 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.2.1 15 2.1 2.1 88.3 5.4 2.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 16 10GR32-75 2.2 2.1 91.4
3.1 1.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 17
19.0 3.0 3.4 63.1 11.5 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A
AFS 6784.2.1 18 10GR32-76 3.3 2.2 86.3 6.1 2.1 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 19 10GR32-77 3.0 2.2 88.9
4.5 1.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 20
10GR32-78 2.7 1.9 88.2 5.4 1.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.2.1 21 2.8 2.3 88.0 5.0 1.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.2.1 22 2.8 2.1 85.1 7.5 2.5
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.2.1 23 5.7 2.1
84.7 5.6 1.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.2.1 24 5.7 2.0 82.7 7.1 2.6 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 1a 3.3 2.0 78.1 14.3 2.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 2a 2.3 2.0 87.0 6.1 2.6
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 3a 2.9 2.4
88.2 4.9 1.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.3.2 4a 12.2 3.1 15.8 59.8 9.2 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 5a 2.3 2.3 87.4 5.4 2.7 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 1 10GR32-82 12.0 2.8 13.6
60.4 11.3 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 2
2.1 2.5 87.8 4.4 3.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.3.2 3 10GR32-84 12.1 3.3 1.5 67.5 15.6 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 4 2.4 2.4 84.2 7.9 3.1
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 5 2.0 2.1
87.2 5.1 3.6 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.3.2 6 2.5 2.5 88.8 4.3 1.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 7 2.0 1.9 86.2 6.1 3.8 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 8 10GR32-87 2.3 2.3 84.7
7.6 3.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 9
1.9 2.2 87.9 4.7 3.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.3.2 10 10.7 2.7 18.9 57.8 9.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 11 2.3 2.2 87.1 5.0 3.5 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 12 2.3 2.4 87.1 5.2 3.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 13 2.7 2.4
76.2 13.8 4.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.3.2 14 10GR32-91 2.0 2.3 88.1 4.7 2.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 15 2.0 2.3 88.8 4.0 2.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 16
10GR32-93 1.9 2.0 89.3 4.3 2.4 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 17 2.0 2.2 88.5 4.3 3.0 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 18 2.1 2.0 87.3 5.4 3.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 19
10GR32-96 1.9 2.4 89.6 4.0 2.1 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 20 2.2 2.2 86.5 5.8 3.4 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 21 2.2 1.8 80.9 11.1 4.1
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.3.2 22
10GR32-99 2.3 2.2 88.6 4.5 2.3 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.3.2 23 2.0 1.8 80.0 7.3 8.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.3.2 24 2.3 2.0 84.3 9.3 2.0
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 1a 13.0 2.7
19.4 56.0 9.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 2a 11.6 2.8 19.4 58.7 7.6 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 3a 10.5 3.4 24.1 52.8 9.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 4a 2.3 2.4 85.1 8.0 2.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 5a 3.0 2.0
76.9 15.8 2.4 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 1 1.9 2.3 86.3 6.8 2.8 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 2 2.2 2.0 87.0 6.4 2.4 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 3 13.6 3.6 1.4 67.2 14.2
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 4 10.3 3.1
11.5 62.1 13.0 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 5 10.1 2.9 18.0 59.0 10.0 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 6 15.2 4.3 19.9 53.3 7.3 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 7 2.1 2.7 80.5 10.9 3.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 8 2.4 2.2
80.7 10.7 3.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 9 2.7 2.1 81.0 11.7 2.6 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 10 10.5 2.9 19.5 59.2 7.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 11 13.5 3.2 1.5 70.8 10.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 12 11.5 3.2
13.5 60.9 10.9 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 13 2.5 2.1 82.6 9.6 3.3 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 14 2.6 2.3 78.4 12.8 3.9 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 15 3.4 2.1 67.0 22.8 4.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 16 10.0 3.0
23.9 54.9 8.2 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 17 10.7 2.9 18.3 58.7 9.4 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 18 3.2 2.0 59.5 30.2 5.2 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 19 2.2 2.7 84.0 8.4 2.7
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS 6784.4.2 20 9.7 2.5
25.7 53.9 8.1 fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS
6784.4.2 21 3.2 2.2 74.3 16.5 39 fatBf/159-fad3c 159-fad2-1b/159-
PHP41784A AFS 6784.4.2 22 10.1 3.2 15.2 61.1 10.4 fatBf/159-fad3c
159-fad2-1b/159- PHP41784A AFS 6784.4.2 23 2.8 2.0 72.7 18.6 3.9
fatBf/159-fad3c 159-fad2-1b/159- PHP41784A AFS6784.4.2 24 2.5 2.0
84.5 8.7 2.2 fatBf/159-fad3c
Analysis of Fatty Acid Profiles of T2 Seed from Events Expressing
amiRNAs Grown in the Greenhouse
[0395] T1 seed from events having phenotypes indicating functional
amiRNAs were planted and plants grown in the greenhouse.
[0396] T1 seed planted from soy transformed with PHP32510,
containing the 369b-fad2-1b amiRNA or PHP32511, containing the
159-fad2-1b amiRNA, or PHP32843, containing the 369b-fad2-1b &
159-fad2-2 amiRNAs were given an 8GR31 experiment designation.
[0397] T1 seed planted from soy transformed with PHP33705,
containing the 396-fad2-1b & 396b-sad3 amiRNAs, or PHP38557,
containing the 159-fad3c amiRNA, or PHP41103, containing the
159-fatBF amiRNA, were given a 10GR13 experiment designation.
[0398] T1 seed planted from soy transformed with PHP41784,
containing the 159-fad2-1b & 159-fatBF& 159-fad3c amiRNAs,
were given a 10GR32 experiment designation.
[0399] T2 seed from each experiment were harvested and
approximately 5 to 10 seed from each T2 seed pack for each event
where the T1 seed indicated a positive phenotype, were chipped and
fatty acids analyzed by GC/FAME as described inter alia.
[0400] Those seed packs where T1 seed indicated a negative
phenotype (null segregant seed), were also analyzed but in that
case, fewer than 5-10 T2 seed were sometimes analyzed.
[0401] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP32510, containing the 369b-fad2-1b amiRNA or
PHP32511, containing the 159-fad2-1b amiRNA is shown in Table
8.
[0402] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP32843, containing the 369b-fad2-1b &
159-fad2-2 amiRNAs is shown in Table 9.
[0403] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP33705, containing the 396-fad2-1b &
396b-sad3 amiRNAs is shown in Table 10.
[0404] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP38557, containing the 159-fad3c amiRNA, is
shown in Table 11.
[0405] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP41103, containing the 159-fatBF amiRNA, is
shown in Table 12.
[0406] The fatty acid profile for T2 seed analyzed from soy
transformed with PHP41784, containing the 159-fad2-1b &
159-fatBF& 159-fad3c amiRNAs is shown in Table 13.
[0407] In Table 8-13, the name of the amiRNA in the construct, as
well as the construct name itself is indicated. Also shown is the
event name for the T2 seed packs that were chosen for analysis.
Each T1 seed planted was also given an experiment name (e.g.
8GR31-1) which was kept for each T2 seed pack harvested. Data shown
in Table 8-13 is from those seed packs where seed fatty acid
profiles indicated the seed pack was from a homozygous plant (i.e.
no null segregants) as well some which were all wild-type-like
(null), where Homoz Pos indicates a homozygous seed pack, and Null
indicates a null segregant pack from that event.
TABLE-US-00023 TABLE 8 Fatty acid profile for T2 seed analyzed from
soy transformed with PHP32510, containing the 369b-fad2-1b amiRNA
or PHP32511, containing the 159- fad2-1b amiRNA grown in the
greenhouse. T2 T2 Seed Seed Seed amiRNA Construct Event Analyzed
No. 16:0 18:0 18:1 18:2 18:3 Comment 396b- PHP32510 AFS 8GR31-16 1
11.9 3.1 26.6 53.6 4.7 Null fad2- 5260.3.3 1b 396b- PHP32510 AFS
8GR31-16 2 12.1 3.3 11.0 62.3 11.2 Null fad2- 5260.3.3 1b 396b-
PHP32510 AFS 8GR31-16 3 11.4 3.2 15.0 62.9 7.5 Null fad2- 5260.3.3
1b 396b- PHP32510 AFS 8GR31-16 4 12.7 3.8 12.5 60.7 10.2 Null fad2-
5260.3.3 1b 396b- PHP32510 AFS 8GR31-16 5 11.2 3.0 17.8 61.5 6.5
Null fad2- 5260.3.3 1b 396b- PHP32510 AFS 8GR31-7 1 14.6 3.5 13.7
60.9 7.2 Null fad2- 5260.3.11 1b 396b- PHP32510 AFS 8GR31-7 2 14.0
3.8 7.7 62.5 12.0 Null fad2- 5260.3.11 1b 396b- PHP32510 AFS
8GR31-7 3 14.1 3.4 35.3 42.9 4.4 Null fad2- 5260.3.11 1b 396b-
PHP32510 AFS 8GR31-7 4 13.5 3.7 21.6 55.2 6.0 Null fad2- 5260.3.11
1b 396b- PHP32510 AFS 8GR31-7 5 13.6 3.2 16.4 60.0 6.8 Null fad2-
5260.3.11 1b 396b- PHP32510 AFS 8GR31-2 1 9.0 3.8 78.6 4.7 3.9
Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-2 2 8.1 3.5
83.6 1.6 3.2 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-2
3 8.0 3.3 83.5 1.6 3.6 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510
AFS 8GR31-2 4 8.2 3.5 83.1 1.6 3.5 Homoz fad2- 5260.2.3 Pos 1b
396b- PHP32510 AFS 8GR31-2 5 7.5 3.5 84.4 1.5 3.2 Homoz fad2-
5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-2 6 7.8 3.0 84.3 1.4 3.6
Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-2 7 8.0 2.8
83.7 1.7 3.8 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-2
8 7.7 3.0 84.5 1.5 3.3 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510
AFS 8GR31-2 9 7.3 3.2 85.0 1.2 3.3 Homoz fad2- 5260.2.3 Pos 1b
396b- PHP32510 AFS 8GR31-2 10 8.0 2.9 84.3 1.4 3.4 Homoz fad2-
5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-3 1 7.6 3.5 84.2 1.4 3.2
Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-3 2 8.1 3.2
84.0 1.3 3.3 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-3
3 8.0 3.3 83.8 1.4 3.5 Homoz fad2- 5260.2.3 Pos 1b 396b- PHP32510
AFS 8GR31-3 4 8.0 3.3 83.2 1.5 4.0 Homoz fad2- 5260.2.3 Pos 1b
396b- PHP32510 AFS 8GR31-3 5 7.6 3.5 84.1 1.3 3.5 Homoz fad2-
5260.2.3 Pos 1b 396b- PHP32510 AFS 8GR31-19 1 7.9 3.4 83.8 1.3 3.7
Homoz fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS 8GR31-19 2 7.5 3.7
83.9 1.4 3.5 Homoz fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS
8GR31-19 3 7.5 4.0 84.2 1.2 3.2 Homoz fad2- 5260.3.6 Pos 1b 396b-
PHP32510 AFS 8GR31-19 4 7.3 3.9 84.6 1.4 2.9 Homoz fad2- 5260.3.6
Pos 1b 396b- PHP32510 AFS 8GR31-19 5 7.5 3.1 84.9 1.3 3.2 Homoz
fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS 8GR31-19 6 8.1 3.2 83.3
1.5 3.8 Homoz fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS 8GR31-19 7
8.0 3.6 80.2 3.3 4.8 Homoz fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS
8GR31-19 8 7.8 3.2 83.7 1.5 3.7 Homoz fad2- 5260.3.6 Pos 1b 396b-
PHP32510 AFS 8GR31-19 9 6.9 4.1 84.8 1.3 3.0 Homoz fad2- 5260.3.6
Pos 1b 396b- PHP32510 AFS 8GR31-19 10 7.7 3.3 84.0 1.3 3.7 Homoz
fad2- 5260.3.6 Pos 1b 396b- PHP32510 AFS 8GR31-24 1 7.6 3.3 85.2
1.2 2.7 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS 8GR31-24 2
8.6 3.0 84.5 1.0 2.9 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS
8GR31-24 3 8.3 3.3 83.4 1.2 3.9 Homoz fad2- 5260.4.5 Pos 1b 396b-
PHP32510 AFS 8GR31-24 4 7.8 3.5 83.8 1.3 3.5 Homoz fad2- 5260.4.5
Pos 1b 396b- PHP32510 AFS 8GR31-24 5 8.0 3.4 84.9 1.2 2.5 Homoz
fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS 8GR31-25 1 7.9 3.1 84.7
1.1 3.2 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS 8GR31-25 2
8.4 3.2 84.0 1.3 3.2 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS
8GR31-25 3 7.7 3.6 84.0 1.2 3.5 Homoz fad2- 5260.4.5 Pos 1b 396b-
PHP32510 AFS 8GR31-25 4 8.2 3.4 84.2 1.0 3.3 Homoz fad2- 5260.4.5
Pos 1b 396b- PHP32510 AFS 8GR31-25 5 7.8 3.2 84.9 1.1 2.9 Homoz
fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS 8GR31-25 6 7.9 3.1 85.2
1.2 2.7 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS 8GR31-25 7
8.2 2.9 85.1 1.0 2.8 Homoz fad2- 5260.4.5 Pos 1b 396b- PHP32510 AFS
8GR31-25 8 7.7 2.9 85.7 1.0 2.6 Homoz fad2- 5260.4.5 Pos 1b 396b-
PHP32510 AFS 8GR31-25 9 7.3 3.1 85.8 0.9 2.8 Homoz fad2- 5260.4.5
Pos 1b 396b- PHP32510 AFS 8GR31-25 10 7.6 3.4 84.4 1.2 3.4 Homoz
fad2- 5260.4.5 Pos 1b 159- PHP32511 AFS 8GR31-58 1 8.3 3.1 83.3 1.4
3.9 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-58 2 8.0
3.5 82.6 1.7 4.3 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS
8GR31-58 3 7.5 4.0 82.1 1.7 4.6 Homoz fad2- 5292.1.4 Pos 1b 159-
PHP32511 AFS 8GR31-58 4 7.4 3.5 84.1 1.4 3.5 Homoz fad2- 5292.1.4
Pos 1b 159- PHP32511 AFS 8GR31-58 5 8.1 3.3 83.7 1.6 3.4 Homoz
fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-59 1 7.9 3.5 84.2 1.5
2.9 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-59 2 7.8
3.3 84.0 1.5 3.4 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS
8GR31-59 3 8.5 3.9 81.6 1.9 4.0 Homoz fad2- 5292.1.4 Pos 1b 159-
PHP32511 AFS 8GR31-59 4 7.8 3.2 84.6 1.5 2.9 Homoz fad2- 5292.1.4
Pos 1b 159- PHP32511 AFS 8GR31-59 5 Homoz fad2- 5292.1.4 Pos 1b
159- PHP32511 AFS 8GR31-59 6 8.2 3.2 81.3 3.4 4.0 Homoz fad2-
5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-59 7 8.1 3.0 84.3 1.6 3.1
Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-59 8 8.0 3.3
82.5 1.8 4.4 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-59
9 8.2 3.2 83.9 1.3 3.5 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511
AFS 8GR31-59 10 8.0 3.2 82.4 2.8 3.7 Homoz fad2- 5292.1.4 Pos 1b
159- PHP32511 AFS 8GR31-61 1 7.7 3.6 82.6 2.0 4.1 Homoz fad2-
5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-61 2 7.8 3.3 83.9 1.6 3.4
Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-61 3 7.8 3.6
83.0 1.7 3.7 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511 AFS 8GR31-61
4 8.1 3.4 83.1 1.7 3.7 Homoz fad2- 5292.1.4 Pos 1b 159- PHP32511
AFS 8GR31-61 5 7.7 3.7 83.6 1.6 3.5 Homoz fad2- 5292.1.4 Pos 1b
159- PHP32511 AFS 8GR31-38 1 7.5 3.5 84.7 1.3 3.1 Homoz fad2-
5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38 2 7.8 3.6 83.9 1.2 3.4
Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38 3 8.1 3.1
84.9 1.3 2.7 Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38
4 8.4 3.8 83.4 1.5 2.9 Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511
AFS 8GR31-38 5 8.4 3.1 83.7 1.1 3.7 Homoz fad2- 5292.5.8 Pos 1b
159- PHP32511 AFS 8GR31-38 6 8.0 3.1 84.3 1.2 3.4 Homoz fad2-
5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38 7 8.4 3.3 83.8 1.4 3.0
Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38 8 8.3 3.1
84.0 1.1 3.4 Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511 AFS 8GR31-38
9 8.7 2.7 85.3 1.4 2.0 Homoz fad2- 5292.5.8 Pos 1b 159- PHP32511
AFS 8GR31-38 10 7.9 3.1 84.5 1.2 3.3 Homoz fad2- 5292.5.8 Pos
1b
159- PHP32511 AFS 8GR31-67 1 9.4 3.3 82.5 1.3 3.5 Homoz fad2-
5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67 2 9.1 3.1 82.9 1.2 3.7
Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67 3 8.5 3.4
81.1 2.3 4.8 Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67
4 8.2 3.0 83.5 1.5 3.8 Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511
AFS 8GR31-67 5 8.7 3.0 84.4 1.0 2.9 Homoz fad2- 5292.6.5 Pos 1b
159- PHP32511 AFS 8GR31-67 6 8.5 2.8 82.3 1.8 4.6 Homoz fad2-
5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67 7 8.5 2.8 83.7 1.3 3.7
Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67 8 8.1 3.1
82.9 1.5 4.4 Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511 AFS 8GR31-67
9 8.2 3.1 81.8 2.1 4.8 Homoz fad2- 5292.6.5 Pos 1b 159- PHP32511
AFS 8GR31-67 10 8.3 2.9 83.9 1.5 3.4 Homoz fad2- 5292.6.5 Pos 1b
159- PHP32511 AFS 8GR31-73 1 7.9 3.3 84.3 1.3 3.2 Homoz fad2-
5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-73 2 8.2 3.3 83.7 1.2 3.6
Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-73 3 8.1 3.4
83.8 1.3 3.4 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-73
4 8.8 3.1 83.2 1.3 3.7 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511
AFS 8GR31-73 5 8.7 3.4 82.9 1.3 3.8 Homoz fad2- 5292.7.2 Pos 1b
159- PHP32511 AFS 8GR31-74 1 Homoz fad2- 5292.7.2 Pos 1b 159-
PHP32511 AFS 8GR31-74 2 7.5 3.3 84.6 1.2 3.4 Homoz fad2- 5292.7.2
Pos 1b 159- PHP32511 AFS 8GR31-74 3 8.4 3.1 83.7 1.3 3.5 Homoz
fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-74 4 8.4 3.0 83.2 1.6
3.8 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-74 5 8.2
3.1 80.6 4.0 4.2 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS
8GR31-74 6 7.7 3.0 85.3 0.9 3.1 Homoz fad2- 5292.7.2 Pos 1b 159-
PHP32511 AFS 8GR31-74 7 8.4 3.2 83.9 1.2 3.3 Homoz fad2- 5292.7.2
Pos 1b 159- PHP32511 AFS 8GR31-74 8 7.8 3.0 84.5 1.2 3.5 Homoz
fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-74 9 8.2 3.1 84.4 1.2
3.1 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-74 10 8.2
2.9 84.7 1.0 3.2 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS
8GR31-75 1 8.9 3.1 82.5 1.4 4.1 Homoz fad2- 5292.7.2 Pos 1b 159-
PHP32511 AFS 8GR31-75 2 7.9 3.1 84.7 1.1 3.2 Homoz fad2- 5292.7.2
Pos 1b 159- PHP32511 AFS 8GR31-75 3 8.6 3.3 83.6 1.2 3.4 Homoz
fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-75 4 8.4 3.7 81.7 1.4
4.8 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-75 5 8.3
3.1 82.8 1.3 4.4 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS
8GR31-76 1 7.7 3.1 85.0 1.1 3.0 Homoz fad2- 5292.7.2 Pos 1b 159-
PHP32511 AFS 8GR31-76 2 8.3 3.3 83.8 1.2 3.4 Homoz fad2- 5292.7.2
Pos 1b 159- PHP32511 AFS 8GR31-76 3 8.3 3.1 83.6 1.2 3.9 Homoz
fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-76 4 7.6 3.4 84.5 1.3
3.3 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-78 1 8.0
3.2 84.0 1.7 3.0 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS
8GR31-78 2 8.3 2.8 85.4 1.0 2.5 Homoz fad2- 5292.7.2 Pos 1b 159-
PHP32511 AFS 8GR31-78 3 8.6 3.3 82.9 1.2 4.1 Homoz fad2- 5292.7.2
Pos 1b 159- PHP32511 AFS 8GR31-78 4 8.3 3.5 83.9 1.1 3.3 Homoz
fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-78 5 9.0 3.4 82.2 1.4
4.0 Homoz fad2- 5292.7.2 Pos 1b 159- PHP32511 AFS 8GR31-42 1 8.0
3.5 83.2 1.5 3.9 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS
8GR31-42 2 7.6 3.5 84.6 1.2 3.0 Homoz fad2- 5292.7.6 Pos 1b 159-
PHP32511 AFS 8GR31-42 3 7.3 3.7 84.9 1.1 3.1 Homoz fad2- 5292.7.6
Pos 1b 159- PHP32511 AFS 8GR31-42 4 8.3 3.7 84.0 1.3 2.8 Homoz
fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS 8GR31-42 5 8.7 3.8 83.2 1.5
2.8 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS 8GR31-44 1 7.8
3.6 83.9 1.2 3.4 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS
8GR31-44 2 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS 8GR31-44 3
8.0 3.4 83.6 1.4 3.6 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS
8GR31-44 4 8.2 3.4 84.1 1.1 3.2 Homoz fad2- 5292.7.6 Pos 1b 159-
PHP32511 AFS 8GR31-44 5 8.2 3.5 83.1 1.9 3.3 Homoz fad2- 5292.7.6
Pos 1b 159- PHP32511 AFS 8GR31-44 6 8.0 3.3 83.8 1.3 3.6 Homoz
fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS 8GR31-44 7 7.7 3.7 84.0 1.2
3.4 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS 8GR31-44 8 7.7
3.6 84.1 1.4 3.3 Homoz fad2- 5292.7.6 Pos 1b 159- PHP32511 AFS
8GR31-44 9 7.6 3.3 84.5 1.2 3.3 Homoz fad2- 5292.7.6 Pos 1b 159-
PHP32511 AFS 8GR31-44 10 7.8 3.3 84.2 1.2 3.4 Homoz fad2- 5292.7.6
Pos 1b
[0408] Table 8 shows that T2 seed from null segregants have oleic
acid contents ranging from 7.7% to 35% which is in the range for
that of Jack seed which ranges from 12.7% to 17.9% (see Table 12).
T2 seed from events expressing the 396b-fad2-1b amiRNA have oleic
acid contents ranging from 78.6% to 85% and T2 seed from events
expressing the 159-fad2-1b amiRNA have oleic acid contents ranging
from 80.2% to 85.8%.
TABLE-US-00024 TABLE 9 Fatty acid profile for T2 seed analyzed from
soy transformed with PHP32843, containing the 369b-fad2-1b &
159-fad2-2 amiRNAs grown in the greenhouse. T2 T2 Seed Seed Seed
amiRNA Construct Event Analyzed No. 16:0 18:0 18:1 18:2 18:3
Comment 396b- PHP32843 AFS 8GR31- 1 7.5 3.0 87.2 0.4 1.7 Homoz
fad2- 5396.2.2 140 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 2
7.9 2.9 85.5 0.5 1.9 Homoz fad2- 5396.2.2 140 Pos 1b/159- fad2-2
396b- PHP32843 AFS 8GR31- 3 7.7 2.8 87.4 0.3 1.7 Homoz fad2-
5396.2.2 140 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 4 8.2 3.0
85.5 0.6 1.9 Homoz fad2- 5396.2.2 140 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 5 7.8 3.1 86.9 0.4 1.6 Homoz fad2- 5396.2.2 140
Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 6 7.6 2.7 86.2 0.4 1.6
Homoz fad2- 5396.2.2 140 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 7 7.8 3.1 86.7 0.4 1.8 Homoz fad2- 5396.2.2 140 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 8 8.2 2.9 85.1 0.4 1.9 Homoz fad2-
5396.2.2 140 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 9 7.3 3.2
87.3 0.3 1.7 Homoz fad2- 5396.2.2 140 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 10 7.7 3.2 85.7 0.5 1.8 Homoz fad2- 5396.2.2
140 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 1 7.5 3.2 86.4 0.8
2.0 Homoz fad2- 5396.2.3 145 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 2 8.0 3.0 85.6 0.4 1.8 Homoz fad2- 5396.2.3 145 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 3 8.0 2.9 87.0 0.4 1.6 Homoz fad2-
5396.2.3 145 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 4 7.6 2.7
86.5 0.4 1.8 Homoz fad2- 5396.2.3 145 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 5 7.4 2.9 87.6 0.3 1.7 Homoz fad2- 5396.2.3 145
Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 6 7.4 2.9 86.5 0.4 1.6
Homoz fad2- 5396.2.3 145 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 7 7.4 3.3 87.0 0.3 1.8 Homoz fad2- 5396.2.3 145 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 8 7.0 2.9 87.2 0.3 1.5 Homoz fad2-
5396.2.3 145 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 9 7.3 2.9
87.4 0.3 1.8 Homoz fad2- 5396.2.3 145 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 10 7.3 3.1 86.2 0.4 1.8 Homoz fad2- 5396.2.3
145 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 1 7.4 3.4 86.4 0.4
2.2 Homoz fad2- 5396.3.8 156 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 2 9.3 3.1 82.3 0.7 3.3 Homoz fad2- 5396.3.8 156 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 3 8.1 2.9 86.6 0.3 2.0 Homoz fad2-
5396.3.8 156 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 4 6.9 3.2
86.6 0.5 1.7 Homoz fad2- 5396.3.8 156 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 5 8.1 3.3 85.3 0.4 2.6 Homoz fad2- 5396.3.8 156
Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 6 7.3 3.1 85.9 0.4 2.0
Homoz fad2- 5396.3.8 156 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 7 6.7 3.1 87.9 0.3 1.8 Homoz fad2- 5396.3.8 156 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 8 7.5 3.5 85.1 0.5 2.0 Homoz fad2-
5396.3.8 156 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 9 6.7 3.3
87.6 0.4 1.8 Homoz fad2- 5396.3.8 156 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 10 8.0 3.4 84.7 0.5 2.3 Homoz fad2- 5396.3.8
156 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 1 6.8 3.3 86.9 0.5
2.2 Homoz fad2- 5396.3.8 157 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 2 7.1 3.2 86.0 0.4 1.8 Homoz fad2- 5396.3.8 157 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 3 7.2 2.7 87.5 0.4 1.9 Homoz fad2-
5396.3.8 157 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 4 7.1 3.3
88.0 0.4 0.0 Homoz fad2- 5396.3.8 157 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 5 7.0 2.9 87.5 0.4 2.0 Homoz fad2- 5396.3.8 157
Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 6 6.6 3.8 86.1 0.4 1.7
Homoz fad2- 5396.3.8 157 Pos 1b/159- fad2-2 396b- PHP32843 AFS
8GR31- 7 7.4 2.9 87.5 0.3 1.8 Homoz fad2- 5396.3.8 157 Pos 1b/159-
fad2-2 396b- PHP32843 AFS 8GR31- 8 6.9 2.5 86.8 0.7 2.1 Homoz fad2-
5396.3.8 157 Pos 1b/159- fad2-2 396b- PHP32843 AFS 8GR31- 9 7.1 2.8
87.7 0.3 1.8 Homoz fad2- 5396.3.8 157 Pos 1b/159- fad2-2 396b-
PHP32843 AFS 8GR31- 10 7.1 3.2 85.6 0.7 2.3 Homoz fad2- 5396.3.8
157 Pos 1b/159- fad2-2
[0409] Table 9 shows that T2 seed from events expressing the
369b-fad2-1b & 159-fad2-2 amiRNAs have oleic acid contents
ranging from 82.3% to 88.0%. These oleic acid contents are higher
than that observed for either the 396b-fad2-1b or 159-fad2-1b
amiRNA when expressed individually.
TABLE-US-00025 TABLE 10 Fatty acid profile for T2 seed analyzed
from soy transformed with PHP33705, containing the 396-fad2-1b
& 396b-sad3 amiRNAs grown in the greenhouse. T2 Seed Seed T2
Seed amiRNA(s) Construct Event Analyzed No. 16:0 18:0 18:1 18:2
18:3 Comment 396-fad2- PHP33705 AFS 10GR13-7 1 10.2 3.9 13.6 60.2
12.0 Null 1b/396b- 5489.3.2 sad3 396-fad2- PHP33705 AFS 10GR13-7 2
10.8 3.6 13.3 62.3 10.0 Null 1b/396b- 5489.3.2 sad3 396-fad2-
PHP33705 AFS 10GR13-8 1 10.5 3.4 16.3 61.0 8.7 Null 1b/396b-
5489.3.2 sad3 396-fad2- PHP33705 AFS 10GR13-8 2 11.0 2.9 17.2 60.7
8.2 Null 1b/396b- 5489.3.2 sad3 396-fad2- PHP33705 AFS 10GR13-6 1
6.4 11.4 75.4 1.8 5.0 Homoz 1b/396b- 5489.3.2 Pos sad3 396-fad2-
PHP33705 AFS 10GR13-6 2 5.8 12.9 73.2 2.0 6.0 Homoz 1b/396b-
5489.3.2 Pos sad3 396-fad2- PHP33705 AFS 10GR13-6 3 6.2 14.2 72.7
1.8 5.1 Homoz 1b/396b- 5489.3.2 Pos sad3 396-fad2- PHP33705 AFS
10GR13-6 4 5.7 17.8 69.1 1.8 5.5 Homoz 1b/396b- 5489.3.2 Pos sad3
396-fad2- PHP33705 AFS 10GR13-6 5 5.5 13.6 72.3 2.5 6.1 Homoz
1b/396b- 5489.3.2 Pos sad3 396-fad2- PHP33705 AFS 10GR13-6 6 6.1
12.8 73.4 2.1 5.6 Homoz 1b/396b- 5489.3.2 Pos sad3 396-fad2-
PHP33705 AFS 10GR13-6 7 5.6 13.6 73.2 2.1 5.5 Homoz 1b/396b-
5489.3.2 Pos sad3 396-fad2- PHP33705 AFS 10GR13-6 8 6.2 11.4 74.1
2.4 5.9 Homoz 1b/396b- 5489.3.2 Pos sad3 396-fad2- PHP33705 AFS
10GR13-6 9 6.4 13.2 73.8 2.1 4.6 Homoz 1b/396b- 5489.3.2 Pos sad3
396-fad2- PHP33705 AFS 10GR13-6 10 6.0 13.2 73.9 1.8 5.2 Homoz
1b/396b- 5489.3.2 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 10.1
3.8 14.8 59.2 12.0 Null 1b/396b- 5489.3.4 17 sad3 396-fad2-
PHP33705 AFS 10GR13- 2 12.3 3.4 16.8 57.9 9.7 Null 1b/396b-
5489.3.4 17 sad3 396-fad2- PHP33705 AFS 10GR13- 1 6.6 10.3 71.9 3.4
7.8 Homoz 1b/396b- 5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.1 11.2 74.1 2.9 5.7 Homoz 1b/396b- 5489.3.4 14 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 5.0 13.5 72.2 3.0 6.4 Homoz
1b/396b- 5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 6.2
10.5 76.0 1.9 5.4 Homoz 1b/396b- 5489.3.4 14 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 5.9 12.1 74.6 1.7 5.7 Homoz 1b/396b-
5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 5.5 13.5 74.6
1.7 4.7 Homoz 1b/396b- 5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 7 5.1 12.9 73.5 2.5 5.9 Homoz 1b/396b- 5489.3.4 14 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 5.4 13.4 73.7 2.2 5.2 Homoz
1b/396b- 5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 5.6
13.0 70.6 3.6 7.1 Homoz 1b/396b- 5489.3.4 14 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 5.7 12.4 74.8 1.7 5.3 Homoz 1b/396b-
5489.3.4 14 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 5.8 10.6 77.2
1.7 4.6 Homoz 1b/396b- 5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.2 11.8 73.4 2.7 5.9 Homoz 1b/396b- 5489.3.4 15 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 5.5 10.7 76.6 1.9 5.3 Homoz
1b/396b- 5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 5.8
10.7 77.5 1.6 4.4 Homoz 1b/396b- 5489.3.4 15 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 5.6 11.3 76.8 1.7 4.7 Homoz 1b/396b-
5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 5.4 13.9 75.1
1.3 4.3 Homoz 1b/396b- 5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 7 5.6 12.7 73.7 2.5 5.4 Homoz 1b/396b- 5489.3.4 15 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 5.9 12.3 72.8 3.0 6.0 Homoz
1b/396b- 5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 5.3
11.7 77.3 1.5 4.2 Homoz 1b/396b- 5489.3.4 15 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 5.5 14.0 72.5 2.8 5.2 Homoz 1b/396b-
5489.3.4 15 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 6.8 8.0 78.8
1.7 4.6 Homoz 1b/396b- 5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.4 9.6 76.7 2.0 5.3 Homoz 1b/396b- 5489.3.4 16 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 6.6 8.5 78.5 1.6 4.8 Homoz
1b/396b- 5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 6.6
8.4 78.7 1.5 4.7 Homoz 1b/396b- 5489.3.4 16 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 6.2 10.7 77.4 1.5 4.2 Homoz 1b/396b-
5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 6.6 8.6 78.9
1.5 4.4 Homoz 1b/396b- 5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 7 6.5 10.1 77.8 1.1 4.5 Homoz 1b/396b- 5489.3.4 16 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 6.3 9.4 78.2 1.5 4.6 Homoz
1b/396b- 5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 6.3
10.5 77.3 1.6 4.4 Homoz 1b/396b- 5489.3.4 16 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 6.4 8.5 78.5 1.5 5.1 Homoz 1b/396b-
5489.3.4 16 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 6.6 9.9 74.5
3.1 5.9 Homoz 1b/396b- 5489.3.4 19 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.8 8.5 74.7 3.5 6.5 Homoz 1b/396b- 5489.3.4 19 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 5.4 10.5 76.2 2.4 5.5 Homoz
1b/396b- 5489.3.4 19 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 6.1
11.1 75.4 2.4 5.0 Homoz 1b/396b- 5489.3.4 19 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 6.8 9.4 73.1 4.0 6.7 Homoz 1b/396b- 5489.3.4
19 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 6.4 9.1 77.6 1.9 4.9
Homoz 1b/396b- 5489.3.4 19 Pos sad3 396-fad2- PHP33705 AFS 10GR13-
7 5.4 11.1 76.9 1.6 5.0 Homoz 1b/396b- 5489.3.4 19 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 6.2 8.6 78.3 1.9 5.0 Homoz
1b/396b- 5489.3.4 19 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 6.1
11.2 73.0 3.4 6.4 Homoz 1b/396b- 5489.3.4 19 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 5.8 11.6 74.9 2.3 5.4 Homoz 1b/396b-
5489.3.4 19 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 5.2 11.3 77.8
1.4 4.3 Homoz 1b/396b- 5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.2 8.1 79.1 1.6 4.9 Homoz 1b/396b- 5489.3.4 20 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 6.0 10.4 77.9 1.5 4.2 Homoz
1b/396b- 5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 6.4
9.7 75.4 2.6 5.9 Homoz 1b/396b- 5489.3.4 20 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 5.2 10.9 78.0 1.5 4.4 Homoz 1b/396b-
5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 6.3 9.9 74.9
2.9 6.1 Homoz 1b/396b- 5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 7 6.3 10.0 76.6 2.0 5.1 Homoz 1b/396b- 5489.3.4 20 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 6.7 9.1 77.4 1.9 5.0 Homoz
1b/396b- 5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 5.3
10.1 76.4 2.7 5.5 Homoz 1b/396b- 5489.3.4 20 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 5.5 13.3 73.7 2.3 5.1 Homoz 1b/396b-
5489.3.4 20 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 6.6 9.8 76.6
2.1 4.9 Homoz 1b/396b- 5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 2 6.3 10.9 76.3 1.8 4.7 Homoz 1b/396b- 5489.3.4 23 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 3 6.7 17.3 68.8 1.7 5.6 Homoz
1b/396b- 5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 5.7
11.2 76.3 1.7 5.1 Homoz 1b/396b- 5489.3.4 23 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 5 6.3 11.5 75.2 1.7 5.3 Homoz 1b/396b-
5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 6 6.7 9.1 76.8
2.1 5.3 Homoz 1b/396b- 5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 7 6.8 10.8 75.1 1.9 5.4 Homoz 1b/396b- 5489.3.4 23 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 8 6.2 12.6 75.4 1.4 4.4 Homoz
1b/396b- 5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 5.8
10.4 77.1 1.7 5.0 Homoz 1b/396b- 5489.3.4 23 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 10 6.1 9.5 77.7 1.7 5.0 Homoz 1b/396b-
5489.3.4 23 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 1 10.4 4.2 16.1
59.0 10.3 Null 1b/396b- 5489.4.1 48 sad3 396-fad2- PHP33705 AFS
10GR13- 2 12.2 3.8 14.4 57.8 11.8 Null 1b/396b- 5489.4.1 48 sad3
396-fad2- PHP33705 AFS 10GR13- 1 10.5 4.4 18.3 57.6 9.1 Null
1b/396b- 5489.4.1 53 sad3 396-fad2- PHP33705 AFS 10GR13- 2 11.8 3.7
15.5 59.3 9.7 Null 1b/396b- 5489.4.1 53 sad3 396-fad2- PHP33705 AFS
10GR13- 1 5.6 14.5 71.5 2.6 5.8 Homoz
1b/396b- 5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 2 5.6
18.0 68.7 1.9 5.8 Homoz 1b/396b- 5489.4.1 54 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 3 5.5 12.7 74.6 1.9 5.3 Homoz 1b/396b-
5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 5.5 17.8 69.1
2.2 5.3 Homoz 1b/396b- 5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 5 5.6 17.2 70.0 1.9 5.3 Homoz 1b/396b- 5489.4.1 54 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 6 5.9 21.0 66.2 1.6 5.3 Homoz
1b/396b- 5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 7 5.1
17.4 69.9 2.1 5.5 Homoz 1b/396b- 5489.4.1 54 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 8 5.7 18.5 68.1 2.2 5.5 Homoz 1b/396b-
5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 5.8 15.7 71.1
2.1 5.3 Homoz 1b/396b- 5489.4.1 54 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 10 5.3 18.9 68.6 1.9 5.4 Homoz 1b/396b- 5489.4.1 54 Pos
sad3 396-fad2- PHP33705 AFS 10GR13- 1 5.7 17.3 70.0 2.0 5.0 Homoz
1b/396b- 5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 2 5.5
16.3 71.2 2.1 4.9 Homoz 1b/396b- 5489.4.1 56 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 3 5.5 19.1 66.7 2.4 6.3 Homoz 1b/396b-
5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 5.4 21.4 64.8
2.4 6.0 Homoz 1b/396b- 5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 5 6.1 15.9 69.5 2.2 6.2 Homoz 1b/396b- 5489.4.1 56 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 6 5.5 13.1 75.3 1.8 4.3 Homoz
1b/396b- 5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 7 4.9
17.6 71.1 2.0 4.4 Homoz 1b/396b- 5489.4.1 56 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 8 5.1 16.1 71.8 2.1 5.0 Homoz 1b/396b-
5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 4.8 17.0 70.0
2.0 6.2 Homoz 1b/396b- 5489.4.1 56 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 10 5.4 17.9 69.2 2.0 5.6 Homoz 1b/396b- 5489.4.1 56 Pos
sad3 396-fad2- PHP33705 AFS 10GR13- 1 6.1 13.3 67.7 5.7 7.2 Homoz
1b/396b- 5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 2 4.9
12.9 76.0 1.6 4.7 Homoz 1b/396b- 5489.4.1 60 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 3 4.5 15.4 72.5 2.0 5.6 Homoz 1b/396b-
5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 4 5.2 12.9 74.7
2.0 5.1 Homoz 1b/396b- 5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 5 6.2 14.2 71.2 2.3 6.1 Homoz 1b/396b- 5489.4.1 60 Pos sad3
396-fad2- PHP33705 AFS 10GR13- 6 5.6 11.3 75.9 1.8 5.4 Homoz
1b/396b- 5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 7 4.8
17.5 71.7 1.6 4.5 Homoz 1b/396b- 5489.4.1 60 Pos sad3 396-fad2-
PHP33705 AFS 10GR13- 8 5.2 15.2 71.4 2.4 5.8 Homoz 1b/396b-
5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS 10GR13- 9 6.1 10.9 74.3
2.2 6.5 Homoz 1b/396b- 5489.4.1 60 Pos sad3 396-fad2- PHP33705 AFS
10GR13- 10 4.7 15.3 73.3 2.0 4.6 Homoz 1b/396b- 5489.4.1 60 Pos
sad3
[0410] Table 10 shows that T2 seed from events expressing the
396-fad2-1b & 396b-sad3 amiRNAs have palmitic acid contents
ranging from 8.0% to 22.4% and have oleic acid contents ranging
from 74.0% to 79.1%. T2 seed from null segregant seed have palmitic
acid contents ranging from 2.9% to 4.4% and have oleic acid
contents ranging from 13.3% to 20.1% which is in the range for that
of Jack seed where palmitic acid ranges from 11.8% to 12.6% and
oleic acid ranges from 12.7% to 17.9% (see Table 12).
TABLE-US-00026 TABLE 11 Fatty acid profile for T2 seed analyzed
from soy transformed with PHP38557, containing the 159-fad3c amiRNA
grown in the greenhouse. T2 T2 Seed Seed Seed amiRNA(s) Construct
Event Analyzed No. 16:0 18:0 18:1 18:2 18:3 Comment 159-fad3c
PHP38557 AFS 10GR13- 1 11.5 3.0 22.2 54.9 8.3 Null 6272.1.4 66
159-fad3c PHP38557 AFS 10GR13- 2 11.1 2.9 20.7 56.7 8.7 Null
6272.1.4 66 159-fad3c PHP38557 AFS 10GR13- 1 10.2 3.4 18.3 59.8 8.2
Null 6272.1.4 70 159-fad3c PHP38557 AFS 10GR13- 2 10.8 2.9 15.3
61.7 9.4 Null 6272.1.4 70 159-fad3c PHP38557 AFS 10GR13- 1 11.7 3.5
18.8 64.9 1.1 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13-
2 10.7 3.9 15.6 68.8 1.0 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557
AFS 10GR13- 3 10.2 3.1 18.6 66.7 1.3 Homoz 6272.1.4 63 Pos
159-fad3c PHP38557 AFS 10GR13- 4 11.6 3.6 17.6 66.0 1.2 Homoz
6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.2 3.3 16.0 68.4
1.2 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.5 2.9
21.4 63.2 1.0 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13-
7 11.5 3.2 16.9 67.2 1.2 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.5 2.9 18.3 66.3 1.0 Homoz 6272.1.4 63 Pos
159-fad3c PHP38557 AFS 10GR13- 9 11.3 3.1 19.8 64.7 1.1 Homoz
6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.8 3.5 16.4
66.1 1.2 Homoz 6272.1.4 63 Pos 159-fad3c PHP38557 AFS 10GR13- 1
12.0 3.2 15.9 66.8 2.1 Homoz 6272.1.4 65 Pos 159-fad3c PHP38557 AFS
10GR13- 2 12.0 3.0 16.8 66.4 1.8 Homoz 6272.1.4 65 Pos 159-fad3c
PHP38557 AFS 10GR13- 3 12.0 3.3 17.2 65.9 1.6 Homoz 6272.1.4 65 Pos
159-fad3c PHP38557 AFS 10GR13- 4 12.1 3.0 18.7 64.6 1.6 Homoz
6272.1.4 65 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.8 3.2 17.4 65.8
1.8 Homoz 6272.1.4 65 Pos 159-fad3c PHP38557 AFS 10GR13- 6 13.2 3.1
17.8 64.6 1.4 Homoz 6272.1.4 65 Pos 159-fad3c PHP38557 AFS 10GR13-
8 12.6 3.2 15.1 66.7 2.3 Homoz 6272.1.4 65 Pos 159-fad3c PHP38557
AFS 10GR13- 9 13.3 3.7 15.2 66.3 1.5 Homoz 6272.1.4 65 Pos
159-fad3c PHP38557 AFS 10GR13- 10 12.6 3.5 15.9 66.6 1.4 Homoz
6272.1.4 65 Pos 159-fad3c PHP38557 AFS 10GR13- 1 11.4 3.4 19.4 64.7
1.1 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13- 2 12.3 3.2
15.9 67.1 1.5 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13-
3 11.4 3.2 16.5 67.7 1.3 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557
AFS 10GR13- 4 12.8 3.5 19.8 62.8 1.1 Homoz 6272.1.4 73 Pos
159-fad3c PHP38557 AFS 10GR13- 5 11.0 3.7 14.8 69.2 1.3 Homoz
6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13- 6 12.3 3.8 19.2 63.7
1.0 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13- 7 11.2 3.7
19.4 64.6 1.1 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13-
8 11.2 3.5 16.7 67.4 1.2 Homoz 6272.1.4 73 Pos 159-fad3c PHP38557
AFS 10GR13- 9 12.3 3.6 18.0 64.8 1.3 Homoz 6272.1.4 73 Pos
159-fad3c PHP38557 AFS 10GR13- 10 11.1 3.6 18.2 66.0 1.1 Homoz
6272.1.4 73 Pos 159-fad3c PHP38557 AFS 10GR13- 1 10.4 2.9 20.6 58.3
7.8 Null 6272.2.1 81 159-fad3c PHP38557 AFS 10GR13- 2 11.0 3.0 19.3
58.2 8.4 Null 6272.2.1 81 159-fad3c PHP38557 AFS 10GR13- 1 12.0 2.9
12.0 59.3 13.7 Null 6272.2.1 82 159-fad3c PHP38557 AFS 10GR13- 2
11.7 3.4 23.5 53.9 7.5 Null 6272.2.1 82 159-fad3c PHP38557 AFS
10GR13- 1 11.8 3.0 16.7 67.3 1.2 Homoz 6272.2.1 83 Pos 159-fad3c
PHP38557 AFS 10GR13- 2 12.1 3.3 15.2 68.3 1.1 Homoz 6272.2.1 83 Pos
159-fad3c PHP38557 AFS 10GR13- 3 11.2 3.1 15.6 68.8 1.3 Homoz
6272.2.1 83 Pos 159-fad3c PHP38557 AFS 10GR13- 4 11.6 3.0 15.6 68.5
1.3 Homoz 6272.2.1 83 Pos 159-fad3c PHP38557 AFS 10GR13- 5 12.0 3.0
18.4 65.4 1.1 Homoz 6272.2.1 83 Pos 159-fad3c PHP38557 AFS 10GR13-
6 12.2 3.7 18.1 64.9 1.0 Homoz 6272.2.1 83 Pos 159-fad3c PHP38557
AFS 10GR13- 7 11.6 3.1 19.0 65.3 1.0 Homoz 6272.2.1 83 Pos
159-fad3c PHP38557 AFS 10GR13- 8 12.0 3.2 15.1 68.3 1.4 Homoz
6272.2.1 83 Pos 159-fad3c PHP38557 AFS 10GR13- 9 12.1 3.1 14.0 69.2
1.6 Homoz 6272.2.1 83 Pos 159-fad3c PHP38557 AFS 10GR13- 10 11.7
3.3 17.0 67.0 1.1 Homoz 6272.2.1 83 Pos 159-fad3c PHP38557 AFS
10GR13- 1 12.6 3.2 19.8 63.4 1.0 Homoz 6272.2.1 90 Pos 159-fad3c
PHP38557 AFS 10GR13- 2 11.8 3.0 19.0 64.9 1.3 Homoz 6272.2.1 90 Pos
159-fad3c PHP38557 AFS 10GR13- 3 10.5 2.6 14.8 70.7 1.4 Homoz
6272.2.1 90 Pos 159-fad3c PHP38557 AFS 10GR13- 4 11.2 3.0 11.9 72.4
1.5 Homoz 6272.2.1 90 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.1 2.9
17.9 67.0 1.2 Homoz 6272.2.1 90 Pos 159-fad3c PHP38557 AFS 10GR13-
6 12.0 2.8 20.1 64.2 1.0 Homoz 6272.2.1 90 Pos 159-fad3c PHP38557
AFS 10GR13- 7 13.6 3.6 13.1 68.2 1.5 Homoz 6272.2.1 90 Pos
159-fad3c PHP38557 AFS 10GR13- 8 10.8 2.9 15.9 69.0 1.4 Homoz
6272.2.1 90 Pos 159-fad3c PHP38557 AFS 10GR13- 9 11.9 2.6 20.3 64.0
1.2 Homoz 6272.2.1 90 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.3
3.2 20.7 62.6 1.2 Homoz 6272.2.1 90 Pos 159-fad3c PHP38557 AFS
10GR13- 1 10.8 3.0 28.2 57.0 1.0 Homoz 6272.2.1 91 Pos 159-fad3c
PHP38557 AFS 10GR13- 2 11.1 3.3 21.3 63.3 1.1 Homoz 6272.2.1 91 Pos
159-fad3c PHP38557 AFS 10GR13- 3 10.9 2.9 19.3 65.8 1.2 Homoz
6272.2.1 91 Pos 159-fad3c PHP38557 AFS 10GR13- 4 11.0 3.0 18.5 66.2
1.2 Homoz 6272.2.1 91 Pos 159-fad3c PHP38557 AFS 10GR13- 5 10.9 2.6
22.4 62.9 1.2 Homoz 6272.2.1 91 Pos 159-fad3c PHP38557 AFS 10GR13-
6 11.1 3.0 22.2 62.7 1.0 Homoz 6272.2.1 91 Pos 159-fad3c PHP38557
AFS 10GR13- 7 10.4 3.5 16.5 68.3 1.3 Homoz 6272.2.1 91 Pos
159-fad3c PHP38557 AFS 10GR13- 8 12.0 3.5 24.1 59.7 0.8 Homoz
6272.2.1 91 Pos 159-fad3c PHP38557 AFS 10GR13- 9 13.8 3.9 23.2 58.2
0.9 Homoz 6272.2.1 91 Pos 159-fad3c PHP38557 AFS 10GR13- 10 11.7
2.8 23.9 60.7 0.9 Homoz 6272.2.1 91 Pos 159-fad3c PHP38557 AFS
10GR13- 1 11.6 2.9 20.8 63.5 1.1 Homoz 6272.2.1 93 Pos 159-fad3c
PHP38557 AFS 10GR13- 2 11.2 2.8 20.2 64.3 1.5 Homoz 6272.2.1 93 Pos
159-fad3c PHP38557 AFS 10GR13- 3 10.3 3.1 18.6 66.3 1.7 Homoz
6272.2.1 93 Pos 159-fad3c PHP38557 AFS 10GR13- 4 10.8 2.7 18.3 66.5
1.7 Homoz 6272.2.1 93 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.2 2.6
20.0 64.8 1.4 Homoz 6272.2.1 93 Pos 159-fad3c PHP38557 AFS 10GR13-
6 10.2 2.9 21.8 63.7 1.4 Homoz 6272.2.1 93 Pos 159-fad3c PHP38557
AFS 10GR13- 7 10.8 2.9 19.5 65.8 1.1 Homoz 6272.2.1 93 Pos
159-fad3c PHP38557 AFS 10GR13- 8 9.9 2.9 21.8 64.2 1.3 Homoz
6272.2.1 93 Pos 159-fad3c PHP38557 AFS 10GR13- 9 11.2 2.8 17.9 66.5
1.7 Homoz 6272.2.1 93 Pos 159-fad3c PHP38557 AFS 10GR13- 10 10.5
2.9 24.5 60.8 1.3 Homoz 6272.2.1 93 Pos 159-fad3c PHP38557 AFS
10GR13- 1 11.1 2.9 20.7 64.3 1.0 Homoz 6272.2.1 94 Pos 159-fad3c
PHP38557 AFS 10GR13- 2 11.4 3.3 16.0 68.0 1.3 Homoz 6272.2.1 94 Pos
159-fad3c PHP38557 AFS 10GR13- 3 11.0 3.0 18.5 66.4 1.1 Homoz
6272.2.1 94 Pos 159-fad3c PHP38557 AFS 10GR13- 4 10.9 3.0 19.5 65.7
1.0 Homoz 6272.2.1 94 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.4 2.8
16.3 68.5 1.2 Homoz 6272.2.1 94 Pos 159-fad3c PHP38557 AFS 10GR13-
6 11.2 3.1 17.7 66.8 1.1 Homoz 6272.2.1 94 Pos 159-fad3c PHP38557
AFS 10GR13- 7 12.0 3.3 14.3 68.9 1.4 Homoz 6272.2.1 94 Pos
159-fad3c PHP38557 AFS 10GR13- 8 11.4 2.9 18.8 65.7 1.1 Homoz
6272.2.1 94 Pos 159-fad3c PHP38557 AFS 10GR13- 9 11.4 3.2 19.7 64.7
1.1 Homoz 6272.2.1 94 Pos 159-fad3c PHP38557 AFS 10GR13- 10 11.6
3.1 20.0 64.3 1.0 Homoz 6272.2.1 94 Pos 159-fad3c PHP38557 AFS
10GR13- 1 11.0 3.8 19.5 58.2 7.6 Null 6272.2.3 107 159-fad3c
PHP38557 AFS 10GR13- 2 11.9 3.5 20.2 56.7 7.7 Null 6272.2.3 107
159-fad3c PHP38557 AFS 10GR13- 1 11.2 3.3 16.3 59.7 9.5 Null
6272.2.3 109 159-fad3c PHP38557 AFS 10GR13- 2 12.1 3.3 14.6 59.3
10.7 Null 6272.2.3 109 159-fad3c PHP38557 AFS 10GR13- 1 10.9 2.8
16.7 68.4 1.2 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13-
2 11.8 3.5 18.7 65.0 1.0 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557
AFS 10GR13- 3 11.7 3.9 18.6 64.9 0.9 Homoz 6272.2.3 95 Pos
159-fad3c PHP38557 AFS 10GR13- 4 11.3 2.4 24.0 61.4 1.0 Homoz
6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.9 2.9 19.3 64.8
1.0 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.7 2.8
13.9 70.3 1.3 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13-
7 11.4 3.0 18.2 66.3 1.2 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.8 3.9 13.6 69.4 1.4 Homoz 6272.2.3 95 Pos
159-fad3c PHP38557 AFS 10GR13- 9 12.5 2.9 16.9 66.4 1.3 Homoz
6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13- 10 11.0 2.9 16.7
68.3 1.1 Homoz 6272.2.3 95 Pos 159-fad3c PHP38557 AFS 10GR13- 1
12.6 3.7 14.6 66.4 2.8 Homoz 6272.2.3 96 Pos 159-fad3c PHP38557 AFS
10GR13- 2 12.4 3.8 13.0 68.1 2.7 Homoz 6272.2.3 96 Pos 159-fad3c
PHP38557 AFS 10GR13- 3 12.3 3.4 12.9 69.2 2.2 Homoz 6272.2.3 96 Pos
159-fad3c PHP38557 AFS 10GR13- 4 12.9 3.9 11.9 67.6 3.7 Homoz
6272.2.3 96 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.9 3.6 12.9 69.0
2.7 Homoz 6272.2.3 96 Pos 159-fad3c PHP38557 AFS 10GR13- 6 12.2 3.5
12.6 67.9 3.8 Homoz 6272.2.3 96 Pos 159-fad3c PHP38557 AFS 10GR13-
7 13.1 4.6 11.5 66.6 4.2 Homoz 6272.2.3 96 Pos 159-fad3c PHP38557
AFS 10GR13- 8 12.7 3.7 12.7 68.6 2.3 Homoz 6272.2.3 96 Pos
159-fad3c PHP38557 AFS 10GR13- 9 12.9 3.6 12.3 68.4 2.7 Homoz
6272.2.3 96 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.0 3.4 15.9
66.5 2.3 Homoz 6272.2.3 96 Pos 159-fad3c PHP38557 AFS 10GR13- 1
11.6 3.8 18.9 64.3 1.5 Homoz 6272.2.3 97 Pos 159-fad3c PHP38557 AFS
10GR13- 2 11.4 3.3 16.2 67.2 1.9 Homoz 6272.2.3 97 Pos 159-fad3c
PHP38557 AFS 10GR13- 3 10.8 3.4 14.8 69.1 1.9 Homoz 6272.2.3 97 Pos
159-fad3c PHP38557 AFS 10GR13- 4 11.6 3.6 18.2 64.7 1.9 Homoz
6272.2.3 97 Pos 159-fad3c PHP38557 AFS 10GR13- 5 11.8 3.4 14.8 67.8
2.2 Homoz 6272.2.3 97 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.1 3.8
15.6 67.6 1.9 Homoz 6272.2.3 97 Pos 159-fad3c PHP38557 AFS 10GR13-
7 11.6 3.4 16.2 66.7 2.1 Homoz 6272.2.3 97 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.8 3.5 13.3 69.1 2.3 Homoz 6272.2.3 97 Pos
159-fad3c PHP38557 AFS 10GR13- 9 12.4 3.7 15.0 66.7 2.2 Homoz
6272.2.3 97 Pos 159-fad3c PHP38557 AFS 10GR13- 10 11.8 3.3 14.6
68.4 1.9 Homoz 6272.2.3 97 Pos
159-fad3c PHP38557 AFS 10GR13- 1 11.6 3.4 16.7 66.6 1.7 Homoz
6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13- 2 11.2 3.7 15.5 67.9
1.7 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13- 3 11.7 3.4
17.1 66.2 1.6 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13-
4 12.2 3.5 17.2 65.6 1.4 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557
AFS 10GR13- 5 11.8 3.4 15.2 67.8 1.7 Homoz 6272.2.3 98 Pos
159-fad3c PHP38557 AFS 10GR13- 6 11.9 3.6 14.9 67.5 2.1 Homoz
6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13- 7 11.4 3.6 16.1 67.2
1.8 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13- 8 11.7 3.6
16.6 66.6 1.5 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557 AFS 10GR13-
9 12.0 3.6 17.7 65.2 1.5 Homoz 6272.2.3 98 Pos 159-fad3c PHP38557
AFS 10GR13- 10 11.7 3.5 15.1 67.7 2.0 Homoz 6272.2.3 98 Pos
159-fad3c PHP38557 AFS 10GR13- 1 11.3 3.6 17.5 66.1 1.6 Homoz
6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 2 11.8 3.5 16.3
66.4 2.0 Homoz 6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 3
11.2 3.5 16.7 66.2 2.4 Homoz 6272.2.3 101 Pos 159-fad3c PHP38557
AFS 10GR13- 4 11.3 3.4 16.8 66.9 1.6 Homoz 6272.2.3 101 Pos
159-fad3c PHP38557 AFS 10GR13- 5 11.8 3.5 16.6 66.1 1.9 Homoz
6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.8 3.6 14.0
68.4 2.3 Homoz 6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 7
11.6 3.5 16.2 66.2 2.5 Homoz 6272.2.3 101 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.5 3.7 14.9 67.2 2.7 Homoz 6272.2.3 101 Pos
159-fad3c PHP38557 AFS 10GR13- 9 12.0 3.6 14.6 66.6 3.1 Homoz
6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.2 3.2 14.6
68.0 2.0 Homoz 6272.2.3 101 Pos 159-fad3c PHP38557 AFS 10GR13- 1
11.4 3.3 14.9 67.5 2.9 Homoz 6272.2.3 102 Pos 159-fad3c PHP38557
AFS 10GR13- 2 11.2 3.5 15.8 67.7 1.8 Homoz 6272.2.3 102 Pos
159-fad3c PHP38557 AFS 10GR13- 3 11.1 3.6 21.6 61.9 1.9 Homoz
6272.2.3 102 Pos 159-fad3c PHP38557 AFS 10GR13- 4 11.2 3.8 18.6
64.2 2.2 Homoz 6272.2.3 102 Pos 159-fad3c PHP38557 AFS 10GR13- 5
11.1 3.6 15.8 67.5 2.0 Homoz 6272.2.3 102 Pos 159-fad3c PHP38557
AFS 10GR13- 6 10.6 3.5 15.3 68.8 1.9 Homoz 6272.2.3 102 Pos
159-fad3c PHP38557 AFS 10GR13- 7 11.2 3.3 17.6 66.3 1.6 Homoz
6272.2.3 102 Pos 159-fad3c PHP38557 AFS 10GR13- 8 10.8 3.2 17.9
65.8 2.2 Homoz 6272.2.3 102 Pos 159-fad3c PHP38557 AFS 10GR13- 9
11.6 3.1 18.4 65.3 1.5 Homoz 6272.2.3 102 Pos 159-fad3c PHP38557
AFS 10GR13- 10 11.6 3.2 16.5 66.9 1.8 Homoz 6272.2.3 102 Pos
159-fad3c PHP38557 AFS 10GR13- 1 11.4 3.4 14.4 68.5 2.2 Homoz
6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 2 11.1 3.5 16.2
67.6 1.6 Homoz 6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 3
11.6 3.5 13.7 67.9 3.3 Homoz 6272.2.3 103 Pos 159-fad3c PHP38557
AFS 10GR13- 4 11.2 3.3 15.6 68.1 1.9 Homoz 6272.2.3 103 Pos
159-fad3c PHP38557 AFS 10GR13- 5 11.7 3.3 14.1 67.1 3.8 Homoz
6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.0 3.7 13.9
68.8 2.6 Homoz 6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 7
11.0 3.6 13.5 68.4 3.5 Homoz 6272.2.3 103 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.4 3.1 14.8 67.7 2.9 Homoz 6272.2.3 103 Pos
159-fad3c PHP38557 AFS 10GR13- 9 11.5 3.6 13.9 67.7 3.3 Homoz
6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.1 3.7 15.1
66.0 3.1 Homoz 6272.2.3 103 Pos 159-fad3c PHP38557 AFS 10GR13- 1
12.3 3.5 12.5 68.8 2.9 Homoz 6272.2.3 104 Pos 159-fad3c PHP38557
AFS 10GR13- 2 10.9 3.3 15.2 68.6 2.0 Homoz 6272.2.3 104 Pos
159-fad3c PHP38557 AFS 10GR13- 3 11.3 3.6 14.0 68.9 2.3 Homoz
6272.2.3 104 Pos 159-fad3c PHP38557 AFS 10GR13- 4 12.2 3.5 13.3
67.0 3.9 Homoz 6272.2.3 104 Pos 159-fad3c PHP38557 AFS 10GR13- 5
12.8 3.8 13.2 66.7 3.4 Homoz 6272.2.3 104 Pos 159-fad3c PHP38557
AFS 10GR13- 6 11.0 3.3 16.1 67.9 1.7 Homoz 6272.2.3 104 Pos
159-fad3c PHP38557 AFS 10GR13- 7 12.6 3.7 12.2 67.0 4.4 Homoz
6272.2.3 104 Pos 159-fad3c PHP38557 AFS 10GR13- 8 12.2 3.6 13.0
68.5 2.7 Homoz 6272.2.3 104 Pos 159-fad3c PHP38557 AFS 10GR13- 9
13.2 3.8 13.3 65.8 4.0 Homoz 6272.2.3 104 Pos 159-fad3c PHP38557
AFS 10GR13- 10 11.4 3.4 13.2 68.5 3.5 Homoz 6272.2.3 104 Pos
159-fad3c PHP38557 AFS 10GR13- 1 11.4 3.5 18.5 64.9 1.8 Homoz
6272.2.3 106 Pos 159-fad3c PHP38557 AFS 10GR13- 2 11.4 3.2 16.7
66.8 1.9 Homoz 6272.2.3 106 Pos 159-fad3c PHP38557 AFS 10GR13- 3
11.1 3.5 16.3 67.1 2.1 Homoz 6272.2.3 106 Pos 159-fad3c PHP38557
AFS 10GR13- 4 12.0 3.6 17.8 64.9 1.7 Homoz 6272.2.3 106 Pos
159-fad3c PHP38557 AFS 10GR13- 5 11.5 3.7 15.0 67.7 2.0 Homoz
6272.2.3 106 Pos 159-fad3c PHP38557 AFS 10GR13- 6 11.0 3.6 14.3
69.0 2.1 Homoz 6272.2.3 106 Pos 159-fad3c PHP38557 AFS 10GR13- 7
11.1 3.3 16.5 67.3 1.9 Homoz 6272.2.3 106 Pos 159-fad3c PHP38557
AFS 10GR13- 8 11.0 3.4 17.2 66.6 1.8 Homoz 6272.2.3 106 Pos
159-fad3c PHP38557 AFS 10GR13- 9 10.9 3.3 19.0 65.4 1.5 Homoz
6272.2.3 106 Pos 159-fad3c PHP38557 AFS 10GR13- 10 12.0 4.0 19.3
62.9 1.7 Homoz 6272.2.3 106 Pos
[0411] Table 11 shows that T2 seed from events expressing the
159-fad3c amiRNA have alpha-linolenic acid contents ranging from
0.8% to 4.4%. T2 seed from null segregant seed have alpha-linolenic
contents ranging from 7.5% to 13.7% which is in the range typically
observed for wild-type soybeans where alpha-linolenic acid ranges
from 8.2% to 11.3% (see Table 12).
TABLE-US-00027 TABLE 12 Fatty acid profile for T2 seed analyzed
from soy transformed with PHP41103, containing the 159-fatBF
amiRNA, grown in the greenhouse. T2 Seed Seed T2 Seed amiRNA
Construct Event Analyzed No. 16:0 18:0 18:1 18:2 18:3 Comment
159-fatBF PHP41103 AFS 10GR13- 1 11.2 3.6 21.3 55.1 8.8 Null
6671.1.4 226 159-fatBF PHP41103 AFS 10GR13- 2 11.3 3.4 19.6 57.0
8.6 Null 6671.1.4 226 159-fatBF PHP41103 AFS 10GR13- 1 11.8 3.8
18.3 56.1 10.0 Null 6671.1.4 237 159-fatBF PHP41103 AFS 10GR13- 2
12.6 4.2 23.7 51.6 7.9 Null 6671.1.4 237 159-fatBF PHP41103 AFS
10GR13- 1 2.0 2.4 28.5 60.2 6.8 Homoz 6671.1.4 222 Pos 159-fatBF
PHP41103 AFS 10GR13- 2 1.9 2.4 31.9 56.9 6.9 Homoz 6671.1.4 222 Pos
159-fatBF PHP41103 AFS 10GR13- 3 2.1 2.8 30.3 57.9 6.9 Homoz
6671.1.4 222 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.0 2.3 24.1 63.4
8.3 Homoz 6671.1.4 222 Pos 159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.5
24.6 63.4 7.5 Homoz 6671.1.4 222 Pos 159-fatBF PHP41103 AFS 10GR13-
6 2.1 3.1 32.8 55.1 6.9 Homoz 6671.1.4 222 Pos 159-fatBF PHP41103
AFS 10GR13- 7 2.0 2.0 28.1 60.9 7.0 Homoz 6671.1.4 222 Pos
159-fatBF PHP41103 AFS 10GR13- 8 2.0 2.7 29.6 58.7 7.1 Homoz
6671.1.4 222 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.1 2.6 29.5 57.9
7.9 Homoz 6671.1.4 222 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.0
2.3 22.6 64.2 8.9 Homoz 6671.1.4 222 Pos 159-fatBF PHP41103 AFS
10GR13- 1 1.9 2.2 25.9 62.5 7.4 Homoz 6671.1.4 229 Pos 159-fatBF
PHP41103 AFS 10GR13- 2 2.0 2.5 27.2 59.7 8.6 Homoz 6671.1.4 229 Pos
159-fatBF PHP41103 AFS 10GR13- 3 1.8 2.1 23.1 65.0 7.9 Homoz
6671.1.4 229 Pos 159-fatBF PHP41103 AFS 10GR13- 4 1.9 2.5 25.2 62.0
8.3 Homoz 6671.1.4 229 Pos 159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.0
24.1 63.4 8.4 Homoz 6671.1.4 229 Pos 159-fatBF PHP41103 AFS 10GR13-
6 1.9 2.1 24.2 64.0 7.9 Homoz 6671.1.4 229 Pos 159-fatBF PHP41103
AFS 10GR13- 7 1.9 2.3 26.1 61.1 8.5 Homoz 6671.1.4 229 Pos
159-fatBF PHP41103 AFS 10GR13- 8 1.9 2.1 27.9 61.2 6.9 Homoz
6671.1.4 229 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.3 23.5 64.0
8.3 Homoz 6671.1.4 229 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.0
2.2 23.6 64.0 8.2 Homoz 6671.1.4 229 Pos 159-fatBF PHP41103 AFS
10GR13- 1 12.1 3.6 24.4 52.7 7.3 Null 6671.1.6 261 159-fatBF
PHP41103 AFS 10GR13- 2 11.1 3.3 23.1 54.8 7.8 Null 6671.1.6 261
159-fatBF PHP41103 AFS 10GR13- 1 11.0 4.5 34.8 44.6 5.2 Null
6671.1.6 262 159-fatBF PHP41103 AFS 10GR13- 2 11.7 4.0 27.1 50.2
7.0 Null 6671.1.6 262 159-fatBF PHP41103 AFS 10GR13- 1 1.9 2.1 26.8
61.7 7.5 Homoz 6671.1.6 242 Pos 159-fatBF PHP41103 AFS 10GR13- 2
1.9 2.0 32.2 56.5 7.3 Homoz 6671.1.6 242 Pos 159-fatBF PHP41103 AFS
10GR13- 3 1.8 2.0 20.8 65.8 9.6 Homoz 6671.1.6 242 Pos 159-fatBF
PHP41103 AFS 10GR13- 4 1.8 2.1 29.7 58.7 7.6 Homoz 6671.1.6 242 Pos
159-fatBF PHP41103 AFS 10GR13- 5 1.9 2.2 23.3 63.9 8.7 Homoz
6671.1.6 242 Pos 159-fatBF PHP41103 AFS 10GR13- 6 1.8 2.1 26.9 60.8
8.4 Homoz 6671.1.6 242 Pos 159-fatBF PHP41103 AFS 10GR13- 7 1.7 1.9
20.7 65.6 10.1 Homoz 6671.1.6 242 Pos 159-fatBF PHP41103 AFS
10GR13- 8 2.0 2.0 33.1 55.2 7.7 Homoz 6671.1.6 242 Pos 159-fatBF
PHP41103 AFS 10GR13- 9 1.9 2.2 33.0 55.9 7.0 Homoz 6671.1.6 242 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.3 23.6 63.5 8.6 Homoz
6671.1.6 242 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.0 2.0 34.9 54.1
7.0 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.1 2.1
37.7 50.8 7.2 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.0 2.0 35.7 52.0 8.3 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.0 2.0 36.3 52.6 7.2 Homoz 6671.1.6 243 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.0 25.6 62.1 8.4 Homoz
6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.1 2.5 46.6 42.3
6.5 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.0 1.9
37.9 51.0 7.2 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13-
8 2.0 2.2 32.5 55.1 8.3 Homoz 6671.1.6 243 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.1 2.4 39.0 49.1 7.5 Homoz 6671.1.6 243 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.2 36.8 52.1 7.0 Homoz
6671.1.6 243 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.0 1.8 25.4 61.9
9.0 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.9 2.2
23.3 64.4 8.2 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13-
3 1.9 1.9 22.9 65.1 8.2 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.9 1.8 33.4 55.1 7.7 Homoz 6671.1.6 244 Pos
159-fatBF PHP41103 AFS 10GR13- 5 1.9 1.8 29.4 60.4 6.5 Homoz
6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13- 6 1.9 2.4 30.3 58.4
7.1 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.0 2.1
25.3 62.6 8.1 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13-
8 1.9 1.7 24.0 64.5 7.8 Homoz 6671.1.6 244 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.0 2.1 33.8 55.6 6.6 Homoz 6671.1.6 244 Pos
159-fatBF PHP41103 AFS 10GR13- 10 1.9 1.9 28.1 60.9 7.2 Homoz
6671.1.6 244 Pos 159-fatBF PHP41103 AFS 10GR13- 1 1.8 2.0 25.0 62.4
8.7 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.8 2.0
30.2 58.3 7.7 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13-
3 1.8 1.9 22.4 65.0 8.9 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.9 1.9 31.5 57.2 7.4 Homoz 6671.1.6 247 Pos
159-fatBF PHP41103 AFS 10GR13- 5 1.9 2.0 24.2 63.0 8.8 Homoz
6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13- 6 1.9 2.0 28.1 59.9
8.0 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13- 7 1.8 2.0
23.2 63.8 9.3 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13-
8 1.8 1.8 27.4 61.3 7.8 Homoz 6671.1.6 247 Pos 159-fatBF PHP41103
AFS 10GR13- 9 1.8 1.9 26.9 61.2 8.2 Homoz 6671.1.6 247 Pos
159-fatBF PHP41103 AFS 10GR13- 10 1.8 2.2 30.4 58.3 7.4 Homoz
6671.1.6 247 Pos 159-fatBF PHP41103 AFS 10GR13- 1 1.8 1.9 25.8 62.7
7.8 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.9 2.0
23.9 63.9 8.4 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.0 2.6 32.7 55.7 7.0 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.9 2.2 23.2 63.9 8.8 Homoz 6671.1.6 249 Pos
159-fatBF PHP41103 AFS 10GR13- 5 1.8 2.0 25.5 62.5 8.1 Homoz
6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13- 6 1.9 2.0 23.6 63.9
8.5 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.0 2.0
23.3 63.7 9.0 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13-
8 1.8 2.0 23.7 64.8 7.8 Homoz 6671.1.6 249 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.0 2.1 24.6 62.9 8.4 Homoz 6671.1.6 249 Pos
159-fatBF PHP41103 AFS 10GR13- 10 1.8 2.3 27.4 61.1 7.5 Homoz
6671.1.6 249 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.0 2.3 26.8 61.1
7.8 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.5
34.2 54.2 7.1 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.0 2.5 37.1 52.0 6.3 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.3 2.9 30.7 57.4 6.6 Homoz 6671.1.6 252 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.4 3.2 39.3 48.8 6.3 Homoz
6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.3 2.3 35.3 53.6
6.5 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.1 2.5
33.0 55.8 6.5 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13-
8 2.2 2.9 29.0 58.9 7.1 Homoz 6671.1.6 252 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.3 3.0 28.4 59.3 7.0 Homoz 6671.1.6 252 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.3 2.7 28.7 59.1 7.2 Homoz
6671.1.6 252 Pos 159-fatBF PHP41103 AFS 10GR13- 1 1.9 2.0 30.8 58.5
6.8 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.1 2.1
20.4 65.8 9.6 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.1 2.1 22.0 64.8 8.9 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.8 2.0 24.2 63.7 8.2 Homoz 6671.1.6 253 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.2 28.4 60.2 7.3 Homoz
6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.1 2.2 24.8 62.0
9.0 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.2 2.3
31.7 57.4 6.5 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13-
8 1.9 2.1 29.1 59.3 7.6 Homoz 6671.1.6 253 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.1 2.4 34.6 54.7 6.2 Homoz 6671.1.6 253 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.0 26.3 62.4 7.3 Homoz
6671.1.6 253 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.0 2.1 23.7 64.1
8.2 Homoz 6671.1.6 256 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.1
27.9 61.1 6.8 Homoz 6671.1.6 256 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.0 2.1 22.1 65.4 8.4 Homoz 6671.1.6 256 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.3 2.2 16.4 66.9 12.2 Homoz 6671.1.6 256 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.1 2.1 30.6 58.6 6.6 Homoz
6671.1.6 256 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 2.2 24.3 63.6
7.9 Homoz 6671.1.6 256 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.1 2.3
17.8 67.4 10.4 Homoz 6671.1.6 256 Pos 159-fatBF PHP41103 AFS
10GR13- 8 1.9 1.7 16.7 70.3 9.4 Homoz 6671.1.6 256 Pos 159-fatBF
PHP41103 AFS 10GR13- 9 2.0 2.1 22.8 65.4 7.7 Homoz 6671.1.6 256 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.2 29.2 60.3 6.3 Homoz
6671.1.6 256 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.4 2.0 23.4 63.3
8.8 Homoz 6671.1.6 258 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.4 2.0
21.1 63.7 10.9 Homoz 6671.1.6 258 Pos 159-fatBF PHP41103 AFS
10GR13- 3 1.9 2.5 31.0 57.8 6.8 Homoz 6671.1.6 258 Pos 159-fatBF
PHP41103 AFS 10GR13- 4 2.5 2.0 21.1 63.6 10.8 Homoz 6671.1.6 258
Pos 159-fatBF PHP41103 AFS 10GR13- 5 2.5 2.0 28.9 59.6 7.1 Homoz
6671.1.6 258 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.7 1.9 17.3 64.5
13.6 Homoz 6671.1.6 258 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.3
2.3 35.2 55.2 5.1 Homoz 6671.1.6 258 Pos 159-fatBF PHP41103 AFS
10GR13- 8 2.1 2.4 32.4 57.0 6.1 Homoz 6671.1.6 258 Pos 159-fatBF
PHP41103 AFS 10GR13- 9 2.3 2.8 28.1 59.4 7.5 Homoz 6671.1.6 258 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.5 1.9 26.8 60.5 8.3 Homoz
6671.1.6 258 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.1 2.1 26.2 61.8
7.7 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.9 1.9
33.4 55.2 7.5 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13-
3 2.0 1.7 32.6 56.6 7.1 Homoz 6671.1.6 260 Pos
159-fatBF PHP41103 AFS 10GR13- 4 2.1 2.1 24.9 62.5 8.5 Homoz
6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.0 29.2 59.8
7.0 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 1.9
26.9 61.3 7.9 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13-
7 1.9 1.8 24.3 63.7 8.2 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103
AFS 10GR13- 8 2.2 2.2 28.9 59.4 7.4 Homoz 6671.1.6 260 Pos
159-fatBF PHP41103 AFS 10GR13- 9 1.9 1.9 34.1 56.1 5.9 Homoz
6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.0 1.9 31.3
58.1 6.6 Homoz 6671.1.6 260 Pos 159-fatBF PHP41103 AFS 10GR13- 1
11.7 3.7 21.4 55.6 7.6 Null 6671.3.2 269 159-fatBF PHP41103 AFS
10GR13- 2 11.9 4.0 23.2 54.6 6.3 Null 6671.3.2 269 159-fatBF
PHP41103 AFS 10GR13- 1 2.1 2.0 25.7 62.4 7.7 Homoz 6671.3.2 270 Pos
159-fatBF PHP41103 AFS 10GR13- 2 1.9 2.3 23.0 64.6 8.2 Homoz
6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.0 2.4 24.6 63.3
7.6 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.2 2.6
29.7 59.7 5.8 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.3 2.5 22.5 63.9 8.8 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.1 2.2 20.0 67.1 8.6 Homoz 6671.3.2 270 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.1 2.4 21.4 66.0 8.1 Homoz
6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.3 2.7 27.4 61.0
6.5 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.2
24.9 64.4 6.5 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.0 2.1 24.9 64.2 6.7 Homoz 6671.3.2 270 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.0 2.0 29.1 59.6 7.3 Homoz 6671.3.2 274 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.1 29.2 58.8 7.9 Homoz
6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.0 2.2 27.3 60.6
7.9 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.0 2.0
27.9 61.0 7.1 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.0 2.4 27.7 60.0 7.9 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.1 2.3 28.4 60.1 7.1 Homoz 6671.3.2 274 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.0 2.6 31.7 55.8 7.8 Homoz
6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13- 8 1.9 2.1 22.6 64.0
9.4 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.4
32.0 56.5 7.1 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.0 2.3 27.4 60.4 7.9 Homoz 6671.3.2 274 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.1 2.3 30.7 58.4 6.6 Homoz 6671.3.2 277 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.3 27.6 60.9 7.2 Homoz
6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.1 2.4 30.6 58.1
6.8 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13- 4 1.9 2.3
28.0 60.4 7.4 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.0 2.4 28.0 60.7 6.9 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.2 2.3 23.8 63.0 8.9 Homoz 6671.3.2 277 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.0 2.5 30.4 58.8 6.3 Homoz
6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.0 2.1 28.0 60.7
7.1 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.1 2.8
27.9 60.1 7.2 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.0 2.0 26.2 62.7 7.1 Homoz 6671.3.2 277 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.0 2.6 33.3 55.5 6.6 Homoz 6671.3.2 278 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.2 25.2 63.4 7.3 Homoz
6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13- 3 1.9 2.3 22.7 65.2
7.9 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13- 4 1.9 2.5
26.0 61.5 8.0 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.1 2.6 28.5 59.8 7.1 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.0 2.9 30.2 57.4 7.5 Homoz 6671.3.2 278 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.0 1.9 28.6 58.8 8.7 Homoz
6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.1 2.3 27.2 61.1
7.4 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.3
29.4 59.4 6.9 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.0 2.3 25.7 62.3 7.7 Homoz 6671.3.2 278 Pos 159-fatBF PHP41103
AFS 10GR13- 1 11.0 3.5 20.3 57.8 7.5 Null 6671.6.1 286 159-fatBF
PHP41103 AFS 10GR13- 2 10.8 3.5 18.3 59.1 8.3 Null 6671.6.1 286
159-fatBF PHP41103 AFS 10GR13- 1 11.5 3.1 19.1 58.4 7.9 Null
6671.6.1 288 159-fatBF PHP41103 AFS 10GR13- 2 11.0 3.2 24.6 53.6
7.6 Null 6671.6.1 288 159-fatBF PHP41103 AFS 10GR13- 1 1.8 2.4 18.7
67.2 9.8 Homoz 6671.6.1 289 Pos 159-fatBF PHP41103 AFS 10GR13- 2
1.9 2.5 27.9 60.1 7.7 Homoz 6671.6.1 289 Pos 159-fatBF PHP41103 AFS
10GR13- 3 1.9 2.4 21.4 65.6 8.7 Homoz 6671.6.1 289 Pos 159-fatBF
PHP41103 AFS 10GR13- 4 1.7 2.1 21.2 66.3 8.7 Homoz 6671.6.1 289 Pos
159-fatBF PHP41103 AFS 10GR13- 5 1.9 2.2 22.5 65.5 7.9 Homoz
6671.6.1 289 Pos 159-fatBF PHP41103 AFS 10GR13- 6 1.8 2.1 26.1 63.2
6.8 Homoz 6671.6.1 289 Pos 159-fatBF PHP41103 AFS 10GR13- 7 1.8 2.3
25.9 63.3 6.7 Homoz 6671.6.1 289 Pos 159-fatBF PHP41103 AFS 10GR13-
8 2.0 2.7 19.8 66.2 9.3 Homoz 6671.6.1 289 Pos 159-fatBF PHP41103
AFS 10GR13- 9 1.9 2.5 20.5 66.4 8.7 Homoz 6671.6.1 289 Pos
159-fatBF PHP41103 AFS 10GR13- 10 1.9 2.3 22.7 65.4 7.7 Homoz
6671.6.1 289 Pos 159-fatBF PHP41103 AFS 10GR13- 1 2.0 2.4 23.7 64.6
7.3 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.0 3.4
33.0 55.3 6.3 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13-
3 1.9 2.6 22.5 65.3 7.6 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.0 2.5 21.6 66.4 7.5 Homoz 6671.6.1 292 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.6 21.7 65.5 8.3 Homoz
6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 3.0 20.9 65.4
8.7 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13- 7 2.0 2.3
25.0 64.1 6.6 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13-
8 2.1 2.7 25.7 63.3 6.2 Homoz 6671.6.1 292 Pos 159-fatBF PHP41103
AFS 10GR13- 9 2.3 3.1 25.6 61.9 7.0 Homoz 6671.6.1 292 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.2 3.1 27.0 61.7 6.1 Homoz
6671.6.1 292 Pos 159-fatBF PHP41103 AFS 10GR13- 1 1.9 2.1 23.7 65.0
7.2 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.9 2.4
31.5 57.9 6.4 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13-
3 1.9 2.2 27.7 60.8 7.4 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.9 2.3 27.6 61.2 7.0 Homoz 6671.6.1 304 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.0 21.5 66.0 8.5 Homoz
6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 2.3 24.9 63.5
7.3 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13- 7 1.9 2.5
39.4 49.9 6.4 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13-
8 1.9 2.2 23.4 65.2 7.3 Homoz 6671.6.1 304 Pos 159-fatBF PHP41103
AFS 10GR13- 9 1.9 2.2 27.3 61.3 7.3 Homoz 6671.6.1 304 Pos
159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.1 34.2 55.5 6.3 Homoz
6671.6.1 304 Pos 159-fatBF PHP41103 AFS 10GR13- 1 11.7 3.3 20.2
57.7 7.1 Null 6671.6.3 309 159-fatBF PHP41103 AFS 10GR13- 2 10.6
3.5 24.3 54.0 7.6 Null 6671.6.3 309 159-fatBF PHP41103 AFS 10GR13-
1 12.4 3.1 21.5 55.1 7.9 Null 6671.6.3 318 159-fatBF PHP41103 AFS
10GR13- 2 12.8 3.4 21.5 54.0 8.3 Null 6671.6.3 318 159-fatBF
PHP41103 AFS 10GR13- 1 3.0 2.6 19.8 65.3 9.3 Homoz 6671.6.3 307 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.7 2.3 23.5 63.0 8.4 Homoz
6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.7 2.7 23.3 62.8
8.5 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.6 2.4
28.0 60.1 6.9 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.8 2.5 20.9 65.8 8.0 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.9 2.8 22.5 63.4 8.4 Homoz 6671.6.3 307 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.6 2.7 25.3 61.9 7.6 Homoz
6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.6 2.4 27.6 60.3
7.1 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.6 2.5
25.2 61.9 7.8 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.4 2.7 21.9 66.1 6.9 Homoz 6671.6.3 307 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.8 2.7 20.5 65.3 8.8 Homoz 6671.6.3 310 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.7 2.4 27.8 60.2 6.9 Homoz
6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.5 2.5 28.9 59.7
6.4 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.8 2.4
22.5 64.7 7.6 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.6 2.3 29.8 59.0 6.2 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.5 2.4 37.0 52.2 6.0 Homoz 6671.6.3 310 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.6 2.2 27.2 60.9 7.2 Homoz
6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.3 2.6 30.0 58.6
6.5 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.6 2.2
26.6 61.6 7.1 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.3 2.4 26.0 61.6 7.7 Homoz 6671.6.3 310 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.5 2.0 28.7 59.5 7.4 Homoz 6671.6.3 313 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.6 2.9 30.7 57.3 6.5 Homoz
6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.4 2.0 30.1 59.0
6.4 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.3 2.3
29.4 59.1 6.9 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.4 1.9 29.4 60.1 6.2 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.2 2.4 33.1 56.2 6.1 Homoz 6671.6.3 313 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.4 2.2 31.8 56.8 6.8 Homoz
6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.1 2.1 30.2 59.8
5.8 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.4 2.2
31.9 57.2 6.4 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103 AFS 10GR13-
10 2.2 2.2 32.6 56.5 6.5 Homoz 6671.6.3 313 Pos 159-fatBF PHP41103
AFS 10GR13- 1 2.5 2.3 29.2 59.0 7.0 Homoz 6671.6.3 321 Pos
159-fatBF PHP41103 AFS 10GR13- 2 2.0 2.1 25.5 62.1 8.3 Homoz
6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 3 2.4 2.1 23.0 64.8
7.7 Homoz 6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.2 2.1
24.7 63.6 7.3 Homoz 6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13-
5 2.4 2.6 25.6 62.2 7.2 Homoz 6671.6.3 321 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.6 2.2 23.0 63.4 8.8 Homoz 6671.6.3 321 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.7 2.2 25.3 61.6 8.2 Homoz
6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.5 2.1 24.2 62.9
8.3 Homoz 6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 9 2.6 2.1
22.7 64.4 8.2 Homoz
6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.2 2.3 27.8
59.9 7.7 Homoz 6671.6.3 321 Pos 159-fatBF PHP41103 AFS 10GR13- 1
10.7 3.5 21.2 56.0 8.5 Null 6671.11.2 328 159-fatBF PHP41103 AFS
10GR13- 2 10.1 4.0 24.3 54.4 7.2 Null 6671.11.2 328 159-fatBF
PHP41103 AFS 10GR13- 1 11.6 4.0 17.7 57.8 9.0 Null 6671.11.2 329
159-fatBF PHP41103 AFS 10GR13- 2 11.5 3.9 17.2 57.9 9.6 Null
6671.11.2 329 159-fatBF PHP41103 AFS 10GR13- 1 11.5 3.7 16.2 59.1
9.5 Null 6671.11.2 330 159-fatBF PHP41103 AFS 10GR13- 2 11.8 3.7
15.9 59.1 9.5 Null 6671.11.2 330 159-fatBF PHP41103 AFS 10GR13- 1
2.3 2.5 30.2 57.0 7.9 Homoz 6671.11.2 331 Pos 159-fatBF PHP41103
AFS 10GR13- 2 2.0 2.3 30.0 58.7 6.9 Homoz 6671.11.2 331 Pos
159-fatBF PHP41103 AFS 10GR13- 3 2.3 2.4 26.7 61.1 7.4 Homoz
6671.11.2 331 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.3 2.2 32.7
56.5 6.3 Homoz 6671.11.2 331 Pos 159-fatBF PHP41103 AFS 10GR13- 5
2.2 2.4 28.1 59.6 7.6 Homoz 6671.11.2 331 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.3 2.4 26.1 61.6 7.6 Homoz 6671.11.2 331 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.3 2.1 29.3 59.6 6.7 Homoz
6671.11.2 331 Pos 159-fatBF PHP41103 AFS 10GR13- 8 1.9 2.1 24.2
63.1 8.7 Homoz 6671.11.2 331 Pos 159-fatBF PHP41103 AFS 10GR13- 9
2.2 2.3 29.3 59.2 6.9 Homoz 6671.11.2 331 Pos 159-fatBF PHP41103
AFS 10GR13- 10 2.5 2.3 20.4 64.8 10.1 Homoz 6671.11.2 331 Pos
159-fatBF PHP41103 AFS 10GR13- 1 2.2 2.4 32.0 55.5 7.8 Homoz
6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.8 2.2 28.8
59.7 7.5 Homoz 6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 3
2.3 2.4 29.3 58.1 7.9 Homoz 6671.11.2 333 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.1 2.5 23.7 63.0 8.8 Homoz 6671.11.2 333 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.1 2.4 26.1 61.8 7.5 Homoz
6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.2 2.9 23.2
63.9 7.8 Homoz 6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 7
2.1 2.3 27.4 60.4 7.8 Homoz 6671.11.2 333 Pos 159-fatBF PHP41103
AFS 10GR13- 8 1.9 2.1 26.3 61.7 7.9 Homoz 6671.11.2 333 Pos
159-fatBF PHP41103 AFS 10GR13- 9 2.1 2.6 27.0 61.6 6.8 Homoz
6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.1 2.4 27.0
60.5 8.0 Homoz 6671.11.2 333 Pos 159-fatBF PHP41103 AFS 10GR13- 1
2.1 2.5 22.6 65.0 7.9 Homoz 6671.11.2 334 Pos 159-fatBF PHP41103
AFS 10GR13- 2 2.0 2.7 24.0 62.9 8.4 Homoz 6671.11.2 334 Pos
159-fatBF PHP41103 AFS 10GR13- 3 2.1 2.5 26.6 61.0 7.8 Homoz
6671.11.2 334 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.2 2.5 26.6
60.8 7.8 Homoz 6671.11.2 334 Pos 159-fatBF PHP41103 AFS 10GR13- 5
2.0 2.6 22.7 64.1 8.5 Homoz 6671.11.2 334 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.1 2.3 22.7 65.0 8.0 Homoz 6671.11.2 334 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.1 2.6 26.5 61.1 7.7 Homoz
6671.11.2 334 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.2 2.6 22.7
63.7 8.8 Homoz 6671.11.2 334 Pos 159-fatBF PHP41103 AFS 10GR13- 9
2.1 2.6 26.1 60.7 8.6 Homoz 6671.11.2 334 Pos 159-fatBF PHP41103
AFS 10GR13- 10 2.1 3.4 30.5 56.7 7.3 Homoz 6671.11.2 334 Pos
159-fatBF PHP41103 AFS 10GR13- 1 2.2 2.3 38.1 50.9 6.4 Homoz
6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 2 2.2 3.9 31.1
56.5 6.4 Homoz 6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 3
2.3 3.3 33.6 54.1 6.7 Homoz 6671.11.2 341 Pos 159-fatBF PHP41103
AFS 10GR13- 4 2.3 2.6 26.2 61.0 7.8 Homoz 6671.11.2 341 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.3 2.9 33.3 54.5 6.9 Homoz
6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 2.6 41.1
48.0 6.4 Homoz 6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 7
2.3 3.3 31.8 55.8 6.7 Homoz 6671.11.2 341 Pos 159-fatBF PHP41103
AFS 10GR13- 8 2.3 3.3 31.2 55.9 7.4 Homoz 6671.11.2 341 Pos
159-fatBF PHP41103 AFS 10GR13- 9 2.4 3.1 36.9 50.9 6.6 Homoz
6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.3 3.0 34.0
52.7 8.1 Homoz 6671.11.2 341 Pos 159-fatBF PHP41103 AFS 10GR13- 1
11.3 3.1 23.3 54.5 7.8 Null 6671.11.3 353 159-fatBF PHP41103 AFS
10GR13- 2 10.8 3.3 19.8 57.7 8.4 Null 6671.11.3 353 159-fatBF
PHP41103 AFS 10GR13- 1 11.4 3.3 21.0 56.1 8.2 Null 6671.11.3 359
159-fatBF PHP41103 AFS 10GR13- 2 12.0 3.5 18.0 57.1 9.4 Null
6671.11.3 359 159-fatBF PHP41103 AFS 10GR13- 1 2.1 1.9 27.0 61.1
7.7 Homoz 6671.11.3 347 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.9
2.5 25.9 61.6 8.1 Homoz 6671.11.3 347 Pos 159-fatBF PHP41103 AFS
10GR13- 3 2.1 2.1 22.9 64.1 8.8 Homoz 6671.11.3 347 Pos 159-fatBF
PHP41103 AFS 10GR13- 4 2.0 2.0 23.7 64.5 7.7 Homoz 6671.11.3 347
Pos 159-fatBF PHP41103 AFS 10GR13- 5 2.0 2.2 23.6 63.1 9.1 Homoz
6671.11.3 347 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.0 2.2 22.7
64.5 8.6 Homoz 6671.11.3 347 Pos 159-fatBF PHP41103 AFS 10GR13- 7
2.0 2.1 25.1 61.9 8.9 Homoz 6671.11.3 347 Pos 159-fatBF PHP41103
AFS 10GR13- 8 2.0 2.2 27.5 59.9 8.4 Homoz 6671.11.3 347 Pos
159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.1 26.7 61.2 8.0 Homoz
6671.11.3 347 Pos 159-fatBF PHP41103 AFS 10GR13- 10 1.9 2.3 24.6
62.8 8.5 Homoz 6671.11.3 347 Pos 159-fatBF PHP41103 AFS 10GR13- 1
2.0 2.4 29.4 58.2 8.0 Homoz 6671.11.3 355 Pos 159-fatBF PHP41103
AFS 10GR13- 2 1.9 2.3 24.5 63.1 8.2 Homoz 6671.11.3 355 Pos
159-fatBF PHP41103 AFS 10GR13- 3 2.0 2.4 27.3 60.3 8.0 Homoz
6671.11.3 355 Pos 159-fatBF PHP41103 AFS 10GR13- 4 2.1 2.4 31.9
56.7 6.9 Homoz 6671.11.3 355 Pos 159-fatBF PHP41103 AFS 10GR13- 5
2.1 2.1 25.5 62.2 8.1 Homoz 6671.11.3 355 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.2 2.6 25.0 63.0 7.1 Homoz 6671.11.3 355 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.1 2.2 27.6 61.7 6.4 Homoz
6671.11.3 355 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.3 2.1 29.6
59.4 6.6 Homoz 6671.11.3 355 Pos 159-fatBF PHP41103 AFS 10GR13- 9
2.1 2.3 22.3 64.7 8.6 Homoz 6671.11.3 355 Pos 159-fatBF PHP41103
AFS 10GR13- 10 2.4 2.8 26.5 61.2 7.1 Homoz 6671.11.3 355 Pos
159-fatBF PHP41103 AFS 10GR13- 1 2.2 2.4 27.4 60.7 7.3 Homoz
6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 2 1.8 2.6 24.6
63.1 7.8 Homoz 6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 3
2.2 2.3 29.9 58.2 7.4 Homoz 6671.11.3 360 Pos 159-fatBF PHP41103
AFS 10GR13- 4 1.8 2.4 27.0 61.6 7.2 Homoz 6671.11.3 360 Pos
159-fatBF PHP41103 AFS 10GR13- 5 2.1 2.8 24.6 63.7 6.8 Homoz
6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 6 2.4 2.3 21.5
66.0 7.7 Homoz 6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 7
2.2 2.4 25.9 61.2 8.3 Homoz 6671.11.3 360 Pos 159-fatBF PHP41103
AFS 10GR13- 8 2.1 2.7 19.8 65.1 10.3 Homoz 6671.11.3 360 Pos
159-fatBF PHP41103 AFS 10GR13- 9 2.0 2.4 24.7 62.3 8.6 Homoz
6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 10 2.0 2.2 28.0
60.1 7.7 Homoz 6671.11.3 360 Pos 159-fatBF PHP41103 AFS 10GR13- 1
2.1 2.4 27.2 60.9 7.3 Homoz 6671.11.3 367 Pos 159-fatBF PHP41103
AFS 10GR13- 2 2.3 2.8 36.7 51.9 6.3 Homoz 6671.11.3 367 Pos
159-fatBF PHP41103 AFS 10GR13- 3 2.1 2.7 28.8 59.1 7.2 Homoz
6671.11.3 367 Pos 159-fatBF PHP41103 AFS 10GR13- 4 1.8 2.1 29.8
59.8 6.5 Homoz 6671.11.3 367 Pos 159-fatBF PHP41103 AFS 10GR13- 5
2.1 2.5 30.3 58.8 6.3 Homoz 6671.11.3 367 Pos 159-fatBF PHP41103
AFS 10GR13- 6 2.3 2.6 35.4 53.0 6.7 Homoz 6671.11.3 367 Pos
159-fatBF PHP41103 AFS 10GR13- 7 2.3 2.7 31.2 57.0 6.8 Homoz
6671.11.3 367 Pos 159-fatBF PHP41103 AFS 10GR13- 8 2.0 2.5 28.9
60.1 6.5 Homoz 6671.11.3 367 Pos 159-fatBF PHP41103 AFS 10GR13- 9
2.3 2.4 30.7 57.6 7.1 Homoz 6671.11.3 367 Pos 159-fatBF PHP41103
AFS 10GR13- 10 2.2 2.2 30.0 58.6 6.9 Homoz 6671.11.3 367 Pos Jack
10GR13- 1 12.5 4.3 14.5 58.3 10.3 61 10GR13- 2 12.6 3.9 14.8 58.5
10.1 61 10GR13- 1 12.3 3.9 17.9 57.7 8.2 62 10GR13- 2 12.1 3.9 15.8
59.0 9.2 62 10GR13- 1 11.8 4.0 13.9 60.5 9.7 111 10GR13- 2 12.0 3.7
12.7 60.4 11.3 111 10GR13- 1 12.3 3.9 15.7 59.2 8.8 112 10GR13- 2
12.0 3.8 15.4 59.4 9.4 112
[0412] Table 12 shows that T2 seed from events expressing the
159-fatBF amiRNA have palmitic acid contents ranging from 1.7% to
3.0%. T2 seed from null segregant seed have palmitic acid contents
ranging from 10.1% to 12.8% which is in the range typically
observed for Jack seed where palmitic acid ranges from 11.8% to
12.6%.
TABLE-US-00028 TABLE 13 Fatty acid profile for T2 seed analyzed
from soy transformed with PHP41784, containing the 159-fad2-1b
& 159-fatBF& 159-fad3c amiRNAs, grown in the greenhouse.
Ami- RNA T2 Seed Seed T2 Seed (s) Construct Event Analyzed No. 16:0
18:0 18:1 18:2 18:3 Comment 159- PHP41784A AFS 10GR32- 1 11.1 2.8
16.9 57.6 11.7 Null fad2- 6784.1.1 43 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 2 11.1 3.2 17.8 57.0 10.9 Null fad2- 6784.1.1
43 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 2.0 2.5
89.6 3.7 2.2 Homoz fad2- 6784.1.1 47 Pos 1b/159- fatBf/159- fad3c
159- PHP41784A AFS 10GR32- 2 2.1 2.8 88.9 3.6 2.6 Homoz fad2-
6784.1.1 47 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
3 1.9 2.4 89.5 3.7 2.5 Homoz fad2- 6784.1.1 47 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 4 2.0 2.5 88.6 3.7 3.1
Homoz fad2- 6784.1.1 47 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 5 1.9 2.6 89.6 3.6 2.4 Homoz fad2- 6784.1.1 47 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 6 2.0 2.6 89.7
3.5 2.2 Homoz fad2- 6784.1.1 47 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 7 1.9 2.3 89.6 3.6 2.6 Homoz fad2- 6784.1.1
47 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 8 2.0
2.7 88.4 3.9 2.9 Homoz fad2- 6784.1.1 47 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 9 1.9 2.3 89.6 3.6 2.6 Homoz fad2-
6784.1.1 47 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
10 2.1 2.6 87.9 4.8 2.7 Homoz fad2- 6784.1.1 47 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 1.9 2.1 89.8 3.3 3.0
Homoz fad2- 6784.1.1 52 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 2 2.2 2.6 85.9 2.5 6.8 Homoz fad2- 6784.1.1 52 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 3 2.0 2.3 88.4
4.1 3.2 Homoz fad2- 6784.1.1 52 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 4 1.9 2.9 87.1 4.9 3.1 Homoz fad2- 6784.1.1
52 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 1.9
2.3 88.3 4.7 2.8 Homoz fad2- 6784.1.1 52 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 6 2.1 2.4 88.3 4.4 2.9 Homoz fad2-
6784.1.1 52 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
7 2.0 2.2 88.5 3.7 3.6 Homoz fad2- 6784.1.1 52 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 8 1.9 1.9 88.2 4.8 3.1
Homoz fad2- 6784.1.1 52 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 9 1.8 2.4 89.4 3.8 2.5 Homoz fad2- 6784.1.1 52 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 10 2.0 2.7 86.7
4.7 4.0 Homoz fad2- 6784.1.1 52 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 1 10.8 3.3 18.3 56.5 11.0 Null fad2- 6784.2.1
62 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 11.3 3.5
15.3 58.0 11.9 Null fad2- 6784.2.1 62 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 1 12.2 3.1 20.5 55.0 9.1 Null fad2- 6784.2.1
71 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 13.1 3.2
14.6 57.6 11.5 Null fad2- 6784.2.1 71 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 1 2.9 2.1 87.9 5.3 1.8 Homoz fad2- 6784.2.1
64 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 2.7
2.1 88.3 5.2 1.7 Homoz fad2- 6784.2.1 64 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 3 2.8 2.1 89.3 4.4 1.4 Homoz fad2-
6784.2.1 64 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
4 6.7 2.5 83.8 5.5 1.5 Homoz fad2- 6784.2.1 64 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 2.7 2.0 89.6 4.4 1.4
Homoz fad2- 6784.2.1 64 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 6 3.0 2.4 88.5 4.6 1.4 Homoz fad2- 6784.2.1 64 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 7 1.8 1.9 90.8
4.2 1.3 Homoz fad2- 6784.2.1 64 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 8 3.1 2.6 87.2 5.3 1.7 Homoz fad2- 6784.2.1
64 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 9 2.9
2.2 88.3 4.9 1.7 Homoz fad2- 6784.2.1 64 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 10 2.8 2.2 89.3 4.4 1.4 Homoz
fad2- 6784.2.1 64 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS
10GR32- 1 6.5 2.5 83.1 6.3 1.6 Homoz fad2- 6784.2.1 66 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 2.0 2.2 90.3 4.2 1.3
Homoz fad2- 6784.2.1 66 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 3 4.9 2.5 86.6 4.5 1.4 Homoz fad2- 6784.2.1 66 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 4 2.6 2.3 89.7
4.2 1.3 Homoz fad2- 6784.2.1 66 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 5 2.9 2.2 89.1 4.7 1.1 Homoz fad2- 6784.2.1
66 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 6 2.6
1.9 89.6 4.4 1.5 Homoz fad2- 6784.2.1 66 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 7 2.6 2.0 89.5 4.6 1.3 Homoz fad2-
6784.2.1 66 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
8 1.9 2.4 90.0 4.3 1.4 Homoz fad2- 6784.2.1 66 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 9 2.8 2.1 90.2 3.7 1.2
Homoz fad2- 6784.2.1 66 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 10 6.0 2.3 85.2 5.1 1.4 Homoz fad2- 6784.2.1 66 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 1.9 2.6 90.2
4.0 1.3 Homoz fad2- 6784.2.1 67 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 2 1.8 2.2 90.8 3.8 1.4 Homoz fad2- 6784.2.1
67 Pos 1b/159- fatBf/159- fad3c
159- PHP41784A AFS 10GR32- 3 1.9 2.0 90.4 4.2 1.5 Homoz fad2-
6784.2.1 67 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
4 1.8 2.0 90.2 4.6 1.5 Homoz fad2- 6784.2.1 67 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 1.8 2.1 90.5 4.2 1.5
Homoz fad2- 6784.2.1 67 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 6 1.9 2.8 89.9 3.9 1.5 Homoz fad2- 6784.2.1 67 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 7 1.8 1.9 89.4
5.0 2.0 Homoz fad2- 6784.2.1 67 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 8 1.9 2.3 90.6 3.8 1.5 Homoz fad2- 6784.2.1
67 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 9 1.8
1.8 90.7 4.3 1.5 Homoz fad2- 6784.2.1 67 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 10 1.8 2.1 90.1 4.5 1.5 Homoz
fad2- 6784.2.1 67 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS
10GR32- 1 3.6 2.7 86.6 5.6 1.5 Homoz fad2- 6784.2.1 77 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 2.0 2.1 87.3 6.8 1.8
Homoz fad2- 6784.2.1 77 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 3 2.7 2.4 88.9 4.7 1.4 Homoz fad2- 6784.2.1 77 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 4 3.3 2.6 89.1
3.7 1.3 Homoz fad2- 6784.2.1 77 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 5 3.6 2.6 87.9 4.3 1.6 Homoz fad2- 6784.2.1
77 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 6 2.3
2.8 89.9 3.8 1.2 Homoz fad2- 6784.2.1 77 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 7 5.8 2.7 86.6 3.8 1.2 Homoz fad2-
6784.2.1 77 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
8 2.9 2.1 89.9 3.7 1.4 Homoz fad2- 6784.2.1 77 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 9 3.4 2.7 87.6 4.9 1.4
Homoz fad2- 6784.2.1 77 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 10 7.1 2.1 84.7 4.6 1.5 Homoz fad2- 6784.2.1 77 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 11.5 3.6 19.1
55.1 10.7 Null fad2- 6784.3.2 82 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 2 11.6 3.6 18.6 55.9 10.3 Null fad2- 6784.3.2
82 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 11.6 3.5
21.2 54.3 9.5 Null fad2- 6784.3.2 84 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 2 11.7 3.8 18.4 56.5 9.6 Null fad2- 6784.3.2
84 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 1.9 2.0
90.2 4.0 2.0 Homoz fad2- 6784.3.2 91 Pos 1b/159- fatBf/159- fad3c
159- PHP41784A AFS 10GR32- 2 2.0 2.2 90.0 3.5 2.3 Homoz fad2-
6784.3.2 91 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
3 1.8 2.5 89.5 3.4 2.8 Homoz fad2- 6784.3.2 91 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 4 1.9 2.5 89.4 3.6 2.5
Homoz fad2- 6784.3.2 91 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 5 1.8 2.3 90.0 3.4 2.5 Homoz fad2- 6784.3.2 91 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 6 1.9 2.3 90.1
3.3 2.4 Homoz fad2- 6784.3.2 91 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 7 1.9 2.4 90.2 3.3 2.3 Homoz fad2- 6784.3.2
91 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 8 2.0
2.5 89.9 3.4 2.2 Homoz fad2- 6784.3.2 91 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 9 1.9 2.3 88.3 4.4 3.0 Homoz fad2-
6784.3.2 91 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
10 1.8 2.1 90.6 3.6 1.9 Homoz fad2- 6784.3.2 91 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 2.1 2.8 87.8 4.5 2.8
Homoz fad2- 6784.3.2 93 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 2 2.2 2.9 87.6 4.5 2.8 Homoz fad2- 6784.3.2 93 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 3 2.2 2.7 87.3
4.6 3.2 Homoz fad2- 6784.3.2 93 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 4 2.3 2.9 86.9 4.8 3.1 Homoz fad2- 6784.3.2
93 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 2.3
3.0 86.2 5.0 3.5 Homoz fad2- 6784.3.2 93 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 6 2.3 3.2 84.5 6.2 3.8 Homoz fad2-
6784.3.2 93 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
7 2.1 2.8 85.2 6.0 3.8 Homoz fad2- 6784.3.2 93 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 8 2.2 2.9 86.3 5.3 3.2
Homoz fad2- 6784.3.2 93 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 9 2.2 2.9 86.4 4.9 3.5 Homoz fad2- 6784.3.2 93 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 10 2.2 2.9 86.7
5.0 3.2 Homoz fad2- 6784.3.2 93 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 1 1.9 2.4 90.0 3.3 2.4 Homoz fad2- 6784.3.2
96 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 2 2.0
2.7 89.0 3.8 2.5 Homoz fad2- 6784.3.2 96 Pos 1b/159- fatBf/159-
fad3c 159- PHP41784A AFS 10GR32- 3 2.1 2.5 90.0 3.3 2.1 Homoz fad2-
6784.3.2 96 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32-
4 2.0 2.8 89.1 3.4 2.7 Homoz fad2- 6784.3.2 96 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 2.0 2.6 89.6 3.5 2.3
Homoz fad2- 6784.3.2 96 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 6 2.1 2.8 89.4 3.5 2.2 Homoz fad2- 6784.3.2 96 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 7 1.8 2.4 90.6
3.1 2.0 Homoz fad2- 6784.3.2 96 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 8 1.9 2.2 90.7 3.4 1.9 Homoz fad2- 6784.3.2
96 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 9 1.9
2.4 90.1 3.5 2.1 Homoz
fad2- 6784.3.2 96 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS
10GR32- 10 1.9 2.3 90.0 3.6 2.3 Homoz fad2- 6784.3.2 96 Pos 1b/159-
fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 1 2.0 2.9 87.3 4.9 2.8
Homoz fad2- 6784.3.2 99 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A
AFS 10GR32- 2 2.0 2.4 88.5 4.8 2.3 Homoz fad2- 6784.3.2 99 Pos
1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 3 2.0 3.1 87.6
4.5 2.8 Homoz fad2- 6784.3.2 99 Pos 1b/159- fatBf/159- fad3c 159-
PHP41784A AFS 10GR32- 4 2.1 3.1 87.5 4.7 2.5 Homoz fad2- 6784.3.2
99 Pos 1b/159- fatBf/159- fad3c 159- PHP41784A AFS 10GR32- 5 2.1
2.7 87.3 5.3 2.6 Homoz fad2- 6784.3.2 99 Pos 1b/159- fatBf/159-
fad3c 93B86 10GR32- 1 11.8 3.8 18.4 56.1 10.0 102 93B86 10GR32- 2
10.9 3.5 19.9 55.6 10.0 102 93B86 10GR32- 1 12.1 4.5 17.4 54.9 11.1
104 93B86 10GR32- 2 11.8 3.9 19.2 55.0 10.2 104
[0413] Table 13 shows that T2 seed from events expressing the
159-fad2-1b & 159-fatBF& 159-fad3c amiRNAs have palmitic
acid contents ranging from 1.8% to 7.1%, oleic acid contents
ranging from 83.1% to 90.8% and alpha-linolenic acid contents
ranging from 1.1% to 6.8%. T2 seed from null segregant seed have
palmitic acid contents ranging from 10.8% to 13.1%, oleic acid
contents ranging from 14.6% to 21.2% and alpha-linolenic acid
contents ranging from 9.1% to 11.9%. The elite variety which was
transformed (93B86) has palmitic acid contents ranging from 10.9%
to 12.1%, oleic acid contents ranging from 16.3% to 19.9% and
alpha-linolenic acid contents ranging from 10.0% to 11.5%.
Analysis of Fatty Acid Profiles of Seed from Events Expressing
amiRNAs Grown in the Field in Iowa
[0414] Seed representative for each experiment from different
events were planted in multiple reps in 4 different locations in
Iowa (Johnston, Stuart, Griswold & Washington) along with
suitable null segregants and Jack or 93B86 controls. Typically,
reps were planted in rows having from 30 seed (short rows) up to
around 220 seed for longer rows.
[0415] Seed were harvested and two 5-seed bulk aliquots from each
row were crushed, lipids were derivitized with TMSH and FAMEs were
analyzed by GC as described inter alia. The resulting two 5-seed
bulk fatty acid profiles were then averaged to obtain the average
fatty acid profile for seeds from that row.
[0416] The average 5-seed bulk fatty acid profiles from all rows
for a given event within an experiment were then averaged to obtain
an overall average fatty acid profile for seed from that event when
grown in the field. This was also done for null segregants within
an event and with the Jack and 93B86 controls.
[0417] The average fatty acid profiles for field grown seed
analyzed from soy transformed with PHP32510, containing the
369b-fad2-1b amiRNA or PHP32511, containing the 159-fad2-1b amiRNA
or PHP32843, containing the 369b-fad2-1b & 159-fad2-2 amiRNAs
or PHP33705, containing the 396-fad2-1b & 396b-sad3 amiRNAs or
PHP38557, containing the 159-fad3c amiRNA or PHP41103, containing
the 159-fatBF amiRNA or PHP41784, containing the 159-fad2-1b &
159-fatBF& 159-fad3c amiRNAs are shown in Table 14.
TABLE-US-00029 TABLE 14 Average fatty acid profiles for seed grown
in the field in four locations in Iowa. AmiRNA Avg. Avg. Avg. Avg.
Avg. T2 Seed (s) Construct Event 16:0 18:0 18:1 18:2 18:3 Comment
396b- PHP32510 AFS 5260.3.3 9.1 4.3 23.4 55.5 7.6 Null fad2-1b A
396b- PHP32510 AFS 5260.3.11 9.1 4.4 31.0 48.5 6.9 Null fad2-1b A
396b- PHP32510 AFS 5260.2.3 6.0 3.7 80.8 4.6 4.7 Homoz fad2-1b A
Pos 396b- PHP32510 AFS 5260.3.6 6.2 3.6 80.6 5.0 4.5 Homoz fad2-1b
A Pos 396b- PHP32510 AFS 5260.4.5 6.1 3.9 82.0 3.5 4.4 Homoz
fad2-1b A Pos 159- PHP32511 AFS 5292.1.4 6.5 4.0 77.1 6.8 5.4 Homoz
fad2-1b A Pos 159- PHP32511 AFS 5292.5.8 6.1 4.0 81.5 3.8 4.4 Homoz
fad2-1b A Pos 159- PHP32511 AFS 5292.6.5 7.0 4.2 71.2 11.7 5.9
Homoz fad2-1b A Pos 159- PHP32511 AFS 5292.7.2 6.3 3.8 75.7 8.4 5.6
Homoz fad2-1b A Pos 159- PHP32511 AFS 5292.7.6 6.1 4.1 81.3 3.9 4.6
Homoz fad2-1b A Pos 396b- PHP32843 EAFS 5396.2.2 5.6 3.5 88.8 0.5
1.5 Homoz fad2- A Pos 1b/159- fad2-2 396b- PHP32843 EAFS 5396.2.3
5.5 3.5 88.7 0.6 1.6 Homoz fad2- A Pos 1b/159- fad2-2 396b-
PHP32843 EAFS 5396.3.8 5.7 3.4 87.8 1.0 2.0 Homoz fad2- A Pos
1b/159- fad2-2 396- PHP33705 AFS 5489.3.2 9.1 4.4 22.7 55.7 7.9
Null fad2- A 1b/396b- sad3 396- PHP33705 AFS 5489.3.4 9.2 4.1 24.2
56.0 6.5 Null fad2- A 1b/396b- sad3 396- PHP33705 AFS 5489.4.1 9.4
4.3 22.4 56.0 7.9 Null fad2- A 1b/396b- sad3 396- PHP33705 AFS
5489.3.2 6.3 11.4 61.6 14.7 5.9 Homoz fad2- A Pos 1b/396b- sad3
396- PHP33705 AFS 5489.3.4 6.2 13.1 63.2 11.8 5.6 Homoz fad2- A Pos
1b/396b- sad3 396- PHP33705 AFS 5489.4.1 5.5 14.2 65.7 8.4 6.1
Homoz fad2- A Pos 1b/396b- sad3 159- PHP38557 AFS 6272.1.4 8.3 4.3
23.8 55.9 7.6 Null fad3c A 159- PHP38557 AFS 6272.2.1 9.3 4.4 24.3
54.5 7.4 Null fad3c A 159- PHP38557 AFS 6272.2.3 9.2 4.3 23.1 55.8
7.5 Null fad3c A 159- PHP38557 AFS 6272.1.4 9.2 4.1 25.4 56.3 4.8
Homoz fad3c A Pos 159- PHP38557 AFS 6272.2.1 9.2 4.2 26.9 57.6 2.1
Homoz fad3c A Pos 159- PHP38557 AFS 6272.2.3 9.4 4.1 24.6 57.4 4.4
Homoz fad3c A Pos 159- PHP41103 AFS 6671.1.4 9.5 3.8 20.6 57.0 9.1
Null fatBF A 159- PHP41103 AFS 6671.1.6 9.9 4.0 21.4 56.5 8.1 Null
fatBF A 159- PHP41103 AFS 6671.3.2 10.4 3.9 19.9 57.3 8.6 Null
fatBF A 159- PHP41103 AFS 6671.6.1 10.0 4.0 21.0 57.1 7.8 Null
fatBF A 159- PHP41103 AFS 6671.6.3 10.6 4.1 21.0 56.3 8.0 Null
fatBF A 159- PHP41103 AFS 6671.11.2 10.0 3.9 20.3 57.3 8.3 Null
fatBF A 159- PHP41103 AFS 6671.11.3 10.2 3.8 20.2 57.1 8.7 Null
fatBF A 159- PHP41103 AFS 6671.1.4 2.2 3.1 23.7 62.4 8.5 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.1.6 1.9 3.1 23.7 62.7 8.6 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.3.2 3.3 3.9 24.8 59.6 8.4 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.6.1 1.9 3.2 23.9 63.3 7.6 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.6.3 2.5 3.3 23.4 62.9 7.9 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.11.2 2.1 3.3 22.5 63.6 8.5 Homoz
fatBF A Pos 159- PHP41103 AFS 6671.11.3 2.2 3.1 23.9 62.1 8.6 Homoz
fatBF A Pos 159-fad2- PHP41784 AFS 6784.1.1 9.9 3.5 18.9 57.7 9.8
Null 1b/159- A fatBf/159- fad3c 159-fad2- PHP41784 AFS 6784.2.1
12.0 3.5 18.1 57.3 9.0 Null 1b/159- A fatBf/159- fad3c 159-fad2-
PHP41784 AFS 6784.3.2 10.2 3.6 19.5 57.9 8.8 Null 1b/159- A
fatBf/159- fad3c 159-fad2- PHP41784 AFS 6784.1.1 2.0 2.7 82.9 9.5
2.7 Homoz 1b/159- A Pos fatBf/159- fad3c 159-fad2- PHP41784 AFS
6784.2.1 2.8 2.6 83.1 9.8 1.5 Homoz 1b/159- A Pos fatBf/159- fad3c
159-fad2- PHP41784 AFS 6784.3.2 1.9 2.7 83.6 9.5 2.2 Homoz 1b/159-
A Pos fatBf/159- fad3c Jack 9.5 4.1 21.6 56.9 7.9 Average 93B86 9.9
3.8 20.5 57.1 8.6 Average
[0418] Table 14 shows that fatty acid profiles in seed expressing
various amiRNAs resemble that obtained for greenhouse grown seed
and are stable when grown in the field.
Example 6
Generation of amiRNA Precursors to Silence Arabidopsis and Brassica
Fatty Acid Biosynthetic Genes
[0419] Key gene family sequences targeted for silencing in
Arabidopsis thaliana and Brassica napus are the fatty acid
desaturase 2 gene families, also known as delta-12 desaturase or
omega-6 desaturase, the fatty acid desaturase 3 (fad3) gene family
[Yadav, N. S., Wierzbicki, A., Aegerter, M., Caster, C. S.,
Perez-Grau, L., Kinney, A. J., Hitz, W. D., Booth, J. R. Jr.,
Schweiger, B., Stecca, K. L., Allen, S. M., Blackwell, M., Reiter,
R. S., Carlson, T. J., Russell, S. H., Feldmann, K. A., Pierce, J.
and Browse, J. Cloning of higher plant omega-3 fatty acid
desaturases Plant Physiol. 103 (2), 467-476 (1993)] and the fatty
acid elongase (fael) gene family (publication number US
2007/0204370 A1, filed Nov. 24, 2004)). A list of fatty acid
biosynthetic genes targeted for silencing by amiRNAs, along with
corresponding Arabidopsis or Brassica genome sequence gene
identifier, nt SEQ ID NO and aa SEQ ID NO are shown in Table
15.
[0420] In Table 15, the gene identifier used for the corresponding
Brassica napus genes is the NCBI Accession number for the coding
sequence of the respective gene.
TABLE-US-00030 TABLE 15 Arabidopsis thaliana and Brassica napus
Fatty Acid Biosynthetic Gene Sequences Targeted for Silencing
Organism nt aa Targeted Gene Family Gene SEQ ID NO SEQ ID NO
Arabidopsis Fad2 At3g12120 44 45 Fad3 At2g29980 46 47 FaeI
At4g34520 48 49 Brassica Fad2 FJ907397 50 51 FJ907398 52 53
FJ907399 54 55 FJ907400 56 57 FJ907401 58 59 AF124360 174 175 Fad3
L01418 60 61 AY599884 62 63 L22962 64 65 FaeI AF274750 66 67
AY888043 68 69 AF009563 70 71 GU325719 72 73 AF490462 74 75
AF490459 76 77 BNU50771 78 79
Design of Artificial microRNAs and Corresponding STAR Sequences
[0421] amiRNAs and corresponding STAR sequences that pair with the
amiRNAs were designed against the Arabidopsis and Brassica
sequences as described in Example 1 and are listed in Table 16
along with their SEQ ID NOs.
TABLE-US-00031 TABLE 16 amiRNAs and corresponding STAR sequences
targeting Arabidopsis and Brassica fatty acid biosynthetic
sequences. amiRNA 159 Precursor STAR 396 Precursor STAR amiRNA SEQ
ID NO: sequence SEQ ID NO: sequence SEQ ID NO: fad2a 80 81 82 fad2b
83 84 85 fad2c 86 87 88 fad3a 89 90 91 fad3b 92 93 94 faeIa 95 96
97 faeIb 98 99 100 faeIc 101 102 103
Generation of In-Fusion.TM. Ready Expression Vectors
[0422] The microRNA GM-159 and GM-396b precursors (described in
Example 1) were altered to include Pme I sites immediately flanking
the star and microRNA sequences to form the In-Fusion.TM. ready
microRNA precursors. These sequences were cloned into the Not I
site of KS332 to form the In-Fusion.TM. ready microRNA GM-159-KS332
and GM-396b-KS332 plasmids (SEQ ID NO: 104 and SEQ ID NO: 105,
respectively).
[0423] In order to remove the DSred cassette, GM-396b-KS332 (SEQ ID
NO: 105) was digested with BamHI and the fragment containing the
GM-396b precursor was re-ligated to produce pKR2007 (SEQ ID NO:
106).
[0424] Plasmid GM-159-KS332 (SEQ ID NO: 104) was digested with
HindIII and the fragment containing the GM-159 precursor was cloned
into the HindIII fragment of pKR2007 (SEQ ID NO: 106), containing
vector backbone DNA, to produce pKR2009 (SEQ ID NO: 107).
[0425] In all of these expression vectors, the expression cassette
(beta-conglycinin promoter:In-Fusion.TM. ready microRNA
precursor:phaseolin terminator) is flanked by AscI sites.
Generation of amiRNA Precursors to Silence Arabidopsis and Brassica
Fatty Acid Biosynthetic Genes
[0426] When synthesizing amiRNA precursors in the GM-159 backbone,
the microRNA GM-159 precursor (Example 1) was used as a PCR
template. Oligonucleotide pairs were designed for each amiRNA/STAR
sequence to be amplified using 5' and 3' oligonucleotide primers
which are identical to the GM-159 precursor region at the 3' end of
the oligonucleotide and which contain either the 21 bp amiRNA or
STAR sequence of interest (as listed in Table 16) and a region
homologous to either side of the PmeI site of pKR2009 (SEQ ID NO:
107) at the 5' end of the oligonucleotide. The oligonucleotide
primers were designed according to the protocol provided by
Clontech and do not leave any footprint of the Pme I sites after
the In-Fusion.TM. recombination reaction.
[0427] A similar approach was used to design oligonucleotides for
amiRNA precursors in the GM-396b backbone except microRNA GM-396b
is used as PCR template and the 5' region of the oligonucleotide is
homologous to either side of the PmeI site of pKR2007 (SEQ ID NO:
106).
[0428] The amplified DNA corresponding to each primer set was
recombined into either pKR2007 or pKR2009, previously digested with
PmeI to linearize the vector, using the manufacturer's protocols
provided with the In-Fusion.TM. kit. In this way, expression
vectors for each of the amiRNA/STAR sequences listed in Table 16
were produced.
[0429] These plasmids were then digested with AscI and the fragment
containing the amiRNA expression cassette was sub-cloned into the
AscI site of pKR92, which was previously described in
WO2007/06110109 (published on May 31, 2007, the contents of which
are herein incorporated by reference). The SEQ ID NOs of sequences
for the resulting precursor amiRNAs, as well as the resulting
plasmids containing amiRNA-396b or amiRNA-159 precursors suitable
for silencing fad2, fad3 and fael genes are listed in Table 17.
TABLE-US-00032 TABLE 17 Precursor amiRNAs and amiRNA Expression
Constructs For Arabidopsis and Brassica Fatty Acid Biosynthetic
Gene Sequences Targeted for Silencing amiRNA Gene amiRNA Precursor
Plasmid Plasmid Family Precursor SEQ ID NO Name SEQ ID NO fad2
159-Atfad2a 136 pKR2032 108 159-Atfad2b 137 pKR2033 109 159-Atfad2c
138 pKR2034 110 396b-Atfad2a 139 pKR2037 111 396b-Atfad2b 140
pKR2038 112 398b-Atfad2c 141 pKR2039 113 fad3 159-Atfad3a 142
pKR2035 114 159-Atfad3b 143 pKR2036 115 396b-Atfad3a 144 pKR2040
116 396b-Atfad3b 145 pKR2041 117 faeI 159-AtfaeIa 146 pKR2076 118
159-AtfaeIb 147 pKR2077 119 159-AtfaeIc 148 pKR2078 120
396b-AtfaeIa 149 pKR2079 121 396b-AtfaeIb 150 pKR2081 122
396b-AtfaeIc 151 pKR2080 123
[0430] From Table 17, the amiRNA precursor 159-Atfad2a (SEQ ID NO:
136) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAD2A
amiRNA (SEQ ID NO: 80) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAD2A Star Sequence (SEQ ID NO: 81).
The genomic miRNA precursor sequences 159 and 396b were described
previously in US20090155910A1 (WO 2009/079532) and their sequences
are set forth in SEQ ID NO: 152 and SEQ ID NO: 153,
respectively.
[0431] From Table 17, the amiRNA precursor 159-Atfad2b (SEQ ID NO:
137) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAD2B
amiRNA (SEQ ID NO: 83) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAD2B Star Sequence (SEQ ID NO:
84).
[0432] From Table 17, the amiRNA precursor 159-Atfad2c (SEQ ID NO:
138) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAD2c
amiRNA (SEQ ID NO: 86) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAD2c Star Sequence (SEQ ID NO:
87).
[0433] From Table 17, the amiRNA precursor 396b-Atfad2a (SEQ ID NO:
139) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAD2A
amiRNA (SEQ ID NO: 80) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAD2A Star Sequence (SEQ ID NO:
82).
[0434] From Table 17, the amiRNA precursor 396b-Atfad2b (SEQ ID NO:
140) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAD2B
amiRNA (SEQ ID NO: 83) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAD2B Star Sequence (SEQ ID NO:
85).
[0435] From Table 17, the amiRNA precursor 396b-Atfad2c (SEQ ID NO:
141) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAD2c
amiRNA (SEQ ID NO: 86) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAD2c Star Sequence (SEQ ID NO:
88).
[0436] From Table 17, the amiRNA precursor 159-Atfad3a (SEQ ID NO:
142) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAD3A
amiRNA (SEQ ID NO: 89) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAD2A Star Sequence (SEQ ID NO:
90).
[0437] From Table 17, the amiRNA precursor 159-Atfad3b (SEQ ID NO:
143) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAD3B
amiRNA (SEQ ID NO: 92) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAD3B Star Sequence (SEQ ID NO:
93).
[0438] From Table 17, the amiRNA precursor 396b-Atfad3a (SEQ ID NO:
144) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAD3A
amiRNA (SEQ ID NO: 89) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAD3A Star Sequence (SEQ ID NO:
91).
[0439] From Table 17, the amiRNA precursor 396b-Atfad2b (SEQ ID NO:
145) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAD3B
amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAD3B Star Sequence (SEQ ID NO:
94).
[0440] From Table 17, the amiRNA precursor 159-Atfaela (SEQ ID NO:
146) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAEIA
amiRNA (SEQ ID NO: 95) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAEIA Star Sequence (SEQ ID NO:
96).
[0441] From Table 17, the amiRNA precursor 159-Atfaelb (SEQ ID NO:
147) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAEIB
amiRNA (SEQ ID NO: 98) and wherein nucleotides 121 to 141 of SEQ ID
NO: 152 are replaced by 159-ATFAEIB Star Sequence (SEQ ID NO:
99).
[0442] From Table 17, the amiRNA precursor 159-Atfaelc (SEQ ID NO:
148) is 958 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 152 wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by ATFAEIc
amiRNA (SEQ ID NO: 101) and wherein nucleotides 121 to 141 of SEQ
ID NO: 152 are replaced by 159-ATFAEIc Star Sequence (SEQ ID NO:
102).
[0443] From Table 17, the amiRNA precursor 396b-Atfaela (SEQ ID NO:
149) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAEIA
amiRNA (SEQ ID NO: 95) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAEIA Star Sequence (SEQ ID NO:
97).
[0444] From Table 17, the amiRNA precursor 396b-Atfaelb (SEQ ID NO:
150) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAEIB
amiRNA (SEQ ID NO: 98) and wherein nucleotides 262 to 282 of SEQ ID
NO: 153 are replaced by 396b-ATFAEIB Star Sequence (SEQ ID NO:
100).
[0445] From Table 17, the amiRNA precursor 396b-Atfaelc (SEQ ID NO:
151) is 574 nt in length and is substantially similar to the
deoxyribonucleotide sequence set forth in SEQ ID NO: 153 wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by ATFAEIc
amiRNA (SEQ ID NO: 101) and wherein nucleotides 262 to 282 of SEQ
ID NO: 153 are replaced by 396b-ATFAEIc Star Sequence (SEQ ID NO:
103).
Example 7
Phenotypic Analysis of Arabidopsis Seed Expressing amiRNAs for
Silencing Fatty Acid Biosynthetic Genes
Transformation of Arabidopsis Plants
[0446] Plasmids listed in Table 17 (Example 6) were introduced into
Agrobacterium tumefaciens NTL4 (Luo et al, Molecular Plant--Microbe
Interactions (2001) 14(1):98-103) by electroporation. Briefly, 1
.mu.g plasmid DNA was mixed with 100 .mu.L of electro-competent
cells on ice. The cell suspension was transferred to a 100 .mu.L
electroporation cuvette (1 mm gap width) and electroporated using a
BIORAD electroporator set to 1 kV, 400.OMEGA. and 25 .mu.F. Cells
were transferred to 1 mL LB medium and incubated for 2 h at
30.degree. C. Cells were plated onto LB medium containing 50
.mu.g/mL kanamycin. Plates were incubated at 30.degree. C. for 60
h. Recombinant Agrobacterium cultures (500 mL LB, 50 .mu.g/mL
kanamycin) were inoculated from single colonies of transformed
agrobacterium cells and grown at 30.degree. C. for 60 h. Cells were
harvested by centrifugation (5000.times.g, 10 min) and resuspended
in 1 L of 5% (W/V) sucrose containing 0.05% (V/V) Silwet.
Arabidopsis plants were grown in soil at a density of 30 plants per
100 cm.sup.2 pot in METRO-MIX.RTM. 360 soil mixture for 4 weeks
(22.degree. C., 16 h light/8 h dark, 100 .mu.E m-2s-1).
[0447] Plants were repeatedly dipped into the Agrobacterium
suspension harboring the expression vectors listed in Table 17 and
kept in a dark, high humidity environment for 24 h. Post dipping,
plants were grown for three to four weeks under standard plant
growth conditions described above and plant material was harvested
and dried for one week at ambient temperatures in paper bags. Seeds
were harvested using a 0.425 mm mesh brass sieve.
[0448] Cleaned Arabidopsis seeds (2 grams, corresponding to about
100,000 seeds) were sterilized by washes in 45 mL of 80% ethanol,
0.01% TRITON.RTM. X-100, followed by 45 mL of 30% (V/V) household
bleach in water, 0.01% TRITON.RTM. X-100 and finally by repeated
rinsing in sterile water. Aliquots of 20,000 seeds were transferred
to square plates (20.times.20 cm) containing 150 mL of sterile
plant growth medium comprised of 0.5.times.MS salts, 0.53% (W/V)
sorbitol, 0.05 MES/KOH (pH 5.8), 200 .mu.g/mL TIMENTIN.RTM., and 50
.mu.g/mL kanamycin solidified with 10 g/L agar. Homogeneous
dispersion of the seed on the medium was facilitated by mixing the
aqueous seed suspension with an equal volume of melted plant growth
medium. Plates were incubated under standard growth conditions for
ten days. Kanamycin-resistant seedlings were transferred to plant
growth medium without selective agent and grown for one week before
transfer to soil. T1 Plants were grown to maturity alongside wt
control plants and T2 seeds were harvested.
Fatty Acid Analysis of Bulk T2 Seed
[0449] Approximately 10-25 Arabidopsis seed were crushed in 50 uL
of TMSH using a plastic inoculating loop and 500 uL of hexane was
added. After vortexing thoroughly, the hexane phase containing
FAMEs were analyzed by GC as described in earlier examples and
results for fatty acid profiles for approximately 10-20 events for
each experiment are presented in Tables 18 to 33 below. In each
Table, fatty acids are identified as 16:0 (palmitate), 18:0
(stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid) and 18:3
(alpha-linolenic acid), 20:0 (eicosanoic acid), 20:1 eicosenoic
acid) 20:2 (eicocadienoic acid) and 20:3 (eicosatrienoic acid) and
results are expressed as a weight percent (wt. %) of total fatty
acids. For constructs expressing a fad2 amiRNA, fatty acid profiles
are sorted from highest 18:1 to lowest. For constructs expressing a
fad3 amiRNA, fatty acids are sorted from lowest 18:3 to highest.
For constructs expressing a fael amiRNA, fatty acids are sorted
from lowest to highest 20:1. A wild-type Columbia seed batch was
also run and shown for comparison in each Table as col-0.
TABLE-US-00033 TABLE 18 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2032 expressing the
159-fad2a amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2032-15 5.5 2.3 52.3 7.8 8.1 1.3 22.5 0.3 pKR2032-4 6.5 2.7 49.3
8.2 7.9 1.4 23.7 0.3 pKR2032-12 6.7 2.7 49.0 8.8 8.2 1.4 23.0 0.3
pKR2032-8 6.6 2.8 48.9 9.2 8.4 1.3 22.6 0.4 pKR2032-10 5.9 2.7 45.7
10.5 9.5 1.5 23.7 0.6 pKR2032-7 6.6 2.9 45.6 10.9 10.0 1.5 21.9 0.5
pKR2032-9 6.4 2.6 43.9 12.7 10.6 1.4 21.9 0.7 pKR2032-2 6.5 2.9
43.4 11.8 11.3 1.3 22.3 0.6 pKR2032-3 6.9 3.0 42.8 12.5 10.6 1.5
21.9 0.7 pKR2032-5 6.7 2.8 42.0 11.8 11.4 1.6 23.1 0.6 pKR2032-13
7.1 2.9 39.8 13.2 12.7 1.6 22.1 0.7 pKR2032-14 7.2 2.7 39.5 13.5
12.6 1.5 22.3 0.7 pKR2032-1 7.0 2.8 37.1 15.7 12.7 1.6 22.2 1.0
pKR2032-6 6.9 2.7 36.4 15.3 13.1 1.7 23.2 0.8 pKR2032-11 7.3 3.0
36.1 15.8 13.8 1.6 21.6 0.9 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8
1.9
TABLE-US-00034 TABLE 19 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2033 expressing the
159-fad2b amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2033-12 5.7 2.6 57.5 4.9 5.9 1.3 21.8 0.1 pKR2033-13 6.8 2.7
56.8 6.6 7.3 1.2 18.3 0.2 pKR2033-9 5.6 2.5 56.2 4.9 6.0 1.3 23.4
0.2 pKR2033-6 5.7 2.5 55.1 5.5 6.6 1.3 22.9 0.2 pKR2033-5 6.3 2.7
53.7 6.3 6.7 1.3 22.8 0.3 pKR2033-8 6.9 2.8 48.5 9.4 8.6 1.4 21.9
0.5 pKR2033-7 6.9 2.9 48.0 10.0 8.9 1.4 21.5 0.5 pKR2033-3 6.7 2.7
46.3 11.0 9.0 1.4 22.4 0.6 pKR2033-2 6.7 2.8 46.2 11.3 9.2 1.5 21.6
0.6 pKR2033-10 6.9 2.8 42.3 12.6 10.9 1.6 22.2 0.7 pKR2033-11 7.0
2.7 41.8 13.0 10.5 1.6 22.5 0.8 pKR2033-1 6.9 2.7 39.3 14.5 11.4
1.6 22.7 0.9 pKR2033-4 6.4 2.4 21.3 27.2 18.5 1.6 20.6 2.0 Col-0
10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00035 TABLE 20 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2034 expressing the
159-fad2c amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2034-13 5.5 2.4 57.3 4.7 6.2 1.2 22.5 0.2 pKR2034-2 5.9 2.6 56.6
5.2 6.3 1.2 22.1 0.2 pKR2034-4 6.2 2.7 54.7 5.1 6.1 1.5 23.6 0.1
pKR2034-1 7.0 2.9 47.7 9.3 8.2 1.6 23.0 0.3 pKR2034-15 6.8 2.9 47.5
9.5 9.4 1.4 21.9 0.5 pKR2034-5 7.0 3.0 46.3 11.0 9.3 1.5 21.2 0.6
pKR2034-6 6.6 2.9 45.5 10.8 9.8 1.4 22.3 0.6 pKR2034-14 6.4 2.7
45.2 10.9 9.4 1.6 23.1 0.6 pKR2034-10 5.8 2.5 45.0 11.3 9.6 1.6
23.5 0.7 pKR2034-9 6.8 2.8 45.0 11.1 9.6 1.6 22.6 0.5 pKR2034-7 6.9
3.0 44.4 11.4 10.2 1.6 22.0 0.7 pKR2034-17 6.9 2.8 43.3 12.3 9.9
1.6 22.4 0.6 pKR2034-8 6.8 2.7 43.1 12.8 9.9 1.6 22.4 0.8
pKR2034-11 7.0 2.9 42.6 13.7 10.4 1.6 21.0 0.7 pKR2034-12 6.9 2.7
41.9 13.5 10.4 1.5 22.3 0.8 pKR2034-16 6.5 2.8 40.3 14.5 11.0 1.8
22.2 0.8 pKR2034-3 7.1 2.7 24.4 35.2 7.1 1.8 19.7 2.0 Col-0 10.2
2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00036 TABLE 21 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2037 expressing the
396b-fad2a amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2037-12 6.4 2.6 43.4 12.5 10.1 1.5 22.8 0.7 pKR2037-14 6.5 2.7
43.3 10.9 11.3 1.6 23.2 0.5 pKR2037-7 6.8 2.9 43.2 12.6 11.0 1.5
21.4 0.6 pKR2037-9 6.8 2.9 43.0 10.3 12.9 1.5 22.3 0.3 pKR2037-11
7.2 2.8 38.0 14.6 13.2 1.5 21.8 0.7 pKR2037-10 7.1 2.9 36.6 15.1
13.6 1.5 22.5 0.8 pKR2037-2 6.5 2.7 36.6 14.3 14.1 1.6 23.7 0.6
pKR2037-13 7.2 2.9 36.2 15.7 13.3 1.7 22.3 0.8 pKR2037-5 6.5 2.8
36.0 14.6 15.8 1.5 22.2 0.6 pKR2037-8 7.3 2.7 36.0 15.0 14.5 1.5
22.3 0.8 pKR2037-6 6.7 2.7 33.5 17.1 14.7 1.5 23.0 0.8 pKR2037-1
7.4 2.9 33.5 18.1 14.7 1.7 20.8 0.9 pKR2037-3 6.9 2.7 25.5 22.5
18.0 1.6 21.6 1.2 pKR2037-4 7.2 2.8 22.1 24.7 18.1 1.6 21.8 1.5
pKR2037-15 7.9 3.0 19.0 26.5 20.0 1.8 20.0 1.7 Col-0 10.2 2.7 14.0
32.4 21.6 1.3 15.8 1.9
TABLE-US-00037 TABLE 22 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2038 expressing the
396b-fad2b amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2038-7 6.7 2.4 55.8 3.8 7.0 1.1 23.0 0.1 pKR2038-12 5.9 2.6 51.2
6.7 9.8 1.3 22.2 0.3 pKR2038-11 6.9 2.9 48.4 8.1 10.0 1.3 22.2 0.3
pKR2038-5 6.8 2.7 47.2 10.5 9.2 1.3 21.8 0.6 pKR2038-4 6.8 2.8 47.0
8.6 10.3 1.6 22.5 0.5 pKR2038-6 7.1 2.6 46.9 9.5 10.6 1.4 21.4 0.5
pKR2038-2 7.0 3.0 45.5 10.5 10.6 1.5 21.3 0.6 pKR2038-9 7.1 2.6
45.0 11.6 10.4 1.2 21.5 0.6 pKR2038-15 6.9 2.5 44.8 10.9 10.8 1.5
21.9 0.6 pKR2038-13 7.1 2.8 44.1 12.0 11.0 1.3 21.0 0.7 pKR2038-8
7.0 2.7 42.4 12.9 11.5 1.5 21.3 0.7 pKR2038-1 7.2 2.8 39.7 13.3
12.4 1.5 22.3 0.9 pKR2038-10 7.1 2.9 39.5 11.6 16.0 1.4 21.2 0.4
pKR2038-3 6.9 2.7 27.5 22.2 17.3 1.6 20.7 1.1 pKR2038-16 8.5 2.8
19.3 25.5 20.9 1.7 19.7 1.6 pKR2038-14 8.3 2.8 14.9 28.7 21.8 1.8
19.6 2.1 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00038 TABLE 23 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2039 expressing the
396b-fad2c amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2039-4 6.7 2.8 55.5 4.7 6.8 1.3 22.0 0.1 pKR2039-14 6.6 2.9 54.1
6.4 7.4 1.3 21.1 0.2 pKR2039-5 6.2 2.5 54.0 5.3 6.9 1.4 23.5 0.2
pKR2039-8 6.7 2.9 53.3 6.8 7.4 1.4 21.2 0.3 pKR2039-11 6.7 2.6 49.4
8.3 8.7 1.4 22.5 0.4 pKR2039-3 6.2 2.4 48.9 9.6 9.5 1.1 21.8 0.5
pKR2039-13 6.6 2.6 48.8 10.3 8.8 1.2 21.1 0.6 pKR2039-7 6.9 2.6
43.6 11.2 10.7 1.5 22.9 0.7 pKR2039-1 7.2 2.7 43.1 12.1 11.2 1.5
21.5 0.7 pKR2039-9 6.9 2.7 42.7 12.6 11.8 1.3 21.3 0.8 pKR2039-10
7.1 2.8 42.2 11.4 12.9 1.4 21.5 0.5 pKR2039-2 7.5 2.8 39.8 14.0
12.8 1.5 21.0 0.6 pKR2039-12 7.6 2.9 31.4 18.0 16.5 1.6 21.2 0.9
pKR2039-6 7.8 2.9 22.2 23.7 20.1 1.7 20.0 1.6 Col-0 10.2 2.7 14.0
32.4 21.6 1.3 15.8 1.9
TABLE-US-00039 TABLE 24 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2035 expressing the
159-fad3a amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2035-15 8.0 2.6 16.8 45.4 2.7 1.7 20.0 2.7 pKR2035-8 8.1 2.8
15.6 46.7 3.0 1.9 19.2 2.7 pKR2035-9 7.5 2.6 18.3 44.0 3.3 1.7 20.1
2.4 pKR2035-10 8.4 3.0 15.7 43.9 5.8 2.0 18.7 2.6 pKR2035-7 8.3 2.8
16.6 42.1 6.3 1.9 19.5 2.5 pKR2035-6 8.3 3.0 16.0 42.5 6.9 1.9 18.9
2.5 pKR2035-12 8.6 2.8 14.5 43.3 6.9 2.2 19.0 2.6 pKR2035-14 7.9
2.9 16.8 42.1 7.1 1.9 18.9 2.4 pKR2035-11 7.9 2.9 16.4 41.1 7.1 1.9
20.3 2.5 pKR2035-16 8.5 3.0 16.4 41.3 7.4 1.9 19.0 2.4 pKR2035-3
7.4 2.8 16.9 40.5 7.4 2.0 20.5 2.4 pKR2035-13 7.8 2.9 17.0 40.7 7.9
2.0 19.5 2.2 pKR2035-17 7.9 2.8 16.3 40.7 7.9 2.0 19.9 2.5
pKR2035-5 8.0 2.9 16.3 41.0 8.0 1.9 19.5 2.4 pKR2035-2 8.6 3.0 14.7
40.4 9.8 1.9 19.0 2.5 pKR2035-1 6.9 2.3 18.2 34.6 13.1 1.7 21.0 2.1
pKR2035-4 8.3 3.0 14.3 30.1 20.8 1.9 19.4 2.1 Col-0 10.2 2.7 14.0
32.4 21.6 1.3 15.8 1.9
TABLE-US-00040 TABLE 25 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2036 expressing the
159-fad3b amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2036-11 7.6 2.9 16.5 46.9 1.5 2.0 20.0 2.6 pKR2036-3 7.8 2.8
17.3 46.6 1.5 1.7 19.8 2.5 pKR2036-10 8.3 2.8 16.5 46.7 1.9 1.9
19.3 2.6 pKR2036-18 8.1 2.9 17.4 46.5 2.1 1.7 19.0 2.4 pKR2036-2
8.0 2.8 17.0 46.4 2.3 1.8 19.2 2.5 pKR2036-12 8.2 2.9 16.6 45.8 2.4
1.8 19.7 2.6 pKR2036-9 8.1 3.1 16.7 44.1 4.8 1.9 18.8 2.3 pKR2036-4
8.2 2.7 16.0 43.3 5.3 1.8 20.1 2.6 pKR2036-13 8.3 3.0 15.9 44.5 5.6
1.7 18.4 2.5 pKR2036-15 8.3 2.8 15.0 44.4 5.6 1.9 19.2 2.7
pKR2036-1 8.0 2.9 15.7 44.0 5.9 1.9 19.1 2.5 pKR2036-17 8.3 2.9
15.5 43.8 5.9 1.9 19.0 2.7 pKR2036-5 8.3 2.9 15.5 43.5 6.1 2.0 19.3
2.6 pKR2036-14 7.3 2.4 17.8 42.7 6.2 1.5 19.9 2.3 pKR2036-8 8.2 2.7
16.1 41.4 7.2 1.7 20.1 2.5 pKR2036-7 8.2 3.0 15.4 42.7 7.5 1.8 18.9
2.5 pKR2036-16 8.1 2.7 14.6 41.7 8.1 1.7 20.3 2.7 pKR2036-6 7.6 2.6
17.2 37.5 10.9 1.7 20.3 2.2 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8
1.9
TABLE-US-00041 TABLE 28 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2040 expressing the
396b-fad3a amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2040-14 8.6 2.9 15.1 47.9 2.4 1.8 18.6 2.8 pKR2040-8 8.8 2.9
16.0 48.0 2.4 1.7 17.8 2.5 pKR2040-17 8.4 2.8 16.5 46.3 2.8 1.8
18.8 2.6 pKR2040-3 8.1 2.8 16.7 46.1 2.9 1.8 19.1 2.5 pKR2040-16
8.5 2.7 15.4 47.4 3.1 1.7 18.5 2.6 pKR2040-10 8.5 2.9 15.8 46.3 3.8
1.8 18.5 2.5 pKR2040-2 8.6 2.9 16.5 45.9 4.1 1.7 17.9 2.4
pKR2040-13 8.3 2.8 16.7 43.7 5.6 1.8 18.7 2.4 pKR2040-5 8.2 2.7
16.2 42.3 6.5 1.7 19.8 2.5 pKR2040-9 8.3 2.8 16.3 42.5 6.8 1.8 19.0
2.4 pKR2040-15 8.3 2.9 16.2 42.5 7.1 2.0 18.5 2.5 pKR2040-1 8.3 2.7
18.5 41.3 7.3 1.6 18.2 2.1 pKR2040-11 8.5 2.8 15.9 42.6 7.4 1.6
18.7 2.4 pKR2040-6 8.4 2.9 15.2 42.9 7.6 1.8 18.7 2.5 pKR2040-7 7.5
2.5 16.8 40.9 8.6 1.8 19.5 2.4 pKR2040-4 7.9 2.8 16.5 40.9 8.8 1.8
19.0 2.3 pKR2040-12 8.0 2.8 15.2 34.3 16.4 1.7 19.4 2.2 Col-0 10.2
2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00042 TABLE 27 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2041 expressing the
396b-fad3b amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2040-14 8.6 2.9 15.1 47.9 2.4 1.8 18.6 2.8 pKR2041-4 8.2 2.8
16.8 46.8 2.3 1.8 18.8 2.5 pKR2041-2 8.1 2.8 16.9 45.6 3.3 1.9 19.1
2.4 pKR2041-11 8.0 2.8 15.9 45.6 3.8 1.9 19.4 2.5 pKR2041-7 8.5 2.9
15.5 45.7 4.8 1.7 18.3 2.5 pKR2041-1 8.4 2.8 16.1 44.8 5.8 1.7 18.0
2.4 pKR2041-6 8.6 2.7 16.4 43.5 6.1 1.7 18.7 2.3 pKR2041-8 8.2 2.9
16.9 42.3 6.4 1.7 19.2 2.4 pKR2041-10 7.8 2.6 17.9 41.9 6.5 1.8
19.3 2.2 pKR2041-5 7.6 2.5 15.6 44.2 7.0 1.7 18.7 2.7 pKR2041-3 8.7
2.9 15.5 43.6 7.2 1.9 17.9 2.4 pKR2041-9 8.3 2.8 17.1 42.0 7.3 1.9
18.5 2.2 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00043 TABLE 28 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2076 expressing the
159-faela amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2076-13 8.4 2.2 32.0 49.5 6.3 0.5 1.1 0.0 pKR2076-12 8.9 2.4
31.4 49.3 5.4 0.5 2.1 0.0 pKR2076-2 8.6 2.6 31.6 49.0 4.9 0.6 2.7
0.0 pKR2076-15 8.9 2.2 31.4 47.1 6.9 0.5 3.0 0.0 pKR2076-9 9.4 2.8
28.2 47.9 8.3 0.0 3.4 0.0 pKR2076-6 8.9 2.4 30.4 48.3 5.7 0.6 3.5
0.3 pKR2076-4 8.2 2.4 32.4 47.4 5.1 0.6 3.7 0.3 pKR2076-14 8.5 2.4
30.3 47.1 7.4 0.5 3.7 0.0 pKR2076-11 8.6 2.5 29.6 47.6 7.2 0.6 3.9
0.0 pKR2076-3 8.6 2.8 29.3 42.7 10.5 0.7 4.9 0.4 pKR2076-10 8.8 2.5
28.0 46.3 8.1 0.6 5.3 0.5 pKR2076-1 8.7 2.4 29.4 45.2 7.9 0.7 5.4
0.5 pKR2076-7 9.0 2.7 27.6 42.4 11.2 0.7 5.8 0.5 pKR2076-5 9.0 2.6
27.5 42.1 11.6 0.7 5.9 0.5 pKR2076-8 9.1 2.6 26.3 43.5 10.9 0.7 6.3
0.6 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00044 TABLE 29 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2077 expressing the
159-faela amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2077-12 8.1 2.8 30.5 34.9 21.6 0.5 1.6 0.2 pKR2077-14 9.3 2.6
27.9 36.7 20.4 0.6 2.2 0.2 pKR2077-2 9.4 2.9 25.6 37.7 21.3 0.6 2.3
0.2 pKR2077-1 9.1 3.1 25.0 37.1 21.8 0.6 3.1 0.3 pKR2077-7 9.4 2.8
23.3 37.2 21.6 0.6 4.6 0.4 pKR2077-9 9.6 2.8 22.8 36.0 23.1 0.6 4.6
0.5 pKR2077-3 9.1 2.7 24.0 36.3 22.2 0.6 4.6 0.5 pKR2077-8 9.6 2.8
23.5 35.6 22.1 0.7 5.2 0.5 pKR2077-10 8.8 2.5 25.2 35.2 21.1 0.8
5.9 0.6 pKR2077-4 9.0 3.0 24.2 35.1 21.2 0.7 6.1 0.6 pKR2077-13 9.8
2.8 21.1 36.5 21.8 0.8 6.5 0.7 pKR2077-11 9.0 3.0 23.3 35.3 20.9
0.8 6.9 0.7 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00045 TABLE 30 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2078 expressing the
159-faelc amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2078-7 9.0 2.5 29.6 36.1 20.5 0.4 1.8 0.0 pKR2078-3 9.1 2.5 30.3
36.8 19.3 0.0 1.9 0.0 pKR2078-10 8.7 2.3 29.8 37.1 19.6 0.5 2.0 0.0
pKR2078-11 8.2 2.2 31.7 37.6 17.7 0.5 2.0 0.0 pKR2078-13 9.1 2.6
29.5 38.1 18.2 0.5 2.1 0.0 pKR2078-8 8.6 2.2 32.2 38.1 15.4 0.5 2.9
0.0 pKR2078-4 8.6 1.9 26.2 35.1 23.9 0.6 3.6 0.0 pKR2078-12 8.9 2.7
29.2 36.7 18.6 0.0 3.9 0.0 pKR2078-14 9.2 2.5 27.7 37.1 19.0 0.6
4.0 0.0 pKR2078-9 8.9 2.6 26.8 37.1 18.8 0.6 4.8 0.4 pKR2078-1 8.3
2.6 28.5 36.9 17.1 0.7 5.6 0.5 pKR2078-5 8.5 2.6 28.3 37.3 16.6 0.7
5.7 0.4 pKR2078-2 9.4 2.9 26.7 34.4 19.1 0.8 6.0 0.6 pKR2078-6 9.1
2.6 22.9 34.8 18.5 1.0 10.1 1.0 Col-0 10.2 2.7 14.0 32.4 21.6 1.3
15.8 1.9
TABLE-US-00046 TABLE 31 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2079 expressing the
396b-faela amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2079-9 8.8 2.3 29.6 47.4 10.4 0.0 1.5 0.0 pKR2079-6 9.5 2.8 28.4
44.3 12.7 0.0 2.2 0.0 pKR2079-15 8.7 2.6 29.7 45.9 10.2 0.5 2.4 0.0
pKR2079-1 8.4 2.6 29.0 43.7 13.1 0.5 2.7 0.0 pKR2079-4 10.3 2.3
28.1 44.7 10.6 0.5 3.4 0.0 pKR2079-12 10.4 2.9 26.5 41.0 15.5 0.0
3.9 0.0 pKR2079-10 9.9 2.7 26.0 41.9 15.4 0.0 4.0 0.0 pKR2079-14
9.4 2.7 23.9 40.2 17.2 0.7 5.4 0.5 pKR2079-7 8.7 2.4 29.2 42.7 11.6
0.0 5.4 0.0 pKR2079-13 8.7 2.9 27.6 43.1 10.8 0.7 5.7 0.5 pKR2079-8
9.5 2.4 23.9 39.6 18.2 0.0 6.4 0.0 pKR2079-2 8.2 2.4 28.1 40.2 13.4
0.6 6.4 0.6 pKR2079-3 9.0 2.5 25.8 39.9 15.1 0.7 6.5 0.6 pKR2079-5
8.9 2.6 25.9 43.4 11.4 0.8 6.5 0.5 pKR2079-11 9.0 2.5 19.4 34.6
17.4 0.0 15.7 1.4 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00047 TABLE 32 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2081 expressing the
396b-faelb amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2081-1 9.3 2.8 28.7 36.7 20.5 0.5 1.6 0.0 pKR2081-5 9.1 2.7 29.7
37.9 18.3 0.5 1.8 0.0 pKR2081-14 8.3 2.7 29.8 36.3 19.1 0.5 3.3 0.0
pKR2081-3 8.9 2.7 26.8 36.4 20.1 0.6 4.5 0.0 pKR2081-9 9.0 2.9 18.0
33.4 17.6 1.4 16.2 1.6 pKR2081-11 8.8 2.6 18.5 33.3 17.4 1.2 16.7
1.5 pKR2081-7 8.3 2.8 18.5 33.0 17.7 1.3 16.7 1.6 pKR2081-13 8.9
2.7 17.5 32.2 18.7 1.2 17.3 1.6 pKR2081-12 8.9 2.4 17.4 32.8 18.2
1.3 17.5 1.6 pKR2081-2 8.9 2.5 16.3 33.5 18.3 1.2 17.7 1.6
pKR2081-6 8.0 2.7 18.5 32.2 18.1 1.3 17.7 1.5 pKR2081-8 9.1 2.6
16.8 33.2 17.5 1.3 17.8 1.6 pKR2081-4 8.2 2.7 17.8 32.5 17.7 1.3
18.2 1.6 pKR2081-10 8.0 2.3 18.0 32.1 18.4 1.2 18.3 1.6 Col-0 10.2
2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00048 TABLE 33 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2080 expressing the
396b-faelc amiRNA Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2
pKR2080-10 8.7 2.5 30.0 39.9 17.0 0.5 1.5 0.0 pKR2080-5 8.7 2.5
30.9 37.2 18.7 0.5 1.5 0.0 pKR2080-2 9.3 2.7 28.7 36.7 20.4 0.5 1.8
0.0 pKR2080-15 9.5 2.7 27.1 37.7 19.5 0.5 2.9 0.0 pKR2080-3 8.9 2.8
28.5 37.3 18.8 0.6 3.1 0.0 pKR2080-6 8.9 2.7 26.9 36.9 20.4 0.5 3.4
0.3 pKR2080-13 9.2 2.6 26.5 37.6 18.4 0.6 4.6 0.5 pKR2080-1 8.9 2.6
27.0 37.0 18.6 0.7 4.9 0.5 pKR2080-16 8.7 2.7 26.7 37.1 18.7 0.6
5.1 0.4 pKR2080-4 8.3 2.5 28.2 35.7 18.9 0.6 5.3 0.4 pKR2080-8 8.1
2.7 27.8 36.5 18.4 0.6 5.5 0.4 pKR2080-14 8.5 2.5 27.1 36.8 18.4
0.7 5.5 0.5 pKR2080-11 8.5 2.5 27.5 36.4 18.3 0.6 5.7 0.5 pKR2080-9
9.2 2.7 25.4 36.8 18.5 0.7 6.2 0.6 pKR2080-12 8.8 2.5 24.6 36.5
19.1 0.7 7.2 0.7 pKR2080-7 8.6 2.4 25.0 35.9 17.2 0.8 9.3 0.9 Col-0
10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
[0450] It is clear from Tables 18 to 23 that events expressing
fad2a, fad2b or fad2c amiRNAs in either the soy 159 or 396b
precursor amiRNA backbone function to suppress fad2 and thus
increase 18:1 content to as high as 57.5%, in bulk T2 seed,
compared to 14.0% for the wild-type seed.
[0451] It is also evident from Tables 24 to 27 that events
expressing fad3a or fad3b amiRNAs in either the soy 159 or 396b
precursor amiRNA backbone function to suppress fad3 and thus
decrease 18:3 to as low as 1.5%, in bulk T2 seed, compared to 21.6%
for wild-type seed.
[0452] Tables 28 to 33 show that events expressing faela, faelb or
faelc amiRNAs in either the soy 159 or 396b precursor amiRNA
backbone function to suppress fael and thus decrease 20:1 to as low
as 1.1%, in bulk T2 seed, compared to 15.8% for wild-type seed.
Fatty Acid Analysis of Bulk T3 Seed from Homozygous Plants
[0453] All T2 events for all constructs were plated on Kanamycin as
described above and those events where kanamycin resistant plants
segregated from kanamycin sensitive plants by a ratio of 3:1,
indicating a single copy insertion were analyzed for fatty acid
profile in individual seed.
[0454] Individual seed were ground in 10 uL of TMSH, 20 uL of
hexane was added and after 30 min, a portion of the heptanes phase
was injected into the GC and FAMEs were analyzed as described
above. Events where expected fatty acid phenotypes segregated 3:1
with wild-type phenotypes having the largest changes in fatty acid
profiles compared to that from wild-type seed were advanced and
plants were grown to obtain T3 seed.
[0455] T3 seed were plated on kanamycin plates as above and those
that no longer segregated for kanamycin sensitive plants were
considered homozygous and in those samples, T3 seed was analyzed
for fatty acid profile in bulk as described above for T2 seed.
Homozygous T3 seed was not obtained for pKR2079 or pKR2081 events.
Also, the pKR2077 events were planted later than the others and
therefore the T3 seed fatty acid profiles were not included
here.
[0456] Results for fatty acid profiles for homozygous T3 seed
batches for one representative event from each experiment are
presented in Tables 34 to 36 below. In each Table, fatty acids are
identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic
acid), 18:2 (linoleic acid) and 18:3 (alpha-linolenic acid), 20:0
(eicosanoic acid), 20:1 eicosenoic acid) 20:2 (eicocadienoic acid)
and 20:3 (eicosatrienoic acid) and results are expressed as a
weight percent (wt. %) of total fatty acids. For constructs
expressing a fad2 amiRNA, fatty acid profiles are sorted from
highest 18:1 to lowest. For constructs expressing a fad3 amiRNA,
fatty acids are sorted from lowest 18:3 to highest. For constructs
expressing a fael amiRNA, fatty acids are sorted from lowest to
highest 20:1. A wild-type Columbia seed batch was also run and
shown for comparison in each Table as col-0.
TABLE-US-00049 TABLE 34 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2032, pKR2033, pKR2034,
pKR2037, pKR2038 or pKR2039 having amiRNAs to fad2 Event amiRNA
16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 pKR2032-10-17 159-fad2a 7.0
2.7 57.6 5.9 6.2 0.9 18.9 0.0 pKR2032-10-10 159-fad2a 6.9 2.7 55.5
6.4 6.8 1.0 19.4 0.2 pKR2032-10-14 159-fad2a 6.8 2.6 55.2 6.4 6.8
1.0 19.7 0.2 pKR2033-8-2 159-fad2b 7.3 2.5 59.4 4.7 5.9 0.9 18.2
0.1 pKR2033-8-14 159-fad2b 7.1 2.7 59.1 4.4 5.6 1.0 19.0 0.1
pKR2033-8-9 159-fad2b 7.1 2.7 58.8 4.6 5.7 1.0 19.1 0.1
pKR2034-15-5 159-fad2c 7.4 2.7 58.6 4.3 6.4 0.9 18.8 0.0
pKR2034-15-3 159-fad2c 7.2 2.8 58.2 4.4 5.9 1.1 19.5 0.0
pKR2034-15-11 159-fad2c 6.9 2.8 58.0 4.8 6.0 1.1 19.6 0.0
pKR2037-12-15 396b-fad2a 6.9 2.5 60.4 6.0 4.5 0.9 18.1 0.0
pKR2037-12-9 396b-fad2a 6.2 2.5 58.6 5.7 4.3 1.1 20.3 0.1
pKR2037-12-11 396b-fad2a 6.7 2.4 58.1 5.9 5.2 1.0 19.8 0.0
pKR2038-4-10 396b-fad2b 0.0 0.0 64.1 0.0 0.0 0.0 35.9 0.0
pKR2038-4-9 396b-fad2b 7.6 2.7 57.3 4.6 6.8 1.0 19.0 0.1
pKR2038-4-6 396b-fad2b 7.1 2.8 57.1 4.7 6.7 1.0 19.6 0.0
pKR2039-11-4 396b-fad2c 6.6 2.7 58.5 3.5 5.3 1.1 21.0 0.1
pKR2039-11-15 396b-fad2c 6.6 2.7 58.0 3.8 5.1 1.2 21.2 0.0
pKR2039-11-14 396b-fad2c 6.2 2.5 56.7 3.7 5.5 1.2 22.7 0.1 Col-0
10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00050 TABLE 35 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2035, pKR2036, pKR2040
and pKR2041 having amiRNAs to fad3 Event amiRNA 16:0 18:0 18:1 18:2
18:3 20:0 20:1 20:2 pKR2035-7-15 159-fad3a 9.8 2.7 19.3 48.9 1.7
1.1 14.1 1.8 pKR2035-7-4 159-fad3a 8.6 2.6 18.0 47.3 1.7 1.3 16.7
2.2 pKR2035-7-9 159-fad3a 8.7 2.6 17.9 48.3 1.7 1.3 16.3 2.1
pKR2036-9-16 159-fad3b 8.0 2.5 18.7 46.7 2.0 1.3 17.2 2.0
pKR2036-9-17 159-fad3b 7.8 2.3 17.9 46.8 2.2 1.3 17.7 2.2
pKR2036-9-9 159-fad3b 7.9 2.4 17.8 46.5 2.2 1.4 17.7 2.2
pKR2040-16-13 396b-fad3a 7.6 2.4 19.5 45.0 1.6 1.5 18.4 2.1
pKR2040-16-12 396b-fad3a 8.1 2.7 18.1 46.2 1.6 1.5 17.8 2.2
pKR2040-16-3 396b-fad3a 8.2 2.4 18.0 46.9 1.7 1.4 17.5 2.2
pKR2041-7-11 396b-fad3b 7.9 2.8 19.5 45.0 1.6 1.6 17.8 2.0
pKR2041-7-3 396b-fad3b 8.2 2.9 18.5 46.1 1.5 1.6 17.2 2.2
pKR2041-7-8 396b-fad3b 7.9 3.0 18.2 45.2 1.5 1.7 18.3 2.3 Col-0
10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9
TABLE-US-00051 TABLE 36 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2076, pKR2078 or
pKR2080 having amiRNAs to fael Event amiRNA 16:0 18:0 18:1 18:2
18:3 20:0 20:1 20:2 pKR2076-15-7 159-faela 10.5 2.5 30.2 50.4 4.8
0.4 1.1 0.0 pKR2076-15-2 159-faela 9.2 3.0 28.7 51.2 5.3 0.6 1.6
0.2 pKR2076-15-1 159-faela 9.3 2.9 28.5 51.3 5.3 0.6 1.6 0.2
pKR2078-8-4 159-faelc 10.0 3.0 27.9 52.1 5.0 0.5 1.3 0.1
pKR2080-5-10 396b-faelc 9.5 3.0 27.5 37.1 20.7 0.5 1.3 0.2 Col-0
10.2 2.7 14.6 32.4 21.8 1.3 15.8 1.9
[0457] It is clear from Table 34 that events expressing fad2a,
fad2b or fad2c amiRNAs in either the soy 159 or 396b precursor
amiRNA backbone function to suppress fad2 and thus increase 18:1
content to as high as 64.1%, in bulk homozygous T3 seed, compared
to 14.0% for the wild-type seed.
[0458] It is also evident from Table 35 that events expressing
fad3a or fad3b amiRNAs in either the soy 159 or 396b precursor
amiRNA backbone function to suppress fad3 and thus decrease 18:3 to
as low as 1.5%, in bulk homozygous T3 seed, compared to 21.6% for
wild-type seed. Table 36 shows that events expressing faela or
faelc amiRNAs in the soy 159 precursor amiRNA or faelc in the soy
396b precursor amiRNA backbone function to suppress fael and thus
decrease 20:1 to as low as 1.1%, in bulk homozygous T3 seed,
compared to 15.8% for wild-type seed.
Example 8
Constructs Expressing amiRNA Combinations for Silencing Arabidopsis
and/or Brassica Fatty Acid Biosynthetic Genes
[0459] From Table 17, the amiRNA precursors 159-Atfad2a (SEQ ID NO:
136), 159-Atfad2b (SEQ ID NO: 137), 159-Atfad2c (SEQ ID NO: 138),
159-Atfad3a (SEQ ID NO: 142), 159-Atfad3b (SEQ ID NO: 143),
159-Atfaela (SEQ ID NO: 146), 159-Atfaelb (SEQ ID NO: 147) and
159-Atfaelc (SEQ ID NO: 148) are 958 nt in length and are
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 152 wherein nucleotides 276 to 296 of SEQ ID NO: 152
are replaced by the fad2a amiRNA (SEQ ID NO: 80), fad2b amiRNA (SEQ
ID NO: 83), fad2c amiRNA (SEQ ID NO: 86), fad3a amiRNA (SEQ ID NO:
89), fad3b amiRNA (SEQ ID NO: 92), fae1a amiRNA (SEQ ID NO: 95),
fae1b amiRNA (SEQ ID NO: 98) or fae1c amiRNA (SEQ ID NO: 101),
respectively and wherein nucleotides 121 to 141 of SEQ ID NO: 152
are replaced by fad2a Star Sequence (SEQ ID NO: 81), fad2b Star
Sequence (SEQ ID NO: 84), fad2c Star Sequence (SEQ ID NO: 87),
fad3a Star Sequence (SEQ ID NO: 90), fad3b Star Sequence (SEQ ID
NO: 93), fae1a Star Sequence (SEQ ID NO: 96), fae1b Star Sequence
(SEQ ID NO: 99) or fae1c Star Sequence (SEQ ID NO: 102),
respectively.
[0460] From Table 17, the amiRNA precursors 396b-Atfad2a (SEQ ID
NO: 139), 396b-Atfad2b (SEQ ID NO: 140), 396b-Atfad2c (SEQ ID NO:
141), 396b-Atfad3a (SEQ ID NO: 144), 396b-Atfad2b (SEQ ID NO: 145),
396b-faela (SEQ ID NO: 149), 396b-faelb (SEQ ID NO: 150) and
396b-faelc (SEQ ID NO: 151) are 574 nt in length and are
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 153 wherein nucleotides 196 to 216 of SEQ ID NO: 153
are replaced by the fad2a amiRNA (SEQ ID NO: 80), fad2b amiRNA (SEQ
ID NO: 83), fad2c amiRNA (SEQ ID NO: 86), fad3a amiRNA (SEQ ID NO:
89), fad3b amiRNA (SEQ ID NO: 92), fae1a amiRNA (SEQ ID NO: 95),
fae1b amiRNA (SEQ ID NO: 98) or fae1c amiRNA (SEQ ID NO: 101),
respectively and wherein nucleotides 262 to 282 of SEQ ID NO: 153
are replaced by the fad2a Star Sequence (SEQ ID NO: 82), fad2b Star
Sequence (SEQ ID NO: 85), fad2c Star Sequence (SEQ ID NO: 88),
fad3a Star Sequence (SEQ ID NO: 91), fad3b Star Sequence (SEQ ID
NO: 94), 396b-fae1a Star Sequence (SEQ ID NO: 97), 396b-fae1b Star
Sequence (SEQ ID NO: 100) or 396b-fae1c Star Sequence (SEQ ID NO:
103), respectively.
[0461] Using cloning methods familiar to one skilled in the art
(e.g. PCR, restriction enzyme digestion, etc.), individual amiRNA
precursors targeting either fad2 and fad3, shown in Table 17, were
combined together into a single transcriptional unit such that both
amiRNA precursors were expressed together downstream of the single
beta-conglycinin promoter. In some cases, a third amiRNA precursor
targeting fae1 was also combined with the fad2 and fad3 amiRNA
precursors to generate triple amiRNA units targeting all three
genes. In each case, the full cassette including the
beta-conglycinin promoter, the multiple amiRNA and the phaseolin
transcription terminator were flanked by AscI sites to enable
cloning into other expression vectors.
[0462] These plasmids were then digested with AscI and the fragment
containing the amiRNA expression cassette was sub-cloned into the
AscI site of expression vector pKR92, which was previously
described in WO2007/06110109 (published on May 31, 2007, the
contents of which are herein incorporated by reference).
[0463] The amiRNA combinations made and the corresponding
expression vector sequences are described in Table 37.
TABLE-US-00052 TABLE 37 Precursor amiRNA combinations and amiRNA
Expression Vectors For Arabidopsis and/or Brassica Fatty Acid
Biosynthetic Gene Sequences Targeted for Silencing amiRNA amiRNA
Precursor Plasmid Plasmid Precursor SEQ ID NO Name SEQ ID NO 159-
154 pKR2232 155 Atfad2a/396b- Atfad3b amiRNA 159- 156 pKR2233 157
Atfad2b/396b- Atfad3b amiRNA 396b- 158 pKR2234 159 Atfad3b/159-
Atfad2a amiRNA 396b- 160 pKR2235 161 Atfad3b/159- Atfad2b amiRNA
159- 162 pKR2248 163 Atfad2a/396b- Atfad3b/159- Atfae1a amiRNA
396b- 164 pKR2249 165 Atfad3b/159- Atfad2a/159- fae1a amiRNA 159-
166 pKR2250 167 Atfad2b/396b- Atfad3b/159- Atfae1a amiRNA 396b- 168
pKR2251 169 Atfad3b/159- Atfad2b/159- Atfae1a amiRNA
[0464] From Table 37, the amiRNA precursor 159-Atfad2a/396b-Atfad3b
(SEQ ID NO: 154), which combines amiRNA precursors 159-Atfad2a (SEQ
ID NO: 136) and 396b-Atfad3b (SEQ ID NO: 143) into one
transcriptional unit, is 1556 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 16 to 974 of 159-Atfad2a/396b-Atfad3b) wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2a amiRNA (SEQ ID NO: 80) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2a amiRNA Star
Sequence (SEQ ID NO: 81). The amiRNA precursor
159-Atfad2a/396b-Atfad3b (SEQ ID NO: 154) is also substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 982 to 1555 of 159-Atfad2a/396b-Atfad3b) wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94).
[0465] From Table 37, the amiRNA precursor 159-Atfad2b/396b-Atfad3b
(SEQ ID NO: 156), which combines amiRNA precursors 159-Atfad2b (SEQ
ID NO: 137) and 396b-Atfad3b (SEQ ID NO: 143) into one
transcriptional unit, is 1556 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 16 to 974 of 159-Atfad2b/396b-Atfad3b) wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2b amiRNA (SEQ ID NO: 83) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2b amiRNA Star
Sequence (SEQ ID NO: 84). The amiRNA precursor
159-Atfad2b/396b-Atfad3b (SEQ ID NO: 156) is also substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 982 to 1555 of 159-Atfad2b/396b-Atfad3b) wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94).
[0466] From Table 37, the amiRNA precursor 396b-Atfad3b/159-Atfad2a
(SEQ ID NO: 158), which combines amiRNA precursors and 396b-Atfad3b
(SEQ ID NO: 143) and 159-Atfad2a (SEQ ID NO: 136) into one
transcriptional unit, is 1556 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 7 to 581 of 396b-Atfad3b/159-Atfad2a) wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94). The amiRNA precursor
396b-Atfad3b/159-Atfad2a (SEQ ID NO: 158) is also substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 588 to 1546 of 396b-Atfad3b/159-Atfad2a) wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2a amiRNA (SEQ ID NO: 80) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2a amiRNA Star
Sequence (SEQ ID NO: 81).
[0467] From Table 37, the amiRNA precursor 396b-Atfad3b/159-Atfad2b
(SEQ ID NO: 160), which combines amiRNA precursors and 396b-Atfad3b
(SEQ ID NO: 143) and 159-Atfad2b (SEQ ID NO: 137) into one
transcriptional unit, is 1556 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 7 to 581 of 396b-Atfad3b/159-Atfad2b) wherein
nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94). The amiRNA precursor
396b-Atfad3b/159-Atfad2b (SEQ ID NO: 160) is also substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 588 to 1546 of 396b-Atfad3b/159-Atfad2b) wherein
nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2b amiRNA (SEQ ID NO: 83) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2b amiRNA Star
Sequence (SEQ ID NO: 84).
[0468] From Table 37, the amiRNA precursor
159-Atfad2a/396b-Atfad3b/159-Atfae1a (SEQ ID NO: 162), which
combines amiRNA precursors 159-Atfad2a (SEQ ID NO: 136),
396b-Atfad3b (SEQ ID NO: 143) and 159-Atfae1a (SEQ ID NO: 146) into
one transcriptional unit, is 2530 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 25 to 983 of 159-Atfad2a/396b-Atfad3b/159-Atfae1a)
wherein nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2a amiRNA (SEQ ID NO: 80) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2a amiRNA Star
Sequence (SEQ ID NO: 81). The amiRNA precursor
159-Atfad2a/396b-Atfad3b/159-Atfae1a (SEQ ID NO: 162) is also
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 153 (from nt 991 to 1564 of
159-Atfad2a/396b-Atfad3b/159-Atfae1a) wherein nucleotides 196 to
216 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA (SEQ ID
NO: 92) and wherein nucleotides 262 to 282 of SEQ ID NO: 153 are
replaced by 396b-Atfad3b amiRNA Star Sequence (SEQ ID NO: 94). The
amiRNA precursor 159-Atfad2a/396b-Atfad3b/159-Atfae1a (SEQ ID NO:
162) is also substantially similar to the deoxyribonucleotide
sequence set forth in SEQ ID NO: 152 (from nt 1571 to 2529 of
159-Atfad2a/396b-Atfad3b/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfae1a amiRNA (SEQ ID
NO: 95) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-Atfae1a amiRNA Star Sequence (SEQ ID NO: 96).
[0469] From Table 37, the amiRNA precursor
396b-Atfad3b/159-Atfad2a/159-Atfae1a (SEQ ID NO: 164), which
combines amiRNA precursors and 396b-Atfad3b (SEQ ID NO: 143),
159-Atfad2a (SEQ ID NO: 136) and 159-Atfae1a (SEQ ID NO: 146) into
one transcriptional unit, is 2530 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 16 to 590 of 396b-Atfad3b/159-Atfad2a/159-Atfae1a)
wherein nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94). The amiRNA precursor
396b-Atfad3b/159-Atfad2a/159-Atfae1a (SEQ ID NO: 164) is also
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 152 (from nt 597 to 1555 of
396b-Atfad3b/159-Atfad2a/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfad2a amiRNA (SEQ ID
NO: 80) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-Atfad2a amiRNA Star Sequence (SEQ ID NO: 81). The
amiRNA precursor 396b-Atfad3b/159-Atfad2a/159-Atfae1a (SEQ ID NO:
164) is also substantially similar to the deoxyribonucleotide
sequence set forth in SEQ ID NO: 152 (from nt 1571 to 2529 of
396b-Atfad3b/159-Atfad2a/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfae1a amiRNA (SEQ ID
NO: 95) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-Atfae1a amiRNA Star Sequence (SEQ ID NO: 96).
[0470] From Table 37, the amiRNA precursor
159-Atfad2b/396b-Atfad3b/159-Atfae1a (SEQ ID NO: 166), which
combines amiRNA precursors 159-Atfad2b (SEQ ID NO: 137),
396b-Atfad3b (SEQ ID NO: 143) and 159-Atfae1a (SEQ ID NO: 146) into
one transcriptional unit, is 2530 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
152 (from nt 25 to 983 of 159-Atfad2b/396b-Atfad3b/159-Atfae1a)
wherein nucleotides 276 to 296 of SEQ ID NO: 152 are replaced by
159-Atfad2b amiRNA (SEQ ID NO: 83) and wherein nucleotides 121 to
141 of SEQ ID NO: 152 are replaced by 159-Atfad2a amiRNA Star
Sequence (SEQ ID NO: 84). The amiRNA precursor
159-Atfad2b/396b-Atfad3b/159-Atfae1a (SEQ ID NO: 166) is also
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 153 (from nt 991 to 1564 of
159-Atfad2b/396b-Atfad3b/159-Atfae1a) wherein nucleotides 196 to
216 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA (SEQ ID
NO: 92) and wherein nucleotides 262 to 282 of SEQ ID NO: 153 are
replaced by 396b-Atfad3b amiRNA Star Sequence (SEQ ID NO: 94). The
amiRNA precursor 159-Atfad2b/396b-Atfad3b/159-Atfae1a (SEQ ID NO:
166) is also substantially similar to the deoxyribonucleotide
sequence set forth in SEQ ID NO: 152 (from nt 1571 to 2529 of
159-Atfad2b/396b-Atfad3b/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfae1a amiRNA (SEQ ID
NO: 95) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-Atfae1a amiRNA Star Sequence (SEQ ID NO: 96).
[0471] From Table 37, the amiRNA precursor
396b-Atfad3b/159-Atfad2b/159-Atfae1a (SEQ ID NO: 168), which
combines amiRNA precursors and 396b-Atfad3b (SEQ ID NO: 143),
159-Atfad2b (SEQ ID NO: 137) and 159-Atfae1a (SEQ ID NO: 146) into
one transcriptional unit, is 2530 nt in length and is substantially
similar to the deoxyribonucleotide sequence set forth in SEQ ID NO:
153 (from nt 16 to 590 of 396b-Atfad3b/159-Atfad2b/159-Atfae1a)
wherein nucleotides 196 to 216 of SEQ ID NO: 153 are replaced by
396b-Atfad3b amiRNA (SEQ ID NO: 92) and wherein nucleotides 262 to
282 of SEQ ID NO: 153 are replaced by 396b-Atfad3b amiRNA Star
Sequence (SEQ ID NO: 94). The amiRNA precursor
396b-Atfad3b/159-Atfad2b/159-Atfae1a (SEQ ID NO: 168) is also
substantially similar to the deoxyribonucleotide sequence set forth
in SEQ ID NO: 152 (from nt 597 to 1555 of
396b-Atfad3b/159-Atfad2b/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfad2b amiRNA (SEQ ID
NO: 83) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-Atfad2b amiRNA Star Sequence (SEQ ID NO: 84). The
amiRNA precursor 396b-Atfad3b/159-Atfad2b/159-Atfae1a (SEQ ID NO:
168) is also substantially similar to the deoxyribonucleotide
sequence set forth in SEQ ID NO: 152 (from nt 1571 to 2529 of
396b-Atfad3b/159-Atfad2b/159-Atfae1a) wherein nucleotides 276 to
296 of SEQ ID NO: 152 are replaced by 159-Atfae1a amiRNA (SEQ ID
NO: 95) and wherein nucleotides 121 to 141 of SEQ ID NO: 152 are
replaced by 159-faea amiRNA Star Sequence (SEQ ID NO: 96).
[0472] In addition to the double amiRNA precursors targeting fad2
and fad3 and the triple amiRNA precursors targeting fad2, fad3 and
fae1, constructs were made where a double amiRNA precursor
targeting fad2 and fad3 was placed downstream of the
beta-conglycinin promoter and a second amiRNA precursor targeting
fae1 was placed in a separate cassette downstream of the soy
glycinin Gy1 promoter (Chen, Z-L, et al., EMBO J. 7:297-302
(1988)),).
[0473] The amiRNA combinations made and the corresponding vector
sequences are described in Table 38.
TABLE-US-00053 TABLE 38 Precursor amiRNA combinations and amiRNA
Expression Vectors For Arabidopsis and/or Brassica Fatty Acid
Biosynthetic Gene Sequences Targeted for Silencing amiRNA amiRNA
Precursor 1 Precursor 2 (beta- amiRNA (glycinin amiRNA conglycinin
Precursor 1 Gy1 Precursor 2 Plasmid Plasmid promoter) SEQ ID NO
promoter) SEQ ID NO Name SEQ ID NO 159- 154 159-faeIa 146 pKR2333
170 Atfad2a/396b- amiRNA Atfad3b amiRNA 159- 156 159-faeIa 146
pKR2334 171 Atfad2b/396b- amiRNA Atfad3b amiRNA 396b- 158 159-faeIa
146 pKR2335 172 Atfad3b/159- amiRNA Atfad2a amiRNA 396b- 160
159-faeIa 146 pKR2336 173 Atfad3b/159- amiRNA Atfad2b amiRNA
Example 9
Phenotypic Analysis of Arabidopsis Seed Expressing amiRNA
Combinations for Silencing Arabidopsis and/or Brassica Fatty Acid
Biosynthetic Genes
Transformation of Arabidopsis Plants
[0474] Plasmids listed in Table 37 or 38 (Example 8) were
introduced into Agrobacterium tumefaciens NTL4 (Luo et al,
Molecular Plant--Microbe Interactions (2001) 14(1):98-103) and
Arabidopsis plants were repeatedly dipped into the respective
Agrobacterium suspensions exactly as described herein. Plants were
maintained, T1 seeds were harvested and transformants selected for
each experiment also exactly as described herein.
[0475] Further, all T2 and T3 seed from events selected were
harvested and analyzed exactly as herein.
Fatty Acid Analysis of Bulk T2 Seed
[0476] Fatty acid profiles for approximately 10-20 events for each
experiment (plants transformed with constructs listed in Table 37)
are presented in Tables 39 to 41 below. In each Table, fatty acids
are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1
(oleic acid), 18:2 (linoleic acid) and 18:3 (alpha-linolenic acid),
20:0 (eicosanoic acid), 20:1 eicosenoic acid) 20:2 (eicocadienoic
acid) and 20:3 (eicosatrienoic acid) and results are expressed as a
weight percent (wt. %) of total fatty acids. Fatty acid profiles
are sorted from highest 18:1 to lowest. A wild-type Columbia seed
batch was also run and shown for comparison in each Table as
col-0.
TABLE-US-00054 TABLE 39 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2232, pKR2233, pKR2234
or pKR2235 expressing the double amiRNAs targeting fad2 and fad3
Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 Col-0 10.2 2.7 14.0
32.4 21.6 1.3 15.8 1.9 pKR2232-1 6.9 2.6 55.9 10.5 3.1 1.0 19.7 0.2
pKR2232-6 6.7 2.6 54.7 10.7 2.7 1.2 21.2 0.2 pKR2232-2 6.4 2.5 53.9
11.3 3.8 1.1 20.8 0.3 pKR2232-3 6.5 2.6 53.4 11.3 4.4 1.0 20.5 0.3
pKR2232-8 6.3 2.2 53.3 12.3 4.6 1.0 20.1 0.3 pKR2232-4 7.2 2.6 49.9
13.1 4.9 1.2 20.8 0.4 pKR2232-5 6.9 2.5 49.1 14.5 5.1 1.1 20.4 0.5
pKR2232-7 7.3 2.6 40.5 19.1 8.9 1.2 19.6 0.8 pKR2233-5 6.4 2.4 60.1
7.2 2.0 1.0 20.6 0.1 pKR2233-3 7.0 2.7 58.1 8.7 2.4 1.0 20.0 0.1
pKR2233-1 6.1 2.7 58.1 8.1 2.0 1.2 21.6 0.2 pKR2233-9 7.4 2.7 54.8
10.6 3.0 1.1 20.2 0.2 pKR2233-8 6.5 2.2 54.2 10.4 4.5 1.0 20.9 0.3
pKR2233-10 6.6 2.5 53.2 11.1 4.6 1.1 20.5 0.4 pKR2233-2 6.4 2.8
52.3 11.0 4.6 1.3 21.2 0.4 pKR2233-11 7.1 2.7 49.2 14.0 4.1 1.2
21.3 0.4 pKR2233-6 6.8 2.7 46.8 15.6 6.5 1.2 19.8 0.6 pKR2233-7 7.3
2.9 45.5 16.2 7.2 1.1 19.2 0.6 pKR2233-4 7.2 2.4 43.0 17.8 7.1 1.2
20.6 0.7 pKR2234-1 5.8 2.5 52.5 13.4 1.4 1.2 22.9 0.3 pKR2234-9 6.8
2.8 49.5 17.3 1.7 1.2 20.4 0.3 pKR2234-5 7.1 2.6 47.4 18.5 2.4 1.2
20.4 0.4 pKR2234-10 7.2 2.9 47.3 17.5 2.7 1.2 20.7 0.4 pKR2234-13
7.3 2.6 45.2 20.5 1.9 1.2 21.0 0.5 pKR2234-6 7.4 3.0 43.7 19.4 5.0
1.2 19.7 0.6 pKR2234-11 7.3 2.8 40.6 21.2 5.3 1.3 20.8 0.7
pKR2234-4 7.6 2.8 37.9 24.3 6.1 1.2 19.3 0.7 pKR2234-7 7.7 2.7 37.3
24.4 6.2 1.3 19.5 0.8 pKR2234-8 7.5 2.8 36.8 24.9 5.9 1.3 19.9 0.8
pKR2234-3 8.1 2.7 33.9 28.5 5.5 1.3 19.1 0.9 pKR2234-12 8.5 2.7
28.4 31.0 7.3 1.2 19.8 1.1 pKR2234-2 8.6 2.8 22.3 34.9 8.4 1.5 20.2
1.3 pKR2234-14 8.0 2.9 18.1 41.9 6.6 1.6 18.9 2.1 pKR2235-6 6.3 2.6
53.0 12.9 1.7 1.2 22.1 0.3 pKR2235-10 6.7 2.7 52.0 15.6 2.5 1.0
19.2 0.3 pKR2235-5 6.1 2.7 52.1 12.9 1.6 1.3 22.9 0.3 pKR2235-4 6.2
2.6 48.0 16.2 2.5 1.3 22.8 0.4 pKR2235-8 8.2 2.8 45.6 20.5 3.0 1.1
18.3 0.4 pKR2235-12 7.6 2.8 45.4 19.1 4.2 1.1 19.3 0.5 pKR2235-7
6.3 3.0 45.5 16.8 4.0 1.5 22.4 0.6 pKR2235-9 7.3 2.5 45.0 19.3 5.0
1.0 19.4 0.5 pKR2235-11 7.4 2.7 44.5 19.6 5.0 1.1 19.1 0.5
pKR2235-13 7.5 2.8 44.2 20.9 3.6 1.2 19.2 0.6 pKR2235-15 7.2 2.8
42.9 18.8 6.6 1.2 19.8 0.7 pKR2235-1 6.7 2.9 41.3 19.8 6.0 1.4 21.0
0.7 pKR2235-16 7.7 2.7 38.6 21.5 7.2 1.2 20.3 0.8 pKR2235-14 7.9
2.9 37.4 22.7 8.8 1.2 18.4 0.8 pKR2235-2 7.6 2.8 36.4 24.9 6.4 1.4
19.8 0.8 pKR2235-3 7.9 2.8 20.0 31.1 16.8 1.5 18.4 1.6
[0477] From Table 39, events expressing double fad2/fad3 amiRNAs
function to suppress both fad2 and fad3 and increase 18:1 content
to as high as 60.1%, in bulk T2 seed, compared to 14.0% for the
wild-type seed, with a decrease in 18:3 content to as low as 2.0%,
compared to 21.6% for the wild-type seed. Further, the increase in
18:1 was higher than in events expressing a fad2 only amiRNA where
the highest 18:1 obtained was 57.5% (Table 19) in T2 seed. Also,
the decrease in 18:3 was more pronounced than observed in the best
fad2 amiRNA-only event, where the lowest 18:3 obtained was 5.9%
(Table 19) and is comparable to the best fad3 amiRNA-only amiRNA
event where the lowest 18:3 obtained was 1.5% Table 25).
[0478] It is interesting to note that in the event having 60.1%
18:1 and 2% 18:3 (Table 39), the 20:1 content is increased to 20.6%
from 15.8% observed in the wt seed.
TABLE-US-00055 TABLE 40 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2248, pKR2249, pKR2250
or pKR2251 expressing the triple amiRNAs targeting fad2, fad3 and
fael. Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 Col-0 10.2 2.7
14.0 32.4 21.6 1.3 15.8 1.9 pKR2248-17 6.8 2.2 71.7 10.7 3.5 0.6
4.3 0.2 pKR2248-6 8.1 2.7 68.6 12.3 3.5 0.6 4.1 0.1 pKR2248-18 8.0
2.7 66.8 12.8 4.2 0.6 4.7 0.2 pKR2248-15 7.5 2.5 65.4 14.1 4.0 0.7
5.5 0.3 pKR2248-4 8.2 2.8 64.4 13.0 4.8 0.8 5.6 0.4 pKR2248-12 8.1
2.6 58.7 16.4 6.0 0.7 6.9 0.5 pKR2248-11 7.7 2.4 58.2 16.3 6.7 0.8
7.4 0.5 pKR2248-1 7.4 2.5 58.0 15.8 6.6 0.8 8.3 0.5 pKR2248-3 7.4
2.7 57.6 15.6 7.1 0.9 8.1 0.6 pKR2248-2 8.3 2.7 56.6 17.7 6.1 0.7
7.4 0.4 pKR2248-13 7.7 2.5 56.3 17.2 6.9 0.9 8.1 0.5 pKR2248-7 7.4
2.7 55.7 16.6 7.5 0.9 8.7 0.6 pKR2248-14 7.9 2.6 54.9 17.0 8.3 0.9
7.8 0.6 pKR2248-8 7.9 2.9 53.3 18.1 7.2 0.9 9.2 0.6 pKR2248-9 7.4
2.6 51.9 18.3 8.3 1.1 9.7 0.8 pKR2248-10 7.6 3.0 48.4 20.4 7.9 1.1
10.8 0.7 pKR2248-16 9.1 2.6 46.1 22.4 8.9 1.0 9.1 0.7 pKR2248-5 8.4
2.5 46.2 22.4 8.6 1.0 10.3 0.7 pKR2249-10 7.3 2.5 67.3 16.0 1.6 0.7
4.4 0.1 pKR2249-6 8.3 2.8 64.0 17.7 3.0 0.6 3.3 0.2 pKR2249-12 6.8
2.8 63.5 16.1 3.0 0.9 6.6 0.3 pKR2249-2 8.4 3.0 59.4 19.8 2.3 0.7
6.2 0.2 pKR2249-1 8.0 2.6 56.2 20.1 5.1 0.7 6.8 0.4 pKR2249-4 7.9
2.8 55.5 19.8 5.6 0.8 7.2 0.5 pKR2249-7 7.9 2.7 52.0 22.0 5.5 0.8
8.5 0.6 pKR2249-3 7.9 2.7 47.8 24.6 6.0 0.9 9.6 0.6 pKR2249-11 7.1
2.9 45.4 24.4 6.0 1.1 12.4 0.7 pKR2249-8 9.2 2.6 43.7 28.1 6.5 0.8
8.5 0.6 pKR2249-9 9.2 2.5 42.9 31.8 4.0 0.7 8.6 0.5 pKR2249-16 8.7
3.0 42.4 29.8 4.1 0.7 10.8 0.6 pKR2249-5 8.2 2.7 39.4 30.5 5.7 0.9
11.9 0.7 pKR2249-14 9.2 3.0 32.3 29.3 11.7 1.1 12.4 1.1 pKR2249-15
8.6 2.6 28.3 36.2 7.3 1.1 14.7 1.1 pKR2249-13 8.0 2.9 16.9 29.9
18.4 1.8 20.1 2.0 pKR2250-1 6.8 2.6 72.2 9.8 2.9 0.7 4.8 0.1
pKR2250-11 7.0 2.7 71.6 9.7 3.4 0.7 4.6 0.2 pKR2250-12 7.1 2.9 70.9
10.0 3.4 0.8 4.8 0.2 pKR2250-15 7.3 2.8 69.7 10.5 3.3 0.8 5.2 0.2
pKR2250-16 7.1 3.0 69.0 11.6 3.2 0.9 5.1 0.2 pKR2250-2 7.8 2.7 66.0
13.5 3.8 0.8 5.1 0.2 pKR2250-4 7.7 3.1 63.1 12.9 5.4 0.9 6.5 0.4
pKR2250-8 7.9 2.8 62.2 14.3 6.2 0.6 5.6 0.4 pKR2250-9 7.2 2.7 60.7
14.5 5.6 0.9 7.9 0.5 pKR2250-14 7.4 3.0 60.5 14.0 6.1 1.0 7.4 0.5
pKR2250-7 7.0 3.0 60.0 14.4 5.9 1.0 8.3 0.5 pKR2250-3 7.0 2.6 59.7
14.7 6.1 1.0 8.2 0.6 pKR2250-5 7.5 2.6 57.1 16.8 6.3 1.0 8.2 0.6
pKR2250-10 9.3 2.7 51.9 19.7 7.9 0.7 7.3 0.5 pKR2250-13 6.9 2.8
50.7 18.2 8.4 1.0 11.1 0.8 pKR2250-6 8.2 2.7 48.7 20.1 8.0 1.1 10.4
0.7 pKR2251-3 6.9 2.2 71.9 12.2 2.2 0.6 3.8 0.1 pKR2251-1 7.0 2.6
67.3 14.5 2.4 0.7 5.2 0.2 pKR2251-11 6.6 2.6 67.3 13.6 2.7 0.7 6.3
0.2 pKR2251-4 7.5 2.7 66.8 14.1 2.4 0.6 5.6 0.2 pKR2251-12 7.7 3.2
62.8 16.0 2.3 0.8 7.1 0.2 pKR2251-5 7.0 2.4 59.4 18.0 3.8 0.7 8.4
0.4 pKR2251-9 7.3 2.7 57.3 17.6 5.3 0.9 8.4 0.5 pKR2251-17 7.8 2.7
55.8 18.6 5.3 0.8 8.5 0.5 pKR2251-14 7.3 2.7 55.5 19.1 4.7 0.9 9.3
0.5 pKR2251-10 7.9 3.0 54.4 18.6 6.3 0.9 8.5 0.6 pKR2251-18 7.8 3.2
53.7 19.8 4.4 0.9 9.8 0.5 pKR2251-13 7.5 2.6 52.0 20.2 6.0 0.9 10.2
0.6 pKR2251-15 7.2 2.9 51.1 21.3 4.9 1.0 11.1 0.5 pKR2251-8 7.8 2.9
49.8 20.7 7.0 1.1 10.0 0.7 pKR2251-16 7.8 3.0 49.1 20.5 7.6 1.1
10.2 0.7 pKR2251-7 7.2 2.9 47.1 22.8 6.5 1.0 11.9 0.7 pKR2251-2 8.1
2.8 45.1 24.2 6.8 0.8 11.4 0.7 pKR2251-6 7.7 3.0 38.9 27.9 6.2 1.1
14.4 0.8
[0479] From Table 40, events expressing triple fad2/fad3/fae1
amiRNAs function to suppress fad2, fad3 and fae1 and increase 18:1
content to as high as 72%, in bulk T2 seed, compared to 14.0% for
the wild-type seed, with a decrease in 18:3 content to as low as
2%, compared to 21.6% for the wild-type seed and decrease in 20:1
to as low as 4% compared to 15.8% in wild-type seed. Further, the
increase in 18:1 was higher than in events expressing a fad2/fad3
double amiRNA where the highest 18:1 obtained was 60.1% in T2 seed.
Also, the decrease in 18:3 was similar to the lowest 18:3 in either
the fad3 amiRNA-only or fad2/fad3 double amiRNA events (1.5%-2%).
In addition, the decrease in 20:1 to 4% is lower than significantly
lower than that observed in the best double fad2/fad3 double amiRNA
event (where 20:1 had actually increased to 20.6%.
TABLE-US-00056 TABLE 41 Fatty acid profiles of lipid from bulk T2
seed samples for events transformed with pKR2333, pKR2334, pKR2335
or pKR2336 expressing one double amiRNAs targeting fad2 and fad3
and another amiRNA targeting fael. Event 16:0 18:0 18:1 18:2 18:3
20:0 20:1 20:2 Col-0 10.2 2.7 14.0 32.4 21.6 1.3 15.8 1.9 pKR2333-1
8.1 3.0 68.3 12.5 4.4 0.6 3.0 0.0 pKR2333-13 9.9 4.6 57.7 14.7 5.9
0.9 5.7 0.6 pKR2333-11 9.1 4.2 57.6 15.2 6.2 0.9 6.7 0.0 pKR2333-18
8.5 3.6 16.9 31.4 18.0 1.8 17.7 2.0 pKR2333-12 9.2 3.7 16.8 32.0
18.3 1.6 16.5 1.9 pKR2333-7 8.3 3.4 16.5 32.4 18.2 1.8 17.5 1.9
pKR2333-5 9.7 4.4 16.3 31.7 17.5 1.7 16.9 1.9 pKR2333-2 9.0 3.2
16.4 32.3 19.0 1.6 16.6 1.9 pKR2333-16 8.4 3.5 16.2 31.3 19.2 1.9
17.4 2.1 pKR2333-6 9.0 3.7 16.2 31.6 18.5 1.6 17.5 2.0 pKR2333-15
9.0 3.6 16.1 32.0 18.2 1.6 17.6 1.9 pKR2333-8 9.6 3.9 16.0 32.5
18.6 1.7 15.9 1.9 pKR2333-3 8.7 3.3 16.0 33.1 18.7 1.7 16.7 1.9
pKR2333-14 9.1 3.3 16.0 32.0 19.2 1.4 16.9 2.0 pKR2333-4 8.8 3.2
15.9 32.6 19.1 1.6 16.9 1.9 pKR2333-10 9.5 3.9 15.9 32.0 18.7 1.7
16.2 2.0 pKR2333-17 9.5 4.4 15.2 31.4 18.0 2.0 17.5 2.1 pKR2333-9
9.6 4.0 14.5 32.3 19.2 2.0 16.5 1.9 pKR2334-13 8.9 3.9 52.0 16.6
7.6 1.1 9.1 0.8 pKR2334-9 0.0 0.0 44.2 55.8 0.0 0.0 0.0 0.0
pKR2334-14 8.3 3.3 17.9 31.2 17.4 1.7 18.2 2.0 pKR2334-10 7.9 3.6
17.9 30.9 16.1 2.1 19.5 1.9 pKR2334-15 8.1 3.7 17.1 31.2 17.2 2.0
19.0 1.8 pKR2334-4 8.3 3.0 16.4 32.2 18.9 1.6 17.7 1.9 pKR2334-6
8.7 3.6 16.4 31.8 18.6 1.8 17.2 2.0 pKR2334-3 8.9 3.9 16.2 32.2
17.7 1.8 17.4 1.9 pKR2334-12 8.6 3.8 16.3 31.0 18.0 2.1 18.2 2.0
pKR2334-16 8.5 3.4 16.0 31.5 18.7 1.7 18.2 2.0 pKR2334-18 8.5 3.5
16.1 31.1 18.7 1.8 18.4 2.0 pKR2334-8 9.2 3.7 15.9 31.9 18.6 1.6
17.1 1.9 pKR2334-1 9.0 3.6 15.8 32.8 18.4 1.9 16.5 1.9 pKR2334-11
8.6 3.8 15.7 31.6 17.9 2.0 18.5 1.9 pKR2334-17 9.3 4.0 15.5 31.8
17.3 1.8 18.4 1.9 pKR2334-7 9.6 4.4 15.2 31.0 18.2 2.0 17.7 1.9
pKR2334-2 8.7 3.3 15.1 32.4 18.8 1.7 18.1 2.0 pKR2334-5 9.1 3.5
15.0 32.9 18.8 1.7 16.9 1.9 pKR2335-6 8.4 3.0 64.8 19.1 2.1 0.6 1.9
0.0 pKR2335-9 8.7 3.6 59.6 22.3 1.7 0.7 3.2 0.2 pKR2335-2 8.9 3.1
50.9 23.1 6.1 0.9 6.3 0.6 pKR2335-12 8.7 3.5 50.7 23.0 5.6 0.9 7.0
0.6 pKR2335-13 8.5 3.4 50.8 22.8 5.8 0.9 7.2 0.6 pKR2335-15 8.6 3.5
48.0 25.2 6.1 1.0 7.1 0.7 pKR2335-5 9.3 3.6 47.9 23.9 6.5 0.9 7.3
0.7 pKR2335-7 8.8 3.2 47.2 26.2 5.8 0.9 7.3 0.6 pKR2335-1 9.5 4.0
47.0 23.3 6.8 1.0 7.5 0.8 pKR2335-10 8.4 3.6 45.9 26.0 6.7 1.1 7.7
0.7 pKR2335-3 9.5 3.7 45.1 29.9 4.6 0.7 5.9 0.6 pKR2335-4 8.6 3.3
44.9 29.8 5.5 0.8 6.4 0.6 pKR2335-14 8.5 3.5 44.9 26.0 6.9 1.1 8.4
0.8 pKR2335-16 8.7 3.5 44.8 25.3 6.8 1.0 8.9 0.8 pKR2335-17 9.3 4.4
43.7 26.2 6.4 1.1 8.2 0.7 pKR2335-8 8.3 3.4 43.7 23.4 9.5 1.1 9.6
1.0 pKR2335-11 8.3 3.4 43.5 28.2 6.7 0.9 8.2 0.7 pKR2336-16 7.5 3.1
64.9 17.8 2.6 0.7 3.2 0.2 pKR2336-6 7.7 2.9 63.7 18.6 2.7 0.6 3.8
0.0 pKR2336-11 7.7 3.1 62.6 18.7 3.3 0.7 3.6 0.2 pKR2336-8 7.0 3.0
60.8 15.7 3.1 0.8 9.2 0.4 pKR2336-9 8.5 3.2 58.0 22.1 2.1 0.7 5.1
0.2 pKR2336-14 8.0 3.5 54.8 20.2 5.7 1.0 6.4 0.5 pKR2336-3 7.8 2.8
52.2 25.1 5.3 0.8 5.6 0.5 pKR2336-2 8.6 3.2 46.2 23.0 8.8 1.1 8.3
0.8 pKR2336-17 8.0 3.2 19.0 32.0 17.0 1.7 17.4 1.7 pKR2336-13 8.1
3.2 18.0 32.3 17.0 1.8 17.8 1.7 pKR2336-5 8.4 3.2 16.3 32.9 18.3
1.7 17.4 1.8 pKR2336-15 8.3 3.3 16.3 32.3 18.1 1.9 18.0 1.8
pKR2336-12 8.2 3.2 16.2 32.4 18.1 1.8 18.2 1.8 pKR2336-7 8.5 3.2
15.6 32.7 18.5 1.8 17.7 1.9 pKR2336-4 9.0 3.3 15.2 33.0 18.6 1.9
17.0 1.9 pKR2336-1 9.3 3.0 15.1 34.4 18.4 1.7 16.3 1.8 pKR2336-10
8.6 3.4 15.2 32.4 18.7 2.1 17.6 1.9
[0480] From Table 41, events expressing double fad2/fad3 and single
fae1 amiRNAs together function to suppress fad2, fad3 and fae1 and
increase 18:1 content to as high as 68%, in bulk T2 seed, compared
to 14.0% for the wild-type seed, with a decrease in 18:3 content to
as low as 4%, compared to 21.6% for the wild-type seed and decrease
in 20:1 to as low as 3% compared to 15.8% in wild-type seed. Other
events had 18:1 at 65% with 18:3 at 2% and 20:1 also at 2%. As
described for the triple amiRNA construct shown in Table 40, these
fatty acid profiles are better than for the double amiRNA
constructs alone.
Fatty Acid Analysis of Bulk T3 Seed from Homozygous Plants
[0481] All T2 events for all constructs were plated on Kanamycin as
described above and those events where kanamycin resistant T2
plants segregated from kanamycin sensitive plants by a ratio of
3:1, indicating a single copy insertion and showing 3:1 segregation
of single T2 seed phenotype (as described herein) were were
advanced to obtain homozygous T3 seed exactly as described herein.
The fatty acid profile for T3 seed from homozygous T2 plants was
also determined exactly as described herein.
[0482] Results for fatty acid profiles for homozygous T3 seed
batches for one representative event from each experiment are
presented in Tables 42 to 44 below. In each Table, fatty acids are
identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic
acid), 18:2 (linoleic acid) and 18:3 (alpha-linolenic acid), 20:0
(eicosanoic acid), 20:1 eicosenoic acid) 20:2 (eicocadienoic acid)
and 20:3 (eicosatrienoic acid) and results are expressed as a
weight percent (wt. %) of total fatty acids. Fatty acid profiles
are sorted from highest 18:1 to lowest. A wild-type Columbia seed
batch was also run and shown for comparison in each Table as
col-0.
TABLE-US-00057 TABLE 42 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2232, pKR2233, pKR2234
or pKR2235 expressing the double amiRNAs targeting fad2 and fad3
Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 pKR2232-3-9 7.2 3.2
55.4 10.8 2.3 1.3 19.9 0.0 pKR2232-3-17 7.0 3.0 51.4 13.7 3.1 1.2
20.2 0.3 pKR2233-10-1 9.3 2.8 64.7 9.9 2.8 0.0 10.5 0.0 pKR2233-10-
7.8 3.0 59.1 9.2 2.3 1.0 17.6 0.0 11 pKR2233-10-9 8.3 2.8 58.8 10.2
2.8 1.0 16.2 0.0 pKR2234-4-13 7.6 3.2 45.5 21.1 1.7 1.4 19.5 0.0
pKR2234-4-2 7.8 3.0 45.4 22.1 1.7 1.2 18.9 0.0 pKR2234-4-5 7.5 3.2
44.3 21.6 2.3 1.4 19.7 0.0 pKR2235-5-9 7.2 3.0 53.6 13.7 1.8 1.2
19.3 0.3 pKR2235-5-3 7.2 2.9 52.4 14.2 1.8 1.2 20.0 0.3 pKR2235-5-7
7.0 2.9 51.4 14.7 1.8 1.2 20.7 0.3
[0483] From Table 42, events expressing double fad2/fad3 amiRNAs
function to suppress both fad2 and fad3 and increase 18:1 content
to as high as 65%, in bulk T3 seed, compared to 14.0% for the
wild-type seed, with a decrease in 18:3 content to 3%, compared to
21.6% for the wild-type seed.
TABLE-US-00058 TABLE 43 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2248, pKR2249, pKR2250
or pKR2251 expressing the triple amiRNAs targeting fad2, fad3 and
fael. Event 16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 pKR2248-4-2 6.8
3.0 75.3 9.2 1.8 0.7 3.1 0.1 pKR2248-4-11 7.3 3.5 70.5 11.1 2.7 0.8
3.9 0.2 pKR2249-1-18 7.9 3.3 58.4 20.7 2.1 0.8 6.4 0.3 pKR2249-1-12
7.9 3.3 58.2 21.1 2.1 0.8 6.2 0.3 pKR2249-1-6 8.3 3.1 56.6 23.3 2.2
0.7 5.5 0.3 pKR2250-4-6 7.7 2.9 74.5 9.2 2.2 0.6 2.9 0.0
pKR2250-4-10 8.3 2.7 73.6 10.2 2.6 0.0 2.7 0.0 pKR2250-4-15 8.5 2.5
72.5 10.6 3.1 0.0 2.7 0.0 pKR2251-17- 8.6 2.6 65.7 15.9 2.4 0.6 4.2
0.0 11 pKR2251-17- 8.4 2.6 65.5 16.0 2.2 0.5 4.7 0.0 16 pKR2251-17-
8.5 2.7 65.0 16.4 2.2 0.5 4.7 0.0 12
[0484] From Table 43, events expressing triple fad2/fad3/fae1
amiRNAs function to suppress fad2, fad3 and fae1 and increase 18:1
content to as high as 75%, in bulk T3 seed, compared to 14.0% for
the wild-type seed, with a decrease in 18:3 content to 2%, compared
to 21.6% for the wild-type seed and decrease in 20:1 to 3% compared
to 15.8% in wild-type seed.
TABLE-US-00059 TABLE 44 Fatty acid profiles of lipid from bulk T3
seed samples for events transformed with pKR2333, pKR2334, pKR2335
or pKR2336 expressing one double amiRNAs targeting fad2 and fad3
and another amiRNA targeting fael. Event 16:0 18:0 18:1 18:2 18:3
20:0 20:1 20:2 2333-13-7 7.6 2.4 74.3 9.3 2.9 0.7 2.8 0.0 2333-13-9
7.6 2.8 74.1 10.6 2.3 0.6 2.0 0.0 2333-13-13 7.8 2.6 74.0 11.0 2.6
0.5 1.6 0.0 2334-13-22 8.1 2.3 73.5 9.9 3.0 0.5 2.7 0.0 2334-13-18
7.9 2.4 72.9 11.7 2.9 0.5 1.7 0.0 2334-13-7 8.2 2.8 71.1 10.5 3.0
0.5 4.0 0.0 2335-12-15 8.1 2.5 64.3 19.8 2.2 0.5 2.6 0.0 2335-12-7
8.1 2.4 64.2 20.1 2.1 0.5 2.5 0.0 2335-12-12 8.4 2.6 63.7 19.5 2.0
0.6 3.1 0.0 2336-3-10 7.2 2.4 66.7 19.4 2.1 0.5 1.7 0.0 2336-3-14
8.4 2.5 63.0 21.1 2.2 0.5 2.1 0.0 2338-3-1 8.1 2.8 62.4 21.3 2.1
0.5 2.8 0.0
[0485] From Table 44, events expressing double fad2/fad3 and single
fae1 amiRNAs together function to suppress fad2, fad3 and fae1 and
increase 18:1 content to as high as 74%, in bulk T3 seed, compared
to 14.0% for the wild-type seed, with a decrease in 18:3 content to
2%, compared to 21.6% for the wild-type seed and decrease in 20:1
to 2% compared to 15.8% in wild-type seed.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150089689A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150089689A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References