NZ618754B2 - Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof - Google Patents
Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof Download PDFInfo
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- NZ618754B2 NZ618754B2 NZ618754A NZ61875412A NZ618754B2 NZ 618754 B2 NZ618754 B2 NZ 618754B2 NZ 618754 A NZ618754 A NZ 618754A NZ 61875412 A NZ61875412 A NZ 61875412A NZ 618754 B2 NZ618754 B2 NZ 618754B2
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/12—Leaves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
Disclosed is a seed of an alfalfa plant comprising event KK179-2 (an insertion of SEQ ID NO: 5 that reduces lignin content, wherein SEQ ID NO: 5 is disclosed within the specification), wherein a representative sample of seed comprising event KK179-2 has been deposited with the American Type Culture Collection (ATCC) with the Patent Deposit Designation PTA-11833. Also disclosed are polynucleotide probes and primers suitable for diagnosing the presence of event KK179-2 DNA. Collection (ATCC) with the Patent Deposit Designation PTA-11833. Also disclosed are polynucleotide probes and primers suitable for diagnosing the presence of event KK179-2 DNA.
Description
ALFALFA PLANT AND SEED CORRESPONDING TO TRANSGENIC EVENT
KK 179-2 AND METHODS FOR ION THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is entitled to priority pursuant to 35 U.S.C. § 119 (e) to US
provisional patent application No. ,373, which was filed on June 30, 2011, and
US provisional patent application No. 61/664,359, which was filed on June, 26, 2012,
the sures of which are incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
The sequence listing file named “57978_seq_listing.txt”, which is 10,564 bytes
(measured in MS—WINDOWS) which was electronically filed and which was created on
May, 1 2012 is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to alfalfa transgenic event KK179—2. The invention
also provides cells, plant parts, seeds, plants, commodity products related to the event,
and DNA les that are unique to the event and were created by the ion of
transgenic DNA into the genome of an alfalfa plant. The invention further es
s for detecting the presence of said alfalfa event nucleotide ces in a sample,
probes and primers for use in detecting nucleotide sequences that are diagnostic for the
presence of said alfalfa event.
BACKGROUND OF THE INVENTION
Alfalfa (Medicago ) is the most cultivated legume worldwide, with the US
being the top alfalfa producer. The methods of biotechnology have been applied to
alfalfa for improvement of agronomic traits and the quality of the product. One such
agronomic trait is lignin content.
Lignin is the second most nt terrestrial biopolymer and accounts for 30%
of the organic carbon. Lignin is crucial for structural integrity of the cell wall and it
imparts stiffness and strength to the stem. Lignin content is inversely correlated with
forage digestibility for diary cattle. A reduction in lignin may be achieved in transgenic
plants by the sion of a RNA suppression construct capable of providing such
decrease while at the same time provide increased alfalfa digestibility. The expression of
foreign genes or suppression molecules in plants is known to be influenced by many
s, such as the regulatory elements used, the chromosomal location of the ene
insert, the proximity of any endogenous regulatory elements close to the transgene
insertion site, and environmental factors such as light and temperature. For example, it
has been ed that there may be variation in the overall level of transgene
suppression or in the l or temporal pattern of transgene suppression between
similarly—produced events. For this reason, it is often necessary to screen hundreds of
independent transformation events in order to ultimately identify one event useful for
cial ltural purposes. Such an event, once identified as having the desired
suppression phenotype, molecular characteristics and the improved trait, may then be
used for introgressing the improved trait into other genetic backgrounds using plant
breeding methods. The resulting progeny would contain the transgenic event and would
therefore have the same characteristics for that trait of the original transformant. This
may be used to produce a number of different crop varieties that se the improved
trait and are suitably adapted to specific local g conditions.
It would be advantageous to be able to detect the presence of ene/genomic
DNA of a particular plant in order to determine whether progeny of a sexual cross
contain the transgene/genomic DNA of interest. In addition, a method for detecting a
particular plant would be helpful when complying with regulations requiring the pre—
market approval and labeling of foods derived from the transgenic crop plants.
The presence or absence of a suppression element may be detected by any well
known c acid detection method such as the polymerase chain reaction (PCR) or
DNA hybridization using nucleic acid probes. These detection methods lly focus
on frequently used genetic elements, such as ers, terminators, marker genes, etc.
As a result, such methods may not be useful for discriminating between different
transformation events, particularly those produced using the same DNA construct unless
the sequence of somal DNA adjacent to the ed DNA (“flanking DNA”) is
known. An specific PCR assay is discussed, for example, by Taverniers et al. (J.
Agric. Food Chem., 53: 3041—3052, 2005) in which an event—specific tracing system for
transgenic maize lines Btll, Btl76, and GA21 and for canola event GT73 was
demonstrated. In this study, event—specific primers and probes were designed based upon
the sequences of the /transgene junctions for each event. enic plant event
specific DNA detection methods have also been described in US Patent Nos. 7,632, 985;
7,566,817; 7,368,241; 7,306,909; 7,718,373; 7,189,514, 7,807,357 and 7,820,392.
SUMMARY OF THE INVENTION
The present invention is an alfalfa transgenic event designated event KK179—2,
having representative seed sample deposited with American Type Culture Collection
(ATCC) under the Accession No. PTA—11833.
The invention provides a plant, seed, cell, progeny plant, or plant part comprising
the event derived from a plant, cell, plant part, or seed comprising event 2. The
invention thus includes, but is not limited to pollen, ovule, flowers, shoots, roots and
leaves.
One aspect of the ion provides compositions and methods for detecting the
presence of a DNA enic/genomic junction region from alfalfa event KK179—2 plant
or seed. DNA molecules are provided that comprise at least one transgene/genomic
junction DNA molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2 and complements thereof, wherein the on molecule spans the ion site.
An alfalfa event KK179—2 and seed comprising these DNA molecules is an aspect of this
invention.
A novel DNA molecule is ed that is a DNA transgene/genomic region SEQ
ID NO:3 or the complement thereof, from alfalfa event KK179—2. An alfalfa plant and
seed comprising SEQ ID NO: 3 in its genome is an aspect of this invention. In another
aspect of the invention, a DNA molecule is provided that is a DNA transgene/genomic
resion SEQ ID NO:4 or the complement thereof, wherein this DNA molecule is novel in
alfalfa event KK179—2. An a plant and seed comprising SEQ ID NO:4 in its
genome is an aspect of this invention.
The invention provides DNA molecules related to event KK179—2. These DNA
molecules may comprise nucleotide sequences representing or derived from the junction
of the transgene insertion and g genomic DNA of event KK179—2, and/or a region
of the genomic DNA flanking the inserted DNA, and/or a region of the integrated
transgenic DNA flanking the insertion site, and/or a region of the integrated transgenic
expression cassette, and/or a contiguous sequence of any of these regions. The invention
also provides DNA molecules useful as primers and probes diagnostic for the event.
Plants, cells, plant parts, commodity products, y, and seeds comprising these
les are provided.
According to one aspect of the invention, compositions and methods are provided
for ing the presence of the transgene/genomic insertion region from a novel a
plant designated KK179—2. DNA sequences are ed that comprise at least one
on sequence of KK179—2 selected from the group consisting of SEQ ID NO: 1
(corresponding to positions 1038 through 1057 of SEQ ID NO: 6, Figure 1 [F]),and SEQ
ID NO: 2 (corresponding to ons 3620 through 3639 of SEQ ID NO: 6, Figure 1 [F]),
and complements thereof; wherein a junction sequence is a nucleotide sequence that
spans the point at which heterologous DNA inserted into the genome is linked to the
alfalfa cell genomic DNA and detection of this sequence in a biological sample
containing alfalfa DNA is diagnostic for the presence of the a event KK179—2 DNA
in said sample (Figure 1). The alfalfa event KK179—2 and alfalfa seed comprising these
DNA molecules is an aspect of this invention.
According to another aspect of the invention, two DNA molecules are provided
for use in a DNA detection method, wherein a first DNA molecule comprises a
polynucleotide having a nucleotide sequence of ient length of consecutive
polynucleotide of any portion of the transgene region of the DNA molecule of SEQ ID
NO: 3 or SEQ ID NO: 5 and a second DNA molecule of similar length of any portion of
a 5’ flanking alfalfa genomic DNA region of SEQ ID NO: 3, where said DNA les
function as DNA primers when used together in an amplification reaction with a template
derived from event KK179—2 to produce an amplicon diagnostic for event KK179—2
DNA in a sample. Any on produced by DNA primers homologous or
complementary to any portion of SEQ ID NO: 3 and SEQ ID NO: 5, and any amplicon
that comprises SEQ ID NO: I is an aspect of the ion.
According to another aspect of the invention, two DNA molecules are provided
for use in a DNA ion method, wherein a first DNA molecule comprises a
polynucleotide having a nucleotide sequence of sufficient length of consecutive
polynucleotide of any portion of the transgene region of the DNA molecule of SEQ ID
NO: 4 or SEQ ID NO: 5 and a second DNA molecule of similar length of any portion of
a 3’ flanking alfalfa genomic DNA of SEQ ID NO: 4, where said DNA molecules
function as DNA primers when used together in an amplification reaction with a template
derived from event KKl79—2 to produce an amplicon diagnostic for event KKl79—2 DNA
in a sample. Any amplicons produced by DNA primers homologous or complementary
to any portion of SEQ ID NO: 4 and SEQ ID NO: 5, and any on that comprises
SEQ ID NO: 2 is an aspect of the invention.
The invention provides methods, compositions, and kits useful for detecting the
presence of DNA derived from a event KKl79—2. Certain methods comprise (a)
contacting a sample comprising DNA with a primer set that when used in a c acid
amplification reaction with genomic DNA from alfalfa event KKl79—2 produces an
amplicon diagnostic for the event; (b) performing a nucleic acid amplification reaction
thereby producing the amplicon; and (c) detecting the amplicon, wherein said amplicon
comprises SEQ ID NO: 1 and/or SEQ ID NO: 2. The invention also provides a method
for detection of the event by (a) contacting a sample comprising DNA with a probe that
when used in a hybridization reaction with genomic DNA from the event hybridizes to a
DNA molecule specific for the event; (b) ting the sample and probe to stringent
hybridization conditions; and (c) detecting the hybridization of the probe to the DNA
molecule. Kits comprising the methods and itions of the invention useful for
detecting the presence of DNA derived from the event are also provided.
The invention further es a method of producing a alfalfa plant comprising:
(a) ng a KKl79—2 alfalfa plant with a second alfalfa plant, thereby producing a
seed; (b) growing said seed to produce a plurality of progeny plants; and (c) ing a
progeny plant that comprises KKl79—2 or a progeny plant with decreased lignin content.
The foregoing and other aspects of the invention will become more apparent from
the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Diagrammatic representation of the enic insert in the genome of alfalfa
event KKl79—2; [A] corresponds to the relative positions of SEQ ID NO: 1 g the
junction between SEQ ID NO: 3 and SEQ ID NO: 5; [B] corresponds to the relative
positions of SEQ ID NO: 2 g the junction between SEQ ID NO: 4 and SEQ ID
NO: 5; [C] corresponds to the relative position of SEQ ID NO: 3, which contains the
alfalfa genomic flanking region and a n of the arbitrarily designated 5’ end of the
transgenic DNA insert; [D] corresponds to the ve position of SEQ ID NO: 4, which
ns the alfalfa genome flanking region and a portion of the arbitrarily designated 3’
end of the transgenic DNA insert; [E] represents SEQ ID NO: 5, which is the sequence of
the transgenic DNA insert including the CCOMT suppression cassette integrated into the
genome of event KKl79—2; [F] represents SEQ ID NO: 6, which is the contiguous
sequence comprising, as ented in the figure from left to right, SEQ ID NO: 3, SEQ
ID NO: 5 and SEQ ID NO: 4, in which SEQ ID NOs: 1 and SEQ ID NOs: 2 are
incorporated as set forth above, as these sequences are present in the genome in event
KKl79—2. LB: refers to the left border of T—DNA; RB: refers to the right border of T—
DNA.
BRIEF DESCRIPTION OF THE SEQUENCES
The sequence listing file named “57978_seq_listing.txt”, which is 10,564 bytes
(measured in MS—WINDOWS) which was electronically filed and which was created on
May, 1 2012 is incorporated herein by nce.
SEQ ID NO: 1 — A 20 bp nucleotide sequence representing the left border
junction between the alfalfa genomic DNA and the integrated DNA insert. This sequence
corresponds to positions 1038 through 1057 of SEQ ID NO: 6, and to ons 1038
through 1047 of SEQ ID NO: 3 ([C] of Figure 1). In addition, SEQ ID NO: 1 corresponds
to the integrated left border of the expression cassette at positions 1 through 10 of SEQ
ID NO: 5 ([E] of Figure 1).
SEQ ID NO: 2 — A 20 bp tide sequence representing the right border
junction between the integrated DNA insert and the alfalfa genomic DNA. This sequence
corresponds to positions 3620 to 3639 of SEQ ID NO: 6, and to positions 91 through 111
of SEQ ID NO: 4 ([D] of Figure 1). In addition, SEQ ID NO: 2 corresponds to positions
2573 through 2582 SEQ ID NO: 5 ([E] of Figure 1).
SEQ ID NO: 3 — A 1147 bp nucleotide sequence including the 5’ alfalfa genomic
sequence (1047 bp) flanking the inserted DNA of event KKl79—2 plus a region (100 bp)
of the integrated DNA. This sequence corresponds to positions 1 through 1047 of SEQ
ID NO: 6.
SEQ ID NO: 4 — A 1356 bp nucleotide sequence including the 3’ a genomic
ce (1256 bp) flanking the inserted DNA of event KKl79—2 plus a region (100 bp)
of the integrated DNA. This sequence corresponds to positions 3529 through 4885 of
SEQ ID NO: 6.
SEQ ID NO: 5 — The sequence of the integrated expression cassette, including the
left and the right border sequences after integration. SEQ ID NO: 5 corresponds to
nucleotide positions 1048 through 3629 of SEQ ID NO: 6.
SEQ ID NO: 6 — A 4885 bp nucleotide sequence representing the contig of the 5’
sequence flanking the ed DNA of KKl79—2 (SEQ ID NO: 3), the sequence of the
integrated DNA insert (SEQ ID NO: 5) and the 3’ ce flanking the inserted DNA of
KKl79-2 (SEQ ID NO: 4).
SEQ ID NO: 7 — The sequence of primer 1 used to identify KKl79—2
event. Production of a 81 bp PCR amplicon using the ation of primers 1
and SQ23728 (SEQ ID NO: 8) is a positive result for the ce of event KKl79—2.
SEQ ID NO 8— The sequence of primer SQ223728 used to identify KKl79—2
event.
SEQ ID NO: 9 — The sequence of probe PB10164 used to identify KKl79—2
event. It is a 6FAMTM—labeled tic oligonucleotide.
SEQ ID NO: 10 — The sequence of primer SQ1532 used as an internal control in
end—point TAQMAN® assays.
SEQ ID NO: ll — The sequence of primer SQ1533 used as an internal control in
end—point TAQMAN® assays.
SEQ ID NO: 12 — The sequence of a labeled synthetic oligonucleotide
probe PB0359 used as an internal control in end—point TAQMAN® assays.
DETAILED DESCRIPTION
The following definitions and methods are provided to better define the present
invention and to guide those of ry skill in the art in the ce of the present
invention. Unless otherwise noted, terms are to be understood according to conventional
usage by those of ordinary skill in the relevant art. Definitions of common terms in
molecular biology may also be found in Rieger et al., Glossary of cs: Classical
and Molecular, 5th edition, Springer—Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994.
The present invention provides transgenic a event KKl79—2. The term
“event” as used herein refers to the plants, seeds, progeny, cells, plant parts thereof, and
DNA molecules produced as a result of transgenic DNA integration into a plant’s
genome at a ular location on a chromosome. Event KKl79—2 refers to the plants,
seeds, progeny, cells, plant parts thereof, and DNA molecules produced as a result of the
insertion of enic DNA having a sequence provided herein as SEQ ID NO: 5 into a
particular chromosomal location in the Medicago sativa genome. A seed sample
containing KKl79—2 has been ted with American Type Culture Collection (ATCC)
under Accession No. PTA—11833.
As used herein, the term “alfalfa” means Medicago sativa and es all plant
varieties that can be bred with alfalfa, including wild alfalfa species. Alfalfa is also
called medic, the name of any plant of the genus Medicago Old World herbs with blue or
yellow flowers similar to those of the related clovers. Unlike corn or soybean, alfalfa
plants are autotetraploid; thus, each trait is determined by genes residing on four
chromosomes instead of two. This complicates c research and also adds to the
difficulty of improving alfalfa. Commercial alfalfa seed is often comprised of a mixture
of clones that may constitute a synthetic ar generated by random interpollination
among the selected clones, followed by one to three generations of open—pollination in
isolation. Additionally, a composite cultivar of alfalfa may also be ped by
blending see of two or more clones or ollinating clones in isolation. When forming
a composite cultivar, equal quantities of seed from each component clone would be
blended to form the initial breeder seed stock.
A transgenic “event” is produced by transformation of plant cells with
heterologous DNA, such as, a nucleic acid construct that comprises the RNA ssion
of a gene of interest, regeneration of a population of independently transformed
transgenic plants resulting from the insertion of the transgene cassette into the genome of
the plant, and ion of a particular plant with desirable molecular characteristics, such
as insertion of the transgene into a particular genome location. A plant sing the
event can refer to the original transformant that includes the transgene inserted into the
particular location in the plant’s genome. A plant comprising the event can also refer to
progeny of the original transformant that retain the transgene at the same ular
location in the plant’s genome. Such progeny may be produced by a sexual outcross
n the transformant, or its progeny, and another plant. Such another plant may be a
transgenic plant comprising the same or a different transgene; or may be a non—transgenic
plant, such as one from a different variety. Even after repeated back—crossing to a
recurrent parent, the event DNA from the transformed parent is present in the y of
the cross at the same genomic location.
A DNA molecule comprising event KKl79—2 refers to a DNA molecule
comprising at least a portion of the inserted transgenic DNA (provided as SEQ ID NO:
) and at least a portion of the flanking genomic DNA immediately adjacent to the
inserted DNA. As such, a DNA molecule comprising event KKl79—2 has a nucleotide
sequence representing at least a portion of the transgenic DNA insert and at least a
portion of the particular region of the genome of the plant into which the transgenic DNA
was inserted. The arrangement of the inserted DNA in event KKl79—2 in relation to the
nding plant genome is specific and unique to event KKl79—2 and as such the
nucleotide sequence of such a DNA le is diagnostic and identifying for event
KKl79—2. Examples of the sequence of such a DNA molecule are provided herein as
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6.
Such a DNA molecule is also an integral part of the chromosome of a plant that
comprises event KKl79—2 and may be passed on to progenies of the plant.
As used herein, a “recombinant DNA molecule” is a DNA molecule comprising a
combination of DNA les that would not lly occur together and is the result
of human intervention, for example, a DNA molecule that is comprised of a combination
of at least two DNA les heterologous to each other, and/or a DNA molecule that is
artificially synthesized and ses a polynucleotide sequence that deviates from the
polynucleotide sequence that would normally exist in nature, and/or a DNA molecule that
comprises a transgene artificially incorporated into a host cell’s genomic DNA and the
ated flanking DNA of the host cell’s genome. An example of a recombinant DNA
molecule is a DNA molecule described herein resulting from the insertion of the
transgene into the go sativa , which may ultimately result in the
suppression of a recombinant RNA and/or protein molecule in that organism. The
nucleotide sequence or any fragment derived therefrom would also be considered a
recombinant DNA molecule if the DNA molecule can be extracted from cells, or tissues,
or homogenate from a plant or seed or plant tissue; or can be produced as an amplicon
from extracted DNA or RNA from cells, or tissues, or homogenate from a plant or seed
or plant tissue, any of which is d from such als d from the event
KKl79—2. For that , the junction sequences as set forth at SEQ ID NO: 1 and SEQ
ID NO: 2, and nucleotide sequences derived from event KKl79—2 that also contain these
junction sequences are considered to be recombinant DNA, whether these sequences are
present within the genome of the cells of event KKl79—2 or present in detectable amounts
in tissues, progeny, ical samples or commodity products derived from the event
KKl79—2. As used herein, the term “transgene” refers to a polynucleotide molecule
artificially incorporated into a host cell’s genome. Such transgene may be heterologous
to the host cell. The term “transgenic plant” refers to a plant comprising such a
transgene. A “transgenic plant” includes a plant, plant part, a plant cell or seed whose
genome has been altered by the stable ation of recombinant DNA. A transgenic
plant includes a plant regenerated from an originally—transformed plant cell and progeny
transgenic plants from later generations or crosses of a transformed plant. As a result of
such genomic alteration, the transgenic plant is distinctly different from the related wild
type plant. An e of a transgenic plant is a plant described herein as comprising
event KKl79—2.
As used herein, the term ologous” refers to a sequence which is not
ly present in a given host genome in the genetic context in which the ce is
currently found. In this respect, the sequence may be native to the host genome, but be
rearranged with respect to other genetic sequences within the host sequence.
The present invention provides DNA molecules and their ponding
nucleotide sequences. As used herein, the terms “DNA sequence”, “nucleotide
sequence” and “polynucleotide sequence” refer to the sequence of nucleotides of a DNA
molecule, usually ted from the 5’ (upstream) end to the 3’ (downstream) end. The
nomenclature used herein is that required by Title 37 of the United States Code of
Federal Regulations § 1.822 and set forth in the tables in WIPO Standard ST.25 (1998),
Appendix 2, Tables I and 3. The present invention is disclosed with reference to only
one strand of the two nucleotide sequence strands that are provided in enic event
KKl79—2. Therefore, by implication and derivation, the mentary sequences, also
referred to in the art as the complete complement or the reverse complementary
ces, are within the scope of the present invention and are therefore also intended to
be within the scope of the subject matter claimed.
The nucleotide sequence corresponding to the complete nucleotide sequence of
the inserted transgenic DNA and substantial ts of the Medicago sativa genomic
DNA flanking either end of the inserted transgenic DNA is provided herein as SEQ ID
NO: 6. A subsection of this is the inserted transgenic DNA provided as SEQ ID NO: 5.
The nucleotide sequence of the genomic DNA flanking the 5’ end of the inserted
enic DNA and a portion of the 5’ end of the inserted DNA is provided herein as
SEQ ID NO: 3. The nucleotide sequence of the genomic DNA flanking the 3’ end of the
inserted transgenic DNA and a portion of the 3’ end of the inserted DNA is provided
herein as SEQ ID NO: 4. The region spanning the location where the transgenic DNA
connects to and is linked to the genomic DNA is referred to herein as the junction. A
“junction sequence” or “junction region” refers to a DNA sequence and/or corresponding
DNA molecule that spans the inserted transgenic DNA and the adjacent flanking genomic
DNA. Examples of a junction ce of event 2 are provided herein as SEQ
WO 03558
ID NO: I and SEQ ID NO: 2. The identification of one of these junction sequences in a
tide molecule derived from a alfalfa plant or seed is conclusive that the DNA was
obtained from event KKl79—2 and is diagnostic for the presence of DNA from event
KKl79—2. SEQ ID NO: I is a 20 bp nucleotide sequence spanning the junction between
the genomic DNA and the 5’ end of the inserted DNA. SEQ ID NO: 2 is a 20 bp
nucleotide sequence spanning the junction between the genomic DNA and the 3’ end of
the inserted DNA. Any segment of DNA derived from transgenic event KKl79—2 that
includes the consecutive tides of SEQ ID NO: I is within the scope of the present
invention. Any segment of DNA derived from transgenic event KKl79—2 that includes
the consecutive nucleotides of SEQ ID NO: 2 is within the scope of the present invention.
In addition, any polynucleotide molecule comprising a sequence complementary to any
of the sequences bed within this paragraph is within the scope of the present
invention. Figure l is an illustration of the transgenic DNA insert in the genome of
alfalfa event KKl79—2, and the relative positions of SEQ ID NOs: l—6 arranged 5’to 3’.
The present invention further provides ary DNA molecules that can be
used either as primers or probes for diagnosing the ce of DNA derived from event
KKl79—2 in a sample. Such primers or probes are specific for a target nucleic acid
sequence and as such are useful for the identification of event KKl79—2 nucleic acid
sequence by the methods of the invention described herein.
A "probe" is an isolated nucleic acid to which is attached a detectable label or
reporter le, for example, a radioactive isotope, ligand, uminescent agent, or
enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the case
of the present invention, to a strand of genomic DNA from alfalfa event KKl79—2
whether from a alfalfa plant or from a sample that comprises DNA from the event.
Probes according to the present ion include not only deoxyribonucleic or
ribonucleic acids but also polyamides and other probe als that bind specifically to a
target DNA sequence and the detection of such binding can be used to
diagnose/determine/confirm the presence of that target DNA sequence in a particular
sample.
A "primer" is typically an isolated polynucleotide that is designed for use in
specific annealing or hybridization s to hybridize to a complementary target DNA
strand to form a hybrid between the primer and the target DNA strand, and then extended
along the target DNA strand by a polymerase, for example, a DNA polymerase. A pair of
primers may be used with template DNA, such as a sample of Medicago sativa genomic
DNA, in a thermal or isothermal amplification, such as polymerase chain reaction (PCR),
or other nucleic acid amplification methods, to produce an amplicon, where the amplicon
produced from such reaction would have a DNA sequence corresponding to sequence of
the template DNA located between the two sites where the primers hybridized to the
template. As used herein, an con” is a piece or nt of DNA that has been
synthesized using amplification techniques, such as the product of an amplification
reaction. In one embodiment of the invention, an amplicon diagnostic for event KKl79—
2 comprises a sequence not naturally found in the Medicago sativa genome. Primer
pairs, as used in the present ion, are intended to refer to use of two primers g
opposite s of a double stranded nucleotide segment for the purpose of amplifying
linearly the cleotide segment between the positions targeted for binding by the
individual members of the primer pair, typically in a thermal or isothermal amplification
on or other c acid amplification methods. Exemplary DNA molecules useful
as primers are provided as SEQ ID NOs: 7—9, may be used as a first DNA molecule and
a second DNA molecule that is ent from the first DNA molecule, and both
molecules are each of sufficient length of consecutive nucleotides of either SEQ ID NO:
4, SEQ ID NO: 5, or SEQ ID NO: 6 or the complements thereof to function as DNA
primers so that, when used together in an amplification reaction with template DNA
derived from event KKl79—2, an on that is specific and unique to transgenic event
KKl79—2 would be produced. The use of the term “amplicon” specifically es
primer—dimers that may be formed in the DNA amplification reaction.
Probes and primers according to the present invention may have complete
sequence identity to the target sequence, although primers and probes differing from the
target sequence that retain the ability to hybridize preferentially to target sequences may
be designed by conventional methods. In order for a nucleic acid molecule to serve as a
primer or probe it needs only be sufficiently complementary in sequence to be able to
form a stable double—stranded structure under the ular solvent and salt
concentrations employed. Any nucleic acid ization or amplification method can be
used to identify the presence of transgenic DNA from event KK179—2 in a sample.
Probes and primers are lly at least about 11 nucleotides, at least about 18
nucleotides, at least about 24 tides, and at least about 30 nucleotides or more in
length. Such probes and primers hybridize specifically to a target ce under high
stringency hybridization conditions.
Methods for preparing and using probes and s are described, for example,
in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1—3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter, "Sambrook
et al., 1989"); Current Protocols in Molecular Biology, ed. l et al., Greene
Publishing and lnterscience, New York, 1992 (with ic updates) (hereinafter,
"Ausubel et al., 1992”); and Innis et al., PCR Protocols: A Guide to Methods and
Applications, Academic Press: San Diego, 1990. PCR—primer pairs can be derived from
a known sequence, for example, by using computer programs intended for that purpose
such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research,
Cambridge, MA).
Primers and probes based on the flanking DNA and insert sequences sed
herein can be used to confirm the disclosed ces by known methods, for example,
by re—cloning and sequencing such sequences.
The nucleic acid probes and primers of the present invention hybridize under
stringent conditions to a target DNA sequence. Any c acid hybridization or
ication method can be used to identify the presence of DNA from a transgenic
event in a sample. Nucleic acid molecules or fragments thereof are capable of
specifically hybridizing to other nucleic acid les under certain circumstances. As
used herein, two nucleic acid molecules are said to be capable of specifically hybridizing
to one another if the two molecules are capable of forming an arallel, double—
stranded nucleic acid structure. A nucleic acid molecule is said to be the “complement”
of another nucleic acid molecule if they t complete complementarity. As used
herein, molecules are said to exhibit “complete complementarity” when every nucleotide
of one of the molecules is complementary to a nucleotide of the other. Two molecules
are said to be “minimally complementary” if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one another under at least "low—
stringency" conditions. Similarly, the molecules are said to be “complementary” if they
can hybridize to one another with sufficient stability to permit them to remain annealed
to one another under "high—stringency" conditions. Stringency conditions are bed
by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, DC (1985). Departures from complete
mentarity are therefore permissible, as long as such departures do not completely
preclude the capacity of the les to form a double—stranded structure. In order for a
nucleic acid molecule to serve as a primer or probe it need only be sufficiently
complementary in sequence to be able to form a stable double—stranded structure under
the particular t and salt concentrations employed.
As used herein, a substantially homologous ce is a nucleic acid sequence
that will specifically hybridize to the complement of the nucleic acid sequence to which
it is being compared under high stringency conditions. riate stringency
conditions that promote DNA ization, for example, 6.0 X sodium chloride/sodium
citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSC at 50°C, are known to
those skilled in the art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, NY. (1989), 6.3.1—6.3.6. For example, the salt concentration in the wash
step can be selected from a low stringency of about 2.0 X SSC at 50°C to a high
stringency of about 0.2 X SSC at 50°C. In addition, the temperature in the wash step can
be increased from low stringency conditions at room temperature, about 22°C, to high
stringency conditions at about 65°C. Both temperature and salt may be varied, or either
the temperature or the salt concentration may be held constant while the other variable is
d. In one embodiment, a nucleic acid of the present invention will specifically
hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, and
SEQ ID NO: 2,or complements or nts thereof under high stringency conditions.
The hybridization of the probe to the target DNA molecule can be detected by any
number of s known to those skilled in the art. These can include, but are not
limited to, fluorescent tags, radioactive tags, antibody based tags, and chemiluminescent
tags.
Regarding the amplification of a target nucleic acid sequence (for eXample, by
PCR) using a particular ication primer pair, gent conditions" are conditions
that permit the primer pair to hybridize only to the target nucleic acid sequence to which
a primer having the corresponding ype sequence (or its complement) would bind
and preferably to produce a unique amplification product, the amplicon, in a DNA
amplification reaction. Examples of DNA amplification methods include PCR,
Recombinase Polymerase Amplification (RPA) (see for example U.S. Pat No.
7,485,428), Strand Displacement Amplification (SDA) (see for example, U.S. Pat. Nos.
,455,166 and 723), Transcription—Mediated Amplification (TMA) (see for
example, li et al., Proc. Natl. Acad. Sci. USA 87:1874—1878 (1990)), Rolling
Circle Amplification (RCA) (see for example, Fire and Xu, Proc. Natl. Acad Sci. USA
92:4641—4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587—1594 (1996); Lizardi, et
al., Nature Genetics 19:225—232 (1998), U.S. Pat. Nos. 5,714,320 and 502)),
Helicase Dependant Amplification (HDA) (see for example Vincent et al., EMBO
Reports 5(8): 795—800 ; U.S. Pat. No. 7,282,328), and Multiple Displacement
Amplification (MDA) (see for example Dean et. al., Proc. Natl. Acad Sci. USA 99:5261—
5266 (2002)).
The term "specific for (a target sequence)" indicates that a probe or primer
hybridizes under stringent hybridization ions only to the target sequence in a
sample comprising the target sequence.
As used herein, the term “isolated” refers to at least partially ting a
molecule from other molecules normally associated with it in its native or natural state.
In one embodiment, the term “isolated” refers to a DNA molecule that is at least partially
ted from the nucleic acids that normally flank the DNA molecule in its native or
natural state. Thus, DNA molecules fused to tory or coding sequences with which
they are not normally associated, for example as the result of recombinant techniques, are
considered ed herein. Such molecules are considered isolated even when integrated
into the some of a host cell or present in a nucleic acid solution with other DNA
molecules.
Any number of methods well known to those skilled in the art can be used to
isolate and manipulate a DNA molecule, or fragment thereof, disclosed in the present
invention. For example, PCR (polymerase chain reaction) technology can be used to
y a particular starting DNA molecule and/or to produce variants of the original
le. DNA molecules, or fragments thereof, can also be obtained by other
techniques such as by directly synthesizing the fragment by chemical means, as is
commonly practiced by using an ted oligonucleotide synthesizer.
The DNA molecules and corresponding nucleotide sequences provided herein are
therefore useful for, among other things, identifying event KKl79—2, selecting plant
ies or hybrids comprising event KKl79—2, detecting the presence of DNA derived
from event KKl79—2 in a sample, and ring samples for the ce and/or
absence of event KKl79—2 or plants and plant parts comprising event KKl79—2.
The present invention provides plants, progeny, seeds, plant cells, plant parts such
as pollen, ovule, pod, flower, root or stem tissue, and leaf. These plants, y, seeds,
plant cells, plant parts, and commodity products contain a detectable amount of a
polynucleotide of the present invention, such as a polynucleotide comprising at least one
of the sequences provided as the consecutive nucleotides of SEQ ID NO: 1, and the
consecutive nucleotides of SEQ ID NO: 2. Plants, y, seeds, plant cells, plant parts
and commodity products of the present invention may also contain one or more
additional suppression targets.
The present invention provides , progeny, seeds, plant cells, and plant part
such as pollen, ovule, pod, flower, root or stem tissue, and leaf derived from a transgenic
plant comprising event KKl79—2. A representative sample of seed comprising event
KKl79—2 has been deposited according to the Budapest Treaty for the purpose of
enabling the present invention. The tory ed for receiving the deposit is the
American Type Culture Collection (ATCC) having an address at 10801 University
Boulevard, Manassas, ia USA, Zip Code 20110. The ATCC repository has
assigned the accession No. PTA—11833 to event KKl79—2 seed.
The present invention provides a microorganism comprising a DNA molecule
having a nucleotide sequence selected from the group consisting of the consecutive
nucleotides of SEQ ID NO: 1, the consecutive tides of SEQ ID NO: 2. An
example of such a microorganism is a transgenic plant cell. Microorganisms, such as a
plant cell of the present invention, are useful in many industrial ations, including
but not limited to: (i) use as research tool for scientific y or industrial research; (ii)
use in culture for producing endogenous or recombinant carbohydrate, lipid, nucleic acid,
enzymes or protein products or small molecules that may be used for subsequent
scientific research or as industrial products; and (iii) use with modern plant tissue e
techniques to produce transgenic plants or plant tissue cultures that may then be used for
agricultural research or production. The production and use of microorganisms such as
transgenic plant cells utilizes modern microbiological techniques and human intervention
to produce a man—made, unique microorganism. In this process, recombinant DNA is
inserted into a plant cell’s genome to create a transgenic plant cell that is separate and
unique from naturally occurring plant cells. This transgenic plant cell can then be
cultured much like bacteria and yeast cells using modern microbiology techniques and
may exist in an undifferentiated, unicellular state. The new plant cell’s c
composition and phenotype is a technical effect created by the integration of the
heterologous DNA into the genome of the cell. Another aspect of the present invention is
a method of using a microorganism of the present invention. Methods of using
microorganisms of the present invention, such as transgenic plant cells, include (i)
s of ing transgenic cells by integrating inant DNA into genome of
the cell and then using this cell to derive additional cells possessing the same
heterologous DNA; (ii) methods of culturing cells that contain recombinant DNA using
modern microbiology techniques; (iii) methods of producing and ing endogenous
or recombinant carbohydrate, lipid, nucleic acid, enzymes or protein products from
cultured cells; and (iv) s of using modern plant tissue culture techniques with
transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.
As used herein, “progeny” includes any plant, seed, plant cell, and/or regenerable
plant part sing the event DNA derived from an ancestor plant and/or a
polynucleotide having at least one of the sequences provided as the consecutive
nucleotides of SEQ ID NO: 1 or the consecutive nucleotides of SEQ ID NO: 2. Plants,
progeny, and seeds may heterozygous for the ce of the transgenic ce.
Progeny may be grown from seeds produced by a plant comprising event KKl79—2
and/or from seeds produced by a plant fertilized with pollen from a plant comprising
event KKl79—2.
y plants may be outcrossed, for example, bred with another plant, to
produce a al or a hybrid seed or plant. The other plant may be transgenic or
nontransgenic. A varietal or hybrid seed or plant of the present ion may thus be
d by crossing a first parent that lacks the specific and unique DNA of event
KKl79—2 with a second parent comprising event KKl79—2, resulting in a hybrid
comprising the specific and unique DNA of event KKl79—2. Each parent can be a hybrid
or an inbred/variety, so long as the cross or breeding results in a plant or seed of the
present invention, such as, a seed having at least one allele comprising the specific and
unique DNA of event KKl79—2 and/or the consecutive nucleotides of SEQ ID NO: I or
SEQ ID NO: 2. Back—crossing to a parental plant and out—crossing with a non—transgenic
plant are also contemplated, as is vegetative ation. Descriptions of other breeding
methods that are commonly used for different traits and crops can be found in one of
several nces, for example, Fehr, in Breeding Methods for Cultivar Development,
Wilcox J. ed., American Society of my, n WI (1987).
Sexually crossing one plant with r plant, such as, cross—pollinating, may be
accomplished or facilitated by human intervention, for example: by human hands
collecting the pollen of one plant and contacting this pollen with the style or stigma of a
second plant; by human hands and/or human actions removing, destroying, or covering
the stamen or anthers of a plant (for example, by manual ention or by application of
a chemical gametocide) so that natural self—pollination is prevented and pollination
would have to take place in order for fertilization to occur; by human placement of
pollinating insects in a position for “directed pollination” (for example, by placing
beehives in orchards or fields or by caging plants with pollinating insects); by human
opening or removing of parts of the flower to allow for ent or t of foreign
pollen on the style or stigma; by selective placement of plants (for example, intentionally
planting plants in pollinating proximity); and/or by application of chemicals to precipitate
flowering or to foster receptivity (of the stigma for pollen).
In practicing this method, the step of sexually crossing one plant with , such
as, self—pollinating or selfing, may be accomplished or facilitated by human intervention,
for example: by human hands collecting the pollen of the plant and contacting this pollen
with the style or stigma of the same plant and then optionally preventing r
fertilization of the plant; by human hands and/or actions removing, destroying, or
covering the stamen or anthers of other nearby plants (for example, by detasseling or by
application of a chemical gametocide) so that natural cross—pollination is prevented and
self—pollination would have to take place in order for fertilization to occur; by human
placement of pollinating insects in a on for “directed pollination” (for example, by
caging a plant alone with ating insects); by human manipulation of the flower or its
parts to allow for self—pollination; by selective placement of plants (for example,
intentionally planting plants beyond pollinating proximity); and/or by application of
chemicals to precipitate flowering or to foster receptivity (of the stigma for pollen).
The present ion provides a plant part that is derived from a plant comprising
event KKl79—2. As used herein, a “plant part” refers to any part of a plant that is
comprised of material derived from a plant comprising event KKl79—2. Plant parts
include but are not limited to pollen, ovule, pod, flower, root or stem tissue, , and
leaf. Plant parts may be viable, nonviable, regenerable, and/or non—regenerable.
The present ion es a commodity product that is derived from a plant
sing event KKl79—2. As used herein, a dity product” refers to any
composition or product that is comprised of material derived from a plant, seed, plant
cell, or plant part comprising event KKl79—2. Commodity products may be sold to
consumers and may be viable or ble. ble commodity products include but
are not limited to nonviable seeds and grains; processed seeds, seed parts, and plant parts;
dehydrated plant tissue, frozen plant tissue, and processed plant tissue; seeds and plant
parts processed for animal feed for terrestrial and/or aquatic animal consumption, oil,
meal, flour, flakes, bran, fiber, and any other food for human consumption; and
biomasses and fuel products. Processed alfalfas are the largest source of forage legume
in the world. A plant comprising event KKl79—2 can thus be used to manufacture any
commodity product typically acquired from an alfalfa plant. Any such commodity
product that is derived from the plants comprising event KKl79—2 may contain at least a
detectable amount of the specific and unique DNA corresponding to event KKl79—2, and
specifically may contain a able amount of a polynucleotide having a tide
sequence of the consecutive nucleotides of SEQ ID NO: 1 and the consecutive
nucleotides of SEQ ID NO: 2. Any rd method of detection for polynucleotide
molecules may be used, including methods of detection disclosed herein. A commodity
product is within the scope of the present invention if there is any detectable amount of
WO 03558
the consecutive tides of SEQ ID NO: I or the consecutive nucleotides of SEQ ID
NO: 2, in the commodity product.
The plant, progeny, seed, plant cell, plant part (such as pollen, ovule, pod, flower,
root or stem tissue, and leaf), and commodity products of the present invention are
therefore useful for, among other things, growing plants for the purpose of producing
seed and/or plant parts comprising event KKl79—2 for agricultural purposes, producing
progeny comprising event 2 for plant breeding and research es, use with
microbiological ques for industrial and research applications, and sale to
consumers.
The present invention provides methods for producing plants with reduced lignin
and plants comprising event KKl79—2. Event KKl79—2 plant was produced by an
Agrobacterium mediated transformation method similar to that bed in US Patent
,914,451, using an inbred alfalfa line with the construct pFGllS. Construct pFGllS
contains a plant suppression cassette for downregulation of the CCOMT enzyme in
alfalfa plant cells. Transgenic alfalfa cells were regenerated into intact alfalfa plants and
individual plants were selected from the population of independently transformed
transgenic plants that showed desirable lar characteristics, such as, the integrity of
the transgene cassette, absence of the construct backbone sequence, loss of the unlinked
kanamycin resistance selection cassette. Furthermore, inverse PCR and DNA sequence
analyses were performed to determine the 5’ and 3’ insert—to—plant genome junctions, to
confirm the organization of the elements within the insert e l), and to determine
the complete DNA ce of the insert in a event KKl79—2 (SEQ ID NO: 5). In
addition, transgenic plants were ed and selected for reduced lignin under field
conditions. An alfalfa plant that contains in its genome the suppression cassette of
pFGl 18 is an aspect of the present invention.
Methods for producing a plant with reduced lignin sing transgenic event
KKl79—2 are provided. Transgenic plants used in these methods may be heterozygous
for the transgene. y plants produced by these s may be varietal or hybrid
plants; may be grown from seeds produced by a plant and/or from seed comprising event
KKl79—2 produced by a plant fertilized with pollen from a plant comprising event
KKl79—2; and may be homozygous or heterozygous for the transgene. Progeny plants
may be subsequently self—pollinated to generate a true ng line of plants, such as,
plants homozygous for the transgene, or alternatively may be ssed, for example,
bred with r unrelated plant, to produce a varietal or a hybrid seed or plant. As used
herein, the term “zygosity” refers to the rity of DNA at a specific chromosomal
location (locus) in a plant. In the present invention, the DNA specifically refers to the
transgene insert along with the junction sequence (event DNA). A plant is homozygous if
the transgene insert with the junction sequence is present at the same on on each
chromosome of a chromosome pair (4 s). A plant is considered heterozygous if the
transgene insert with the junction sequence is present on only one chromosome of a
some pair (1 allele). A Wild—type plant is null for the event DNA.
Progeny plants and seeds encompassed by these methods and produced by using
these methods are ct from other plants, for example, because the progeny plants and
seeds are inant and as such created by human intervention; contain at least one
allele that consists of the transgenic DNA of the present invention; and/or contain a
detectable amount of a polynucleotide sequence selected from the group consisting of
consecutive nucleotides of SEQ ID NO: 1, or consecutive nucleotides of SEQ ID NO: 2.
A seed may be selected from an individual progeny plant, and so long as the seed
comprises SEQ ID NO: 1, or SEQ ID NO: 2, it will be Within the scope of the present
invention.
The plants, progeny, seeds, plant cells, plant parts (such as pollen, ovule, pod,
flower, root or stem tissue, and leaves), and commodity ts of the present invention
may be evaluated for DNA ition, gene expression, and/or n expression.
Such evaluation may be done by using various methods such as PCR, sequencing,
northern blotting, southern analysis, western blotting, immuno—precipitation, and ELISA
or by using the methods of detection and/or the detection kits provided herein.
Methods of detecting the presence of itions specific to event KKl79—2 in a
sample are provided. One method consists of detecting the presence of DNA specific to
and derived from a cell, a tissue, a seed, a plant or plant parts comprising event KKl79—2.
The method provides for a template DNA sample to be contacted with a primer pair that
is capable of producing an on from event KKl79—2 DNA upon being subjected to
conditions appropriate for amplification, particularly an amplicon that comprises SEQ ID
NO: 1, and/or SEQ ID NO: 2, or the ments thereof. The amplicon is produced
from a template DNA le derived from event KKl79—2, so long as the template
DNA molecule incorporates the ic and unique nucleotide sequences of SEQ ID NO:
1, or SEQ ID NO: 2. The amplicon may be single or double stranded DNA or RNA,
depending on the polymerase selected for use in the production of the amplicon. The
method provides for detecting the amplicon molecule produced in any such amplification
reaction, and confirming within the sequence of the amplicon the presence of the
nucleotides corresponding to SEQ ID NO: 1, or SEQ ID NO: 2, or the complements
thereof. The detection of the nucleotides corresponding to SEQ ID NO: 1, and/or SEQ
ID NO: 2, or the complements thereof in the on are determinative and/or
stic for the presence of event KKl79—2 specific DNA and thus biological material
comprising event KKl79—2 in the sample.
Another method is provided for detecting the presence of a DNA molecule
corresponding to SEQ ID NO: 3 or SEQ ID NO: 4 in a sample consisting of material
derived from plant or plant tissue. The method consists of (i) obtaining a DNA sample
from a plant, or from a group of different , (ii) contacting the DNA sample with a
DNA probe molecule comprising the tides as set forth in either SEQ ID NO: I or
SEQ ID NO: 2, (iii) allowing the probe and the DNA sample to hybridize under stringent
ization conditions, and then (iv) detecting a ization event between the probe
and the target DNA sample. Detection of the hybrid composition is diagnostic for the
presence of SEQ ID NO: 3 or SEQ ID NO: 4, as the case may be, in the DNA sample.
e of hybridization is alternatively diagnostic of the absence of the transgenic event
in the sample if the appropriate positive controls are run concurrently. Alternatively,
determining that a particular plant contains either or both of the sequences corresponding
to SEQ ID NO: I or SEQ ID NO: 2, or the complements thereof, is determinative that the
plant contains at least one allele corresponding to event KKl79—2.
It is thus possible to detect the presence of a nucleic acid molecule of the present
invention by any well known nucleic acid amplification and detection methods such as
rase chain reaction (PCR), recombinase polymerase amplification (RPA), or DNA
hybridization using nucleic acid . An event—specific PCR assay is discussed, for
example, by Taverniers et al. (J. Agric. Food Chem., 53: 3041—3052, 2005) in which an
2012/044590
event—specific tracing system for transgenic maize lines Btll, Bt176, and GA21 and for
transgenic event RT73 is demonstrated. In this study, event—specific primers and probes
were designed based upon the sequences of the genome/transgene junctions for each
event. Transgenic plant event specific DNA detection methods have also been described
in US Patent Nos. 7,632, 985; 817; 7,368,241; 7,306,909; 7,718,373; 7,189,514,
7,807,357 and 7,820,392.
DNA detection kits are provided. One type of kit ns at least one DNA
molecule of sufficient length of contiguous nucleotides of SEQ ID NO: 3, SEQ ID NO:
, or SEQ ID NO: 6 to function as a DNA primer or probe specific for ing the
presence of DNA derived from transgenic event 2 in a sample. The DNA
molecule being detected with the kit comprises contiguous nucleotides of the sequence as
set forth in SEQ ID NO: 1. Alternatively, the kit may contain at least one DNA molecule
of sufficient length of contiguous nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ
ID NO: 6 to function as a DNA primer or probe ic for detecting the presence of
DNA derived from transgenic event KK179—2 in a sample. The DNA molecule being
detected with the kit comprises uous nucleotides as set forth in SEQ ID NO: 2.
An alternative kit employs a method in which the target DNA sample is contacted
with a primer pair as described above, then performing a nucleic acid amplification
on sufficient to produce an amplicon comprising the consecutive nucleotides of
SEQ ID NO: 1, and SEQ ID NO: 2. Detection of the amplicon and determining the
presence of the consecutive nucleotides of SEQ ID NO: 1, and SEQ ID NO: 2or the
complements thereof within the sequence of the amplicon is diagnostic for the ce
of event KK179—2 specific DNA in a DNA sample.
A DNA molecule sufficient for use as a DNA probe is provided that is useful for
ining, detecting, or for diagnosing the presence or even the absence of DNA
specific and unique to event KK179—2 DNA in a sample. The DNA molecule ns
the consecutive nucleotides of SEQ ID NO: 1, or the complement thereof, and the
consecutive nucleotides of SEQ ID NO: 2, or the complement thereof.
Nucleic acid amplification can be accomplished by any of the various nucleic
acid amplification methods known in the art, ing thermal and isothermal
amplification methods. The ce of the heterologous DNA insert, junction
2012/044590
sequences, or flanking sequences from event KK179—2 (with representative seed samples
comprising event KK179—2 deposited as ATCC PTA—11883) can be verified by
amplifying such sequences from the event using primers derived from the sequences
ed herein followed by standard DNA sequencing of the amplicon or of the cloned
DNA.
The amplicon produced by these methods may be detected by a plurality of
ques. One such method is Genetic Bit is (Nikiforov, et al. Nucleic Acid
Res. 22:4167—4175, 1994) where a DNA oligonucleotide is ed which overlaps both
the adjacent flanking genomic DNA sequence and the ed DNA sequence. The
oligonucleotide is immobilized in wells of a microwell plate. Following thermal
amplification of the region of interest (using one primer in the inserted sequence and one
in the adjacent flanking genomic sequence), a single—stranded amplicon can be hybridized
to the immobilized oligonucleotide and serve as a template for a single base extension
reaction using a DNA polymerase and labelled ddNTPs specific for the ed next
base. Readout may be fluorescent or ELISA—based. ion of a fluorescent or other
signal indicates the presence of the insert/flanking sequence due to successful
amplification, hybridization, and single base extension.
r method is the Pyrosequencing technique as described by Winge .
Pharma. Tech. 00:18—24, 2000). In this method an ucleotide is designed that
overlaps the adjacent genomic DNA and insert DNA junction. The oligonucleotide is
hybridized to a single—stranded amplicon from the region of interest (one primer in the
inserted ce and one in the flanking genomic sequence) and ted in the
presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5’
phosphosulfate and luciferin. ddNTPs are added individually and the incorporation
results in a light signal which is measured. A light signal indicates the presence of the
transgene insert/flanking sequence due to sful amplification, hybridization, and
single or multi—base extension.
Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492—498,
1999) is a method that can be used to detect the amplicon. Using this method an
oligonucleotide is designed which overlaps the genomic flanking and inserted DNA
junction. The oligonucleotide is hybridized to single—stranded amplicon from the region
of interest (one primer in the inserted DNA and one in the g c DNA
sequence) and incubated in the presence of a DNA polymerase and a cent—labeled
ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can
be measured as a change in polarization using a fluorometer. A change in polarization
indicates the presence of the transgene insert/flanking sequence due to sful
amplification, hybridization, and single base extension.
TAQMAN® (PE Applied Biosystems, Foster City, CA) may also be used to
detect and/or quantifying the presence of a DNA sequence using the instructions provided
by the manufacturer. Briefly, a FRET oligonucleotide probe is designed which overlaps
the c flanking and insert DNA junction. The FRET probe and amplification
primers (one primer in the insert DNA sequence and one in the flanking genomic
sequence) are cycled in the ce of a thermostable polymerase and dNTPs.
Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety
away from the quenching moiety on the FRET probe. A fluorescent signal indicates the
presence of the flanking/transgene insert sequence due to successful amplification and
hybridization.
Molecular Beacons have been described for use in sequence detection as
described in Tyangi, et al. (Nature Biotech.l4:303—308, 1996). Briefly, a FRET
oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA
junction. The unique structure of the FRET probe results in it containing secondary
structure that keeps the fluorescent and quenching moieties in close ity. The
FRET probe and amplification primers (one primer in the insert DNA sequence and one
in the flanking genomic sequence) are cycled in the presence of a thermostable
polymerase and dNTPs. Following successful amplification, hybridization of the FRET
probe to the target sequence results in the l of the probe ary structure and
l separation of the fluorescent and quenching moieties resulting in the production of
a fluorescent signal. The cent signal indicates the presence of the
flanking/transgene insert sequence due to successful amplification and hybridization.
Other described methods, such as, uidics (US Patent Publication No.
2006068398, US Patent No. 6,544,734) provide methods and devices to separate and
amplify DNA samples. l dyes are used to detect and measure specific DNA
molecules (WC/05017181). Nanotube devices (WC/06024023) that comprise an
electronic sensor for the detection of DNA molecules or nanobeads that bind specific
DNA molecules and can then be detected.
DNA detection kits can be developed using the compositions disclosed herein and
the methods well known in the art of DNA detection. The kits are useful for the
identification of event KKl79—2 in a sample and can be applied to methods for breeding
plants containing the appropriate event DNA. The kits may contain DNA primers or
probes that are similar or complementary to SEQ ID NO: 1—6, or fragments or
complements thereof.
The kits and detection methods of the present invention are therefore useful for,
among other , identifying event KKl79—2, selecting plant ies or hybrids
comprising event KKl79—2, detecting the presence of DNA derived from event KKl79—2
in a sample, and monitoring samples for the presence and/or absence of event KKl79—2
or , plant parts or commodity products comprising event KKl79—2.
The following es are included to demonstrate examples of certain
embodiments of the invention. It should be appreciated by those of skill in the art that the
techniques disclosed in the examples that follow represent approaches the inventors have
found function well in the practice of the invention, and thus can be considered to
constitute examples of preferred modes for its practice. r, those of skill in the art
should, in light of the t disclosure, appreciate that many changes can be made in
the specific embodiments that are disclosed and still obtain a like or r result without
departing from the spirit and scope of the invention.
EXAMPLES
Example 1: Isolation of Flanking Sequences Using Inverse PCR And fication of
Flanking Sequences by Sequencing
This e describes isolation of the alfalfa genomic DNA sequences flanking
the enic DNA insert using inverse PCR for event KKl79—2, and identification of
the flanking genomic sequences by sequencing.
Sequences flanking the T—DNA ion in event KK179—2 were determined
using inverse PCR as bed in Ochman et al., 1990 (PCR Protocols: A guide to
Methods and Applications, Academic Press, Inc.). Plant genomic DNA was ed
from both ype R2336 and the transgenic line from tissue grown under greenhouse
conditions. Frozen leaf tissue was ground with a mortar and a pestle in liquid nitrogen or
by mechanical grinding, followed by DNA extraction using methods known in the art.
This method can be modified by one skilled in the art to extract DNA from any tissue,
including, but not limited to seed.
An aliquot of DNA from each sample was digested with ction endonucleases
selected based on restriction analysis of the transgenic DNA. After self—ligation of the
restriction fragments, PCR amplification was performed using primers designed from the
transgenic sequence that would amplify sequences extending away from the 5’ and 3’
ends of the transgenic DNA. A variety of Taq polymerases and amplification systems
may be used. Table 2 shows an example of PCR amplification for flanking sequence
isolation using Phusion High Fidelity DNA rase (Cat. No. F5318 or F531L, New
d Biolabs), and Thermalcyclers Applied Biosystems GeneAmp 9700, ABI 9800
Fast Thermal Cycler and MJ Opticon.
Table I. An example of inverse PCR amplification for flanking ce isolation.
PCR master mix (per Volume Component
reaction) 2.9 ul water
0.05 ul Primer l (100 MM)
0.05 ul Primer l (100 MM)
.0 ul 2X Phusion Taq
2.0 ul ligated DNA
ul Total
DNA amplification in a fast Step Condition
thermocycler
1 98°C 30 sec
2 98°C 5 sec
3 60°C 10 sec
4 72°C 2 min
Go to step 2 30
times
6 72°C 4 min
7 10°C forever
8 End
PCR ts were ted by agarose gel electrophoresis and purified using a
QIAGEN gel purification kit (Qiagen, Valencia, CA). The subsequent products were
sequenced directly using standard sequencing protocols. Using these two methods, the 5’
flanking sequence, which extends into the left border sequence of the integrated DNA
insert including the CCOMT suppression cassette, was identified and is presented as SEQ
ID NO: 3 ([C] of Figure l). The 3’ flanking sequence, which extends into the right border
sequence of the integrated DNA insert including the CCOMT suppression cassette, was
identified and is ted as SEQ ID NO: 4 ([D] of Figure l). The transgenic DNA
integrated into the R2336 c DNA is presented as SEQ ID NO: 5 ([E] of Figure l).
The ed sequences were compared to the T—DNA sequence to identify the
flanking sequences and the co—isolated T—DNA nts. Confirmation of the presence
of the expression cassette was achieved by PCR with primers designed based upon the
deduced flanking sequence data and the known T—DNA sequence. The R2336 wild type
sequence corresponding to the same region in which the T—DNA was integrated in the
transformed line was isolated using primers designed from the flanking sequences in
KKl79—2. The flanking sequences in KKl79—2 and the R2336 wild type sequence were
ed against multiple nucleotide and protein databases. This information was used to
examine the relationship of the transgene to the plant genome and to look at the insertion
site integrity. The flanking sequence and wild type sequences were used to design
primers for TAQMAN® endpoint assays used to identify the events as described in
e 2.
Example 2: Event—Specific Endpoint TAQMAN®
This example describes an event—specific endpoint TAQMAN® thermal
amplification method for identification of event KKl79—2 DNA in a .
Examples of conditions useful with the event KKl79—2—specific nt
® method are described in Table 2 and Table 3. The DNA primers used in the
endpoint assay are primers SQ20901 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8) and
6—FAMTM labeled oligonucleotide probe PBlOl64 (SEQ ID NO: 9). 6FAMTM is a
fluorescent dye product of Applied Biosystems (Foster City, CA) attached to the DNA
probe. For TAQMAN® MGB (Minor Groove Binding) probes, the 5’exonuclease
activity of Taq DNA polymerase cleaves the probe from the 5’—end, n the
fluorophore and quencher. When hybridized to the target DNA strand, quencher and
fluorophore are separated enough to e a fluorescent signal.
Primers SQ20901 (SEQ ID NO: 7) and SQ23728 (SEQ ID NO: 8) when used as
described with probe PBlOl64 (SEQ ID NO: 9) produce an amplicon of 81 nt that is
diagnostic for event KKl79—2 DNA. The is includes a positive l from alfalfa
known to contain event KKl79—2 DNA, a negative control from non—transgenic alfalfa
and a negative control that contains no template DNA.
These assays are optimized for use with Applied Biosystems GeneAmp PCR
System 9700, ABI 9800 Fast Thermal Cycler and MJ Research DNA Engine FTC—225.
Other methods and apparatus known to those skilled in the art may be used to produce
amplicons that identify the event KKl79—2 DNA.
Table 2. Alfalfa KK179—2 Event—Specific Endpoint TAQMAN® PCR Conditions
Step
Il—‘ volume of 10 1
2X Universal Master Mix 1X final tration
(dNTPs, enzyme and buffer) of dNTPs, enzyme and
buffer
Event Primers SQ20901 and
SQ23728 Mix (resuspended 0 5 ul 1.0 MM final
in 18 megohm water to a concentration
concentration of 20 MM for
each primer)
Example: In a
microcentrifuge tube, the
following are added to
e 500 Ml at a final
concentration of 20 MM:
100 pl of Primer SQ20901 at
a concentration of 100 MM
100 ul of Primer SQ23728 at
a concentration of 100 MM
300 pl of 18 megohm water
Event 6—FAM MGB Probe
PBlOl64 0.2 M1 0.2 MM final
(resuspended in 18 megohm concentration
water to a concentration of 10
Internal control Primer—1 and
internal l —2 Mix 05 n1 1 HM final
(resuspended in 18 megohm concentration
water to a tration of 20
M for each nrimer)
Internal control VIC probe
(resuspended in 18 megohm 0.2 n1 0.2 MM final
water to a concentration of 10 tration
7 Extracted DNA (template):
1. Leaf or seed samples to be 30 n1
analyzed
2. Negative control
(non—transgenic DNA)
3. Negative water control
(no template control)
4. Positive control
(KK179—2 DNA)
WO 03558
Table 3. Endpoint TAQMAN® thermocycler conditions
Cycle N0. Settings
1 50°C 2 minutes
1 95°C 10 minutes
95°C 15 seconds
64°C 1 minute (—1°C/cycle)
95°C 15 seconds
54°C 1 minute
°C Forever
A deposit of entative alfalfa event KK179—2 seed sed above and
recited in the claims, has been made under the st Treaty with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA. 20110. The
ATCC accession number is PTA—11833. The deposit will be maintained in the
tory for a period of 30 years, or 5 years after the last request, or for the effective
life of the patent, Whichever is longer, and will be replaced as necessary during that
period.
Having illustrated and described the principles of the present invention, it should
be apparent to s skilled in the art that the invention can be modified in arrangement
and detail Without departing from such principles. We claim all modifications that are
Within the spirit and scope of the appended claims.
Example 3.
Example 3: ADL Measurements in the Lower Stem of Reduced Lignin Alfalfa Events
Table 4. Lower stem ADL measurements for the 6 reduced lignin alfalfa events in two
fall dormant (FD) germplasms from 3 ons in 2008
Dormancy P—value
germplasm Mean UCI @ Diff.
FD <.001
KK376 FD 7.37 —2.29 —2.55 —2.04 -23.75 <.001
KK465 FD 7.30 —2.36 —2.59 —2.13 —24.44 <.001
JJ041 FD 7.71 —1.77 -2.01 —1.53 —18.70 <.001
JJ266 FD 6.98 —2.50 —2.74 —2.26 —26.38 <.001
KK136 FD 7.38 —2.10 —2.34 _—22.14 <.001
KK179 FD 7.56 —1.92 —2.16 —1.68 —20.24 <.001
KK376 FD 6.51 —2.97 -3.21 -2.73 —31.33 <.001
KK465 FD 7.33 —2.15 —2.39 -1.91 —22.68 <.001
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
FD: Fall Dormant
KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / l * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Event positive plants in Table 4 showed a significant (p30.05) decrease in lower
stem ADL which ranged from 18—31% when compared to the pooled negative control.
KK179—2 alfalfa event has the reduced lignin phenotype fied by the “sweet spot”
selection method.
WO 03558
Table 5. Lower stem ADL measurements for the 6 reduced lignin alfalfa lead events in
fall dormant (FD) germplasms grown in 4 locations in 2009.
Control % Diff. P—value
Mean
.79 -12.26 <.001
.79
.79
.79
8.49 —2.29 —2.54 —2.04
8.55 10.79 —2.24 —2.47 -2.00
iations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
FD: Fall t
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = ility of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Table 6. Lower stem ADL measurements for the 6 reduced lignin alfalfa lead events in
fall dormant (FD) germplasms grown at 2 locations in 2009
Control % Diff. P—value
Mean
11.73 —19.72 <.001
KK376 8.73 11.73 —3.00 —3.30 —2.70 —25.57 <.001
KK465 9.17 11.73 -2.56 —2.85 —2.27 —21.84 <.001
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
FD: Fall t
KK179=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
e = probability of a greater absolute ence under the null esis (2—tailed
test for significance).
Table 7. Lower stem ADL measurements for the 6 d lignin alfalfa lead events in
non t (ND) germplasms grown at 4 locations in 2009
Control % Diff. P—value
Mean
.89 —l4.50 <.001
KK376 8.26 10.89 —2.63 —2.86 —2.40 —24.14 <.OOl
KK465 9.09 10.89 —l.81 —2.03 —l.58 —l6.58 <.OOl
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
ND: Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 t difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence al of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Table 8. Lower stem ADL measurements for 6 reduced lignin alfalfa lead events in non
dormant (ND) germplasms grown at 2 locations in 2009.
Control % Diff. e
Mean
ll.l6 -20.2l <.001
KK376 8.35 11.16 —2.81 —3.20 —2.42 —25.16 <.001
KK465 9.14 11.16 -2.03 —2.41 —l.64 —18.15 <.001
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant ence
ND: Non Dormant
KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Tables 6—8 show 2009 data for lower stem ADL in a fall dormant (ED) and non
dormant (ND) asms at 4 and 2 locations respectively. The 6 event positive
lines showed a significant (ng.05) reduction in ADL ranging from 12—26% when
compared to the pooled negative control, with the lead event KKl79 showing a reduction
in ADL of 18—22%.
Example 4.
e 4: NDFD ements in the Lower Stem of Reduced Lignin Alfalfa Events
Table 9. Lower stem NDFD measurements for the 6 d lignin alfalfa lead events in
fall dormant (FD) germplasms grown at 3 locations in 2008
Dormancy Control
germplasm Mean UCI @ Diff.
FD 27.70
FD 34.12 27.70 6.43 5.57 7.28 23.20
KK465 FD 35.33 27.70 7.63 6.88 8.38 27.55
FD 33.27 27.70 5.56 4.69 6.44 20.08
JJ266 FD 34.98 27.70 7.27 6.40 8.15 26.25
KK136 FD 3429. 27.70 659. 5 .7 1 7.46 23 .77 <. 001
KKl79 FD 33.13 27.70 5.42 4.54 6.30 19.57 <.001
KK376 FD 37.44 27.70 9.74 8.86 10.61 35.14 <.001
KK465 FD 35.34 27.70 7.64 6.76 8.52 27.57 <.001
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral ent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
KKl79=KK179—2 reduced lignin alfalfa lead event
Delta 2 ence between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a r absolute difference under the null hypothesis (2—tailed
test for icance).
Lower stem NDFD for the 6 reduce lignin events in fall dormant (FD)
germplasms at 3 locations. Event positive plants showed a icant (p30.05) increase
in lower stem NDFD which ranged from 18—35% when compared to the pooled negative
control.
Table 10. Lower stem NDFD measurements for the 6 reduced lignin alfalfa lead events
in fall dormant (FD) germplasms grown at 4 locations in 2009
Dormancy Control p-
germplasm Mean value
JJ04l FD 28.09 22.31 5.79 4.89 6.69 25.95 <.001
JJ266 FD 28.58 22.31 6.27 5.46 7.08 28.11 <.001
KK136 28.88 22.31 6.57 5.58 7.56 29.46
KKl79 FD 27.20 22.31 4.90 4.01 5.78 21.95 <.001
KK376 FD 28.65 22.31 6.34 5.38 7.31 28.43 <.001
KK465 FD 28.21 22.31 5.91 4.99 6.83 26.49 <.001
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, llulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — l)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence al of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute ence under the null hypothesis led
test for significance).
Table ll. Lower stem NDFD measurements for the 6 reduced lignin alfalfa lead events
in non t (ND) germplasms grown at 2 locations in 2009
Dormancy Control
germplasm Mean Mean
ND 2088
ND 20.88 6.31
KK465 ND 2088 6.14
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly digestible ents in plant cell wall
lose, hemicellulose, lignin (units 2 % of dry matter))
ND: Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence al of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
e = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
WO 03558
Table 12. Lower stem NDFD measurements for the 6 reduced lignin alfalfa lead events
in fall dormant (FD) germplasms grown at 4 locations in 2009
Dormancy Control
germplasm Mean Mean
FD 22.1 1
ND 22.11 7.70
KK465 ND 2211 5.26
Abbreviations used in the tables that follow:
NDFD 2 Neutral ent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
ND 2 Non t
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and l (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence al of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
WO 03558
Table 13. Lower stem NDFD measurements for the 6 reduced lignin a lead events
in non dormant (ND) germplasms grown at 2 locations in 2009
Event Dormancy Control
germplasm Mean
JJ266 ND 28.73 22.39 6.34
6-27
KK376 ND 29.87 22.39 7.48
KK465 ND 28.95 22.39 56.56 5.00 8.11 29.29 <.001
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
ND 2 Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference n Event and Control (Delta / l * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute ence under the null hypothesis (2—tailed
test for significance).
Table ll—l3 show 2009 data for lower stem NDFD in fall dormant (ED) and non
dormant (ND) germplasm at 4 and 2 ons respectively. The 6 event positive reduced
lignin alalfa events showed a significant (ng.05) increase in NDFD ranging from 22—
36% when compared to the pooled negative control, with the lead event KKl79—2
showing an increase in NDFD of 22—28%.
Example 5.
Example 5: Vigor Rating for Reduced Lignin Alfalfa Events
2012/044590
Table 14. Vigor ratings for the 2 reduced lignin alfalfa events, JJ266 and KK179—2
compared to commercial checks and the null controls in 3 locations. The reduced lignin
event KK179—2 resulted in no off—types for vigor rating scale.
Location Location 2 Location 3
8.0 7.4 7.8
J]266, null 7.8 7.4 8.0
KK179—2 8.0 7.6 7.6
KK179, null 7.4 7.7 8.1
cial Check 1 6. 9 6.7 7.1
cial Check 2 7.1 7.0 6.7
Commercial Check 3 7 8 8 1 8 1
Commercial Check 4 7.3 7.6 7.9
Data collected for these trials are as follows: plant vigor (scored 1—10, 10 being
best) taken 21 days after previous t and the second week of May for the spring
score, lodging tolerance (scored 1—10, 10 being perfectly upright) taken 1—5 days prior to
harvest per season. Plant yield (grams of dry matter (DM) per plant) taken after plants
were dried, NDFD (using CAI NIR calibration for RL alfalfa) and ADL (using NIR
calibration for RL alfalfa).
Example 6.
Example 6: ADL Measurements in the Whole Plant for Reduced Lignin Alfalfa Events
Table 15. Whole plant hay ADL measurements for the 6 reduced lignin a lead
events in fall dormant (FD) germplasms grown in 4 locations in 2009
Control % Diff. P—value
Mean
.66 —12.27 <.001
JJ266 4.85 5.66 —1.04 —2.47 —0.59
KK136 4.81 5.66 —1.12 —2.53 —0.59
KK179 5.11 _5.66 —0.80 —2.19 —0.31
KK376 4.73 5.66 —1.19 —2.54 —0.66
KK465 5.18 5.66 —0.74 —2.47 —0.22
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
FD: Fall Dormant
KK179=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute ence under the null hypothesis (2—tailed
test for significance).
Table 16. Whole plant hay ADL measurements for the 6 reduced lignin a lead
events in non dormant (ND) germplasms grown in 2 locations in 2009
Event Event Control Delta Delta Delta % Diff. P—value
Mean Mean LCI @ UCI @
90% 90%
.40 6.16 —0.77 —1.22 —0.31 —12.43
.27 6.16 —0.89 —1.30 —0.48 44.47
KK465 5.57 6.16 —0.60 —1.00 —0.19 —9.69 0.016
Abbreviations used in the tables that :
ADL 2 Acid Detergent , % of dry matter
LSD: Least Significant Difference
ND: Non Dormant
KK179=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a r te difference under the null hypothesis (2—tailed
test for significance).
Table 17. Whole plant hay ADL measurements for the 6 reduced lignin alfalfa lead
events in fall dormant (FD) germplasms grown in 4 ons in 2009
Control % Diff. P—value
Mean
.77 —l4.64 <0.001
.77 —1.16 —20.09
.77 —0.49 —0.7l —0.26 —8.43
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
FD: Fall Dormant
KKl79=KKl79—2 d lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper ence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Whole plant ADL data from 2009 across 4 ons is shown in Table 17 and 19.
The 6 reduced lignin ve events in fall dormant germplasm showed a significant
(p30.05) decrease in ADL ranging from 8—l9% when compared to the pooled negative
control. Event KKl79—2 had a 9.8% and a 9.45 reduction in ADL in the fall dormany
germplasms respectively.
Table 18. Whole plant hay ADL measurements for the 6 reduced lignin alfalfa lead
events in non dormant (ND) germplasms grown in 2 locations in 2009
Control % Diff. P—value
Mean
.94 —ll.59 0.006
.02 5.94 —0.92 —15.47
.37 5.94 —0.57 —0.96 —0.18 —9.6l
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
LSD: Least Significant Difference
ND: Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference n Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence al of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for icance).
Whole plant ADL data from 2009 across 2 locations is shown in Table 18 and 20.
The 6 reduced lignin positive events in the non t germplasm showed a significant
(p30.05) decrease in ADL ranging from 10—l6% when compared to the pooled ve
control. Five of the 6 events showed a significant se in ADL ranging from 10—18%
when compared to the pooled negative control. Event KKl79—2 had 12.3% and 10.9%
reduction in ADL in the non dormant germplasms respectively.
Table 19. Whole plant hay ADL measurements for the reduced lignin a event
KKl79—2 in two fall dormant (FD) germplasms grown in 4 locations in 2009 compared to
cial checks
Commercial Dormancy KKl79
Check Germplasm
FDl <.001
FDl 5.22 5.69 —0.47 —0.76 —0.18 —8.31 0.008
FDl 5.22 5.38 —0.17 —0.46 0.13 —3.08 0.350
.22 5.59 —0.38 —6.75 0.034
FD2 5.10 6.12 —1.02 —1.31 —0.73 — 6.67 <.001
FD2 0-001
FD2 5.10 5.38 —0.28
FD2 5.10 5.59 —0.49 —0.79 —0.20 —8.83 0.006
Abbreviations used in the tables that follow:
ADL 2 Acid ent Lignin, % of dry matter
FD: Fall Dormant
KKl79=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null esis (2—tailed
test for significance).
Table 20. Whole plant hay ADL measurements for the reduced lignin alfalfa event
KKl79—2 in two non dormant (ND) germplasm grown in 2 locations in 2009 compared to
commercial checks
Commercial Germplasm KKl79—
Check 2
.29
ND1 5.29 5.81 0.01 —8.92
ND1 5.29 5.77 0.05 —8.34
NDl 5.29 —10.61
ND2 5.39 —5.77
ND2 5.81 —0.41 —0.96 0.13 —7.11
ND2 5.39 5.77 —0.38 —0.92 0.17 —6.51
ND2 5.39 5.92 —0.52 —1.07 0.02 —8.82
Abbreviations used in the tables that follow:
ADL 2 Acid Detergent Lignin, % of dry matter
ND: Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — l)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower ence Interval of Delta value using an alpha level of
gifia UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
fill/glue = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Tables 19 and 20 contain whole plant ADL data for the reduced lignin alfalfa
event KKl79—2 compared to commercial checks. The KKl79—2 event showed a
significant (p301) decrease in ADL when compared to 3 of the 4 fall t
commercial checks which ranged from 6.8 — 16.7% (Table 19, data from 4 locations).
KKl79—2 event in non dormant ound germplasm (ND1) showed a decrease (p302)
in ADL compared to all 4 non dormant commercial checks ranging from 7.6 — 10.6%
(Table 20, data from 2 locations). The KKl79—2 event in non t background
germplasm (ND2) showed a overall decrease (p302) in ADL compared to all 4 non
dormant commercial checks with a significant (p301) decrease of 8.8% ed to
cial event 4 (ND2, data from 2 locations).
Example 7. NDFD Measurements in the Whole Plant for Reduced Lignin Alfalfa Events
Table 21. Whole plant hay NDFD ements for the 6 reduced lignin a lead
events in fall dormant (FD) germplasms grown in 4 locations in 2009.
Event Event Control Delta Delta Delta % Diff. P—value
LCI @ UCI @
Mean Mean
90% 90%
KK465 42.13 39.47
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly ible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower ence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Table 22. Whole plant hay NDFD measurements for the 6 reduced lignin alfalfa lead
events in non dormant (ND) germplasms grown in 2 locations in 2009
Control % Diff. P—value
Mean
.41 5.23 14.76 0.011
.41 5.41 8.46
.41 3.25 6.70
.41 4.96 8.19
39.75 35.41 4.35 0.96 7.73 12.28
38.72 35.41 3.32 0.26 6.37 9.37
Abbreviations used in the tables that follow:
NDFD 2 l Detergent Fiber Digestibility, % of NDF (NDF 2 l detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
ND: Non Dormant
KK179=KK179—2 d lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 t difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater te difference under the null hypothesis (2—tailed
test for significance).
Table 23. Whole plant hay NDFD measurements for the 6 reduced lignin alfalfa lead
events in fall dormant (FD) germplasms grown in 4 locations in 2009.
Event Event l Delta Delta Delta % Diff. P—value
Mean Mean LCI @ UCI @
90% 90%
JJ041 44.42 38.96 5.46 3.92 7.00
JJ266 45.19 38.96 6.22 4.72 7.73
KK136 43.63 _38.96 4.66 3.16 6.17
KK179 42.56 38.96 3.60 2.10 5.10
KK376 45.41 38.96 6.45 4.90 7.99
KK465 41.52 38.96 2.55 1.05 4.06 6.55 0.005
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly ible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
KK179=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
e = ility of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Whole plant NDFD data from 2009 across 4 locations is shown in Table 23 and
. The 6 reduced lignin positive events in fall dormant germplasm showed a significant
(p30.05) increase in NDFD g from 7—16% when ed to the pooled negative
control. Event KKl79—2 had a 7.5% and 9.2% increase in NDFD in the fall dormant
germplasm respectively.
Table 24. Whole plant hay NDFD measurements for the 6 reduced lignin alfalfa lead
events in non dormant (ND) germplasms grown in 2 locations in 2009.
Control % Diff. P—value
Mean
37.21 10.06 0.045
37.21 13.05 0.007
41.48 37.21 4.27 1.34 7.21 11.49
KK376 42.22 37.21 5.01 1.95 8.08 13.47 0.007
KK465 40.35 37.21 3.15 0.21 6.08 8.46 0.078
Abbreviations used in the tables that follow:
NDFD 2 Neutral ent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
ND: Non Dormant
KKl79=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — l)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = ility of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Whole plant NDFD data from 2009 across 2 locations is shown in Table 24 and
26. The 6 reduced lignin positive events in non dormant germplasm showed a significant
(p301) increase in NDFD ranging from 8—15% when compared to the pooled negative
control. Event KKl79—2 had a 14.0% and 11.5% increase in NDFD in the non dormant
germplasm respectively.
Table 25. Whole plant hay NDFD measurements for the reduced lignin alfalfa event
KKl79—2 in two fall dormant (FD) germplasms grown in 4 locations in 2009 compared to
commercial checks
cial Germplasm KKl79 % P—
Check Diff. value
FD1 42.17 6.07 4.27 7.86 16.80 <.001
FD1 42.17 1.83 0.00 3.66 4.53 0.101
FD1 2.72 2.16 0.423
FD1 5.12 8.47 0.003
FD2 7.76 16.44 <.001
FD2 42.03 1.70 -0.17 3.56 4.21 0.134
FD2 42.03 0.76 -1.10 2.63 1.84 0.502
4 FD2 42.03 38.87 3.16 1.30 5.03 8.13 0.005
Abbreviations used in the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 neutral detergent
fiber. ents the stible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
FD: Fall Dormant
KKl79=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — l)
% Diff 2 Percent ence between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence al of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
WO 03558
Table 26. Whole plant hay NDFD measurements for the reduced lignin alfalfa event
KKl79—2 in two non dormant (ND) asm grown in 2 locations in 2009 compared to
commercial checks
Commercial asm KKl79 % P—
Check Diff. value
. . . . . 9.75 0.126
ND1 41.46 37.12 4.34 0.39 8.30 11.70 0.071
ND1 41.46 34.71 6.74 2.79 10.70 19.43 0.005
ND1 41.46 35.70 5.75
40.38 37.77 2.60 —1.49 6.70 6.89 0.296
40.38 34.71 5.66
ND2 40.38 35.70 4.67 0.58 8.77 13.09 0.061
Abbreviations usedin the tables that follow:
NDFD 2 Neutral Detergent Fiber Digestibility, % of NDF (NDF 2 l detergent
fiber. Represents the indigestible and slowly digestible components in plant cell wall
(cellulose, hemicellulose, lignin (units 2 % of dry matter))
ND: Non Dormant
KKl79=KK179—2 reduced lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
Tables 25 and 26 contain whole plant NDFD data for the reduced lignin alfalfa
event KKl79—2 compared to commercial checks. The KKl79—2 event showed an
increase (p302) in NDFD when compared to 3 of the 4 fall dormant commercial checks
which ranged from 4.2 — 16.8% (Table 25, data from 4 locations). 2 event
showed an se (p302) in NDFD compared to all 4 non dormant commercial checks
(ND1) ranging from 9.8—19.4% (Table 26, data from 2 locations). The KKl79—2 event
showed an increase (p302) in NDFD compared to all 4 non dormant commercial checks
(ND2), which ranged from 8.8 —16.3% (Table 26, data from 2 locations).
Example 8.
Example 8: Yield Across Location Analysis for Reduced Lignin Alfalfa Events
Table 27. Yield across location analysis for 6 reduced lignin events for in fall dormant
(ED) and non—dormant (ND) backgrounds compared to pooled negative controls
l P-
UCI @ Diff. value
Mean
2008 0.532
FD 2008 364.72 15.40 -12.94 43.74 4.41 0.370
[\3OO 00 -12.21 0.048
2008 5.49 0.309
FD 2008 354.92 5.60 -29.85 41.05 1.60 0.794
[\3OO 00 358.74 349.32 . 2.70 0.617
2009 1148.41 8 -9.00 0.083
2009 156.50 1591.58 0.724
2009 1468.30 1591.58 0.164
2009 1577.84 1591.58 0.863
2009 1371.19 1591.58 0.011
2009 1459.44 8 . 0.121
2009 591.17 764.86 0.018
2009 758.32 0.923
2009 771.81 . 135.10 0.91 0.928
2009 754.11 —10.75 —130.04 108.54 -0.881
NOO\O 0.014
2009 637.67 -16.63 0.064
iations used in the tables that follow:
Yield: Yield calculated on a per plant basis in grams
FD: Fall Dormant
ND: Non Dormant
KKl79=KKl79—2 d lignin alfalfa lead event
Delta 2 difference between Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis (2—tailed
test for significance).
The data in Table 27 shows the across on yield analysis for the 6 reduced
lignin events in the fall dormant (ED) and non dormant (ND) germplasms compared to
the pooled negative control. There were no significant decrease in yield is detected for
KKl79—2 when compared to the pooled negative controls.
WO 03558
Table 28. Yield across location analysis for Event KKl79—2 compared to cial
checks
Com- Control % Diff. P-
mercial value
Mean
Check
54.00 <.001
19.62 0.008
368.51 349.47 5.45 0.397
368.51 301.09 22.39 0.003
1361.60 1106.96 23.00 0.003
1361.60 1289.66 5.58 0.412
1361.60 1396.58 -2.51 0.690
1361.60 1225.01 11.15 0.120
129.80 2.31 0.802
61.42 -6.39 0.451
698.51 54.12 -58.66 166.89 7.75 0.427
8 ND 2009 752.63 618.75 133.89 21.11 246.66 21.64 0.052
Abbreviations used in the tables that follow:
Yield: Yield calculated on a per plant basis in grams
FD: Fall Dormant
ND: Non Dormant
KKl79=KKl79—2 reduced lignin alfalfa lead event
Delta 2 difference n Event and Control means (Event — Control)
% Diff 2 Percent difference between Event and Control (Delta / Control * 100)
Delta LCI @90% = Lower Confidence Interval of Delta value using an alpha level of
0.10
Delta UCI @90% 2 Upper Confidence Interval of Delta value using an alpha level of
0.10
P—value = probability of a greater absolute difference under the null hypothesis led
test for significance).
Yield data for d lignin alfalfa lead event in fall dormant (FD) and non—dormant
(ND) germplasms resulted in no significant yield decrease when compared to 8
commercial checks.
BUDAPEST RESTRICTED CERTIFICATE OF DEPOSIT
BUDAPEST TREATY ON THE INTERNATIONAL ITION OF
THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7.3
AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2
The American Type Culture Collection (ATCC®) has received your t of seeds/strain(s)/strain(s) in connection
with the filing of an application for patent. The following information is provided to fulfill Patent Office requirements.
Ms. Monica Ravanello
to Company
1920 5th Street
Davis, CA 95616
Deposited on Behalf of: Monsanto Company
Date of Receipt of seeds/strain(s) by the ATCC®: April 18, 2011
Identification Reference by Depositor: ATCC ®Patent Deposit Designation: Quantity Received:
Alfalfa, Medicago sativa, KK179-2 PTA-11833 100packets/25 seeds in each packet
The ATCC® understands that:
1. The deposit of these seeds/strain(s) does not grant ATCC® a license, either express or implied, to ge the
patent, and our release of these seeds/strain(s) to others does not grant them a license, either express or
implied, to infringe the patent.
2. If the t should die or be destroyed during the effective term of the patent, it shall be your responsibility
to replace it with viable material. It is also your responsibility to supply a sufficient quantity for distribution for
the deposit term. ATCC® will distribute and maintain the material for 30 years or 5 years following the most
recent t for the deposit, whichever is longer. The United States and many other countries are signatory
to the Budapest Treaty.
Prior to the issuance of a U.S. Patent, the ATCC® agrees in consideration for a me e charge, not to
distribute these seeds/strain(s) or any information relating o or to their deposit except as instructed by the
depositor or nt patent office. After nt patent issues we are responsible to release the seeds/strain(s) and
they will be made available for distribution to the public without any restrictions. We will inform you of requests for the
seeds/strain(s) for 30 years from date of deposit.
Doc ID: 20127
Revision: 2
ive Date: 10/13/2009
Page 1 of 2
The deposit was tested May 03, 2011 and on that date, the seeds/strain(s) were viable
International Depository Authority: American Type Culture Collection (ATCC®), Manassas, VA, USA
Signature of person having authority to ent ATCC®:
Ü·¹·¬¿´´§ ?·¹²»¼ ¾§ Ô¿¬¸¿ ο³¿µ®·?¸²¿²
ÜÒæ ¸¿ ο³¿µ®·?¸²¿²ô ±ãßÌÝÝô
Latha Ramakrishna n ±«ã×ÐÔÍ Ü»°¿®¬³»²¬ô
»³¿·´ã´®¿³¿µ®·?¸²¿²à¿¬½½ò±®¹ô ½ãËÍ
Ü¿¬»æ îðïïòðëòðí ïíæîçæðï óðìùððù
May 3, 2011
ATCC® Patent Depository Date
cc: Nikki Davis, Asst. GC
Ref: Docket or Case No: 57978
Doc ID: 20127
Revision: 2
Effective Date: 10/13/2009
Page 2 of 2
Claims (16)
1. A seed of an alfalfa plant comprising event 2, wherein a representative sample of seed comprising event KK179-2 has been deposited with the an Type e Collection (ATCC) with the Patent Deposit Designation PTA-11833.
2. An alfalfa plant or parts f comprising event KK179-2 produced by growing the seed of claim 1.
3. The alfalfa plant or parts thereof of claim 2, comprising pollen, ovule, flowers, shoots, roots or leaves.
4. A progeny plant or parts thereof an alfalfa plant of claim 2 wherein said progeny plant or parts thereof comprise alfalfa event KK179-2.
5. The progeny plant or parts thereof, of claim 4 r comprising pollen, ovule, flower, shoots, roots or leaves.
6. The alfalfa plant or seed or parts f of claim 4, the genome of which produces an amplicon comprising a DNA molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
7. A recombinant DNA molecule comprising a. a polynucleotide molecule selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; b. a polynucleotide le with at least 90% identity to the full length of SEQ ID NO: 6; or c. a polynucleotide molecule complementary to a) or b).
8. The DNA molecule of claim 7, wherein a representative sample of seed comprising event KK179-2 has been deposited under ATCC Accession NO. PTA-11833.
9. The DNA molecule of claim 7, wherein said DNA molecule is an amplicon ed from a template molecule derived from DNA from event KK179-2.
10. A polynucleotide probe diagnostic for the presence of event 2 DNA, n said polynucleotide probe is of ient length to bind to a nucleic acid molecule comprising SEQ ID NO: 1, and SEQ ID NO: 2, or ments thereof, and wherein said polynucleotide probe hybridizes under stringent ization conditions with a DNA molecule comprising SEQ ID NO: 1, or SEQ ID NO: 2, or complements thereof and does not hybridize under stringent hybridization conditions with a DNA molecule not comprising SEQ ID NO: 1, SEQ ID NO: 2, or complements thereof.
11. A method of detecting the presence of a DNA molecule d from event KK179-2 in a DNA sample, the method sing: a. contacting said DNA sample with the cleotide probe of claim 6; b. subjecting said DNA sample and said polynucleotide probe to stringent hybridization conditions; and c. detecting ization of said polynucleotide probe to said DNA molecule derived from event KK179-2 in said DNA sample.
12. A pair of DNA molecules consisting of a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein said first and second DNA molecules comprise a polynucleotide molecule having a nucleotide sequence of sufficient length of utive nucleotides of SEQ ID NO: 6, or a complement thereof, to function as DNA primers when used together in an amplification reaction with a template derived from event KK179-2 to produce an amplicon diagnostic for event KK179-2 DNA in a sample.
13. A DNA detection kit comprising at least one polynucleotide molecule of sufficient length of consecutive nucleotides of SEQ ID NO: 6, or complements thereof, to function as a DNA primer or polynucleotide probe specific for detecting the presence of DNA derived from event KK179-2, wherein detection of said DNA is diagnostic for the presence of said KK179-2 DNA in a sample.
14. The DNA detection kit of claim 13, wherein at least one polynucleotide molecule is selected from the group ting of SEQ ID NO: 1, and SEQ ID NO: 2.
15. A microorganism comprising the DNA molecule of claim 7.
16. The microorganism of claim 15, n said microorganism is a plant cell.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161503373P | 2011-06-30 | 2011-06-30 | |
US61/503,373 | 2011-06-30 | ||
US201261664359P | 2012-06-26 | 2012-06-26 | |
US61/664,359 | 2012-06-26 | ||
PCT/US2012/044590 WO2013003558A1 (en) | 2011-06-30 | 2012-06-28 | Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ618754A NZ618754A (en) | 2014-12-24 |
NZ618754B2 true NZ618754B2 (en) | 2015-03-25 |
Family
ID=
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