U.S. patent application number 16/023851 was filed with the patent office on 2018-12-27 for molecular markers for low palmitic acid content in sunflower (helianthus annus), and methods of using the same.
The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Jan E. Backlund, James T. Gerdes, Xueyi Hu, Mandy Sullivan-Gilbert.
Application Number | 20180371483 16/023851 |
Document ID | / |
Family ID | 49213625 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371483 |
Kind Code |
A1 |
Hu; Xueyi ; et al. |
December 27, 2018 |
MOLECULAR MARKERS FOR LOW PALMITIC ACID CONTENT IN SUNFLOWER
(HELIANTHUS ANNUS), AND METHODS OF USING THE SAME
Abstract
This disclosure concerns methods and compositions for
identifying sunflower plants that have a low palmitic acid content
phenotype. Some embodiments concern molecular markers to identify,
select, and/or construct low palmitic acid content plants and
germplasm, or to identify and counter-select relatively high
palmitic acid content plants. This disclosure also concerns
sunflower plants comprising a low palmitic acid content phenotype
that are generated by methods utilizing at least one marker
described herein.
Inventors: |
Hu; Xueyi; (Westfield,
IN) ; Sullivan-Gilbert; Mandy; (Lebanon, IN) ;
Backlund; Jan E.; (Indianapolis, IN) ; Gerdes; James
T.; (London, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Family ID: |
49213625 |
Appl. No.: |
16/023851 |
Filed: |
June 29, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13837877 |
Mar 15, 2013 |
10036029 |
|
|
16023851 |
|
|
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61613383 |
Mar 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
A01H 1/04 20130101; C12N 15/8247 20130101; A01H 5/10 20130101; C12Q
1/6895 20130101; A01H 1/02 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/10 20060101 A01H005/10; A01H 1/04 20060101
A01H001/04; C12Q 1/6895 20060101 C12Q001/6895; A01H 1/02 20060101
A01H001/02 |
Claims
1. A method for identifying and producing a sunflower plant or
germplasm that comprises low palmitic acid content, the method
comprising: isolating a nucleic acid from a first sunflower plant
or germplasm; detecting whether a marker allele that positively
correlates with low palmitic acid content is present in the
isolated nucleic acid by amplifying the isolated nucleic acid with
an amplification primer or primer pair that is complementary or
partially complementary to at least a portion of the marker
selected from the group consisting of HA0031B, HA0908, HA1665,
HA0304A_H757B, HA0850_H757B, and HA0870, to generate at least one
amplicon; and detecting the at least one marker amplicon;
identifying that the first sunflower plant or germplasm comprises
low palmitic acid content when the at least one marker amplicon is
detected; and crossing the first sunflower plant or germplasm
identified for low palmitic acid content with a second sunflower
plant or germplasm, to produce a sunflower plant or germplasm
progeny comprising low palmitic acid content.
2. The method according to claim 1, wherein the isolated nucleic
acid is a DNA molecule or RNA molecule.
3. The method according to claim 1, wherein the amplifying
comprises utilizing a polymerase chain reaction (PCR) or ligase
chain reaction (LCR) using the nucleic acid isolated from the first
sunflower plant or germplasm as a template in the PCR or LCR.
4. The method according to claim 1, wherein detecting the at least
one marker amplicon comprises using software selected from
TASSEL.TM., GeneFlow.TM., and MapManager-QTX.TM..
5. The method according to claim 1, wherein the method comprises
selecting the first sunflower plant or germplasm.
6. The method according to claim 1, wherein the method comprises
selecting the sunflower plant or germplasm progeny.
7. The method according to claim 1, wherein the second sunflower
plant or germplasm is a plant or germplasm from an elite sunflower
variety or an exotic sunflower variety.
8. A method for identifying and producing a sunflower plant or
germplasm that comprises low palmitic acid content, the method
comprising: amplifying from genomic DNA of the sunflower plant or
germplasm at least one marker linked to low palmitic acid content,
wherein the at least one marker is selected from the group
consisting of HA0031B, HA0908, HA1665, HA0304A_H757B, HA0850_H757B,
and HA0870, to yield a marker amplicon, wherein the amplifying
comprises: admixing an amplification primer or amplification primer
pair with a nucleic acid isolated from the sunflower plant or
germplasm, wherein the primer or primer pair is complementary or
partially complementary to at least a portion of the marker, and is
capable of initiating DNA polymerization by a DNA polymerase using
the sunflower nucleic acid as a template, and extending the primer
or primer pair in a DNA polymerization reaction comprising a DNA
polymerase and a template nucleic acid to generate at least one
amplicon; detecting the at least one marker amplicon; and crossing
the sunflower plant or germplasm with a different sunflower plant
or germplasm.
9. The method according to claim 8, wherein the different sunflower
plant or germplasm is a plant or germplasm from an elite sunflower
variety or an exotic sunflower variety.
10. A method for producing an introgressed sunflower plant or
germplasm, the method comprising: introgressing at least one marker
allele that is positively correlated with low palmitic acid content
from a first sunflower plant or germplasm into a second sunflower
plant or germplasm, to produce an introgressed sunflower plant or
germplasm, wherein the at least one marker is selected from the
group consisting of HA0031B, HA0908, HA1665, HA0304A, HA0850,
HA0743, HA0870, HA0907, HA0612A, and a marker linked to at least
one of HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870,
HA0907, and HA0612A.
11. The method according to claim 10, wherein the second sunflower
plant or germplasm displays a higher palmitic acid content as
compared to the first sunflower plant or germplasm, and wherein the
introgressed sunflower plant or germplasm displays an decreased
palmitic acid content as compared to the second plant or
germplasm.
12. The introgressed sunflower plant or germplasm produced by the
method according to claim 10.
13. The introgressed sunflower plant or germplasm of claim 12,
wherein the introgressed sunflower plant or germplasm comprises
about 3.3% or less total combined palmitic acid (16:0) and stearic
acid (18:0) content.
14. The introgressed sunflower plant or germplasm of claim 12,
wherein the introgressed sunflower plant or germplasm comprises
less than about 2.5% total palmitic acid content.
15. The introgressed sunflower plant or germplasm of claim 14,
wherein the introgressed sunflower plant or germplasm comprises
less than 2.5% total palmitic acid content.
16. The introgressed sunflower plant or germplasm of claim 15,
wherein the introgressed sunflower plant or germplasm comprises
less than 2.2% total palmitic acid content.
17. The introgressed sunflower plant or germplasm of claim 16,
wherein the introgressed sunflower plant or germplasm comprises
less than 2.1% total palmitic acid content.
18. The introgressed sunflower plant or germplasm of claim 17,
wherein the introgressed sunflower plant or germplasm comprises
less than 2.0% total palmitic acid content.
19. The method according to claim 10, wherein the second sunflower
plant or germplasm is a plant or germplasm from an elite sunflower
variety or an exotic sunflower variety.
20. A system for identifying a sunflower plant predicted to have a
low palmitic acid content phenotype, the system comprising: a set
of marker probes or primers configured to detect at least one
marker linked to low palmitic acid content, wherein the marker is
selected from the group consisting of HA0031B, HA0908, HA1665,
HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and a marker
linked to at least one of HA0031B, HA0908, HA1665, HA0304A, HA0850,
HA0743, HA0870, HA0907, and HA0612A; a detector that is configured
to detect one or more signal outputs from the set of marker probes
or primers, or an amplicon thereof, thereby identifying the
presence or absence of the marker; and system instructions that
correlate the presence or absence of the marker with the low
palmitic acid content phenotype.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/837,877 filed Mar. 15, 2013, which claims the benefit
of U.S. Provisional Patent Application Ser. No. 61/613,383, filed
Mar. 20, 2012, the disclosure of which is hereby incorporated
herein in its entirety by this reference.
TECHNICAL FIELD
[0002] The present disclosure relates to compositions and methods
for identifying sunflower plants that have low palmitic acid
content, where the methods use molecular genetic markers to
identify, select and/or construct low palmitic acid content plants.
The disclosure also relates to sunflower plants that display low
palmitic acid content that are generated by the methods of the
invention.
BACKGROUND
[0003] The cultivated sunflower (Helianthus annuus L.) is a major
worldwide source of vegetable oil. In the United States,
approximately 4 million acres of sunflower are planted annually,
primarily in the Dakotas and Minnesota.
[0004] The very rapid expansion over the last decade of acreage
planted in sunflower in the United States is due in part to several
important developments in the field of sunflower breeding and
varietal improvement, including the discovery of cytoplasmic male
sterility and genes for fertility restoration. This discovery that
allowed the production of hybrid sunflowers. The hybrids thus
produced were introduced during the early 1970s. A description of
cytoplasmic male sterility (CMS) and genetic fertility restoration
in sunflowers is presented by Fick, "Breeding and Genetics," in
Sunflower Science and Technology 279-338 (J. F. Carter ed. 1978),
the contents of which are incorporated herein by reference.
[0005] Sunflower oil is comprised primarily of palmitic (16:0),
stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3)
fatty acids. While other unusual fatty acids exist in plants,
palmitic, stearic, oleic, linoleic, and linolenic acids comprise
about 88% of the fatty acids present in the world production of
vegetable oils. J. L. Harwood, "Plant Acyl Lipids: Structure,
Distribution and Analysis," 4 Lipids: Structure and Function, P. K.
Stumpf and E. E. Conn ed. (1988). Palmitic and stearic acids are
saturated fatty acids that have been demonstrated in certain
studies to contribute to an increase in the plasma cholesterol
level, a factor contributing to the development of coronary heart
disease. According to recent studies, vegetable oils high in
unsaturated fatty acids (such as oleic and linoleic acid) may have
the ability to lower plasma cholesterol.
[0006] Saturated fatty acids generally also have higher melting
points than unsaturated fatty acids of the same carbon number,
which contributes to cold tolerance problems in foodstuffs, and can
further contribute to a waxy or greasy feel in the mouth of the
foodstuff during ingestion. It is also known that food products
made from fats and oils having less than about 3% saturated fatty
acids will typically contain less than 0.5 grams saturated fat per
serving, and as a result can be labeled as containing "zero
saturated fat" under current labeling regulations.
[0007] There are numerous steps in the development of any novel,
desirable plant germplasm. Plant breeding programs combine
desirable traits from two or more cultivars or various broad-based
sources into breeding pools, from which cultivars are developed by
selfing and selection of desired phenotypes. The new cultivars are
evaluated to determine which have commercial potential. Plant
breeding begins with the analysis and definition of problems and
weaknesses of the current germplasm, the establishment of program
goals, and the definition of specific breeding objectives. The next
step is selection of germplasm that possess the traits to meet the
program goals. The goal is to combine in a single variety an
improved combination of desirable traits from the parental
germplasm. These important traits may include higher seed yield,
resistance to diseases and insects, better stems and roots,
tolerance to drought and heat, and better agronomic quality.
[0008] Choice of breeding or selection methods depends on the mode
of plant reproduction, the heritability of the trait(s) being
improved, and the type of cultivar used commercially (e.g., F.sub.1
hybrid cultivar, pureline cultivar, etc.). For highly heritable
traits, a choice of superior individual plants evaluated at a
single location may be effective, whereas for traits with low
heritability, selection should be based on mean values obtained
from replicated evaluations of families of related plants. Popular
selection methods commonly include pedigree selection, modified
pedigree selection, mass selection, and recurrent selection.
[0009] The complexity of inheritance influences the choice of the
breeding method. Backcross breeding is used to transfer one or a
few favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes. The use of recurrent selection in self-pollinating
crops depends on the ease of pollination, the frequency of
successful hybrids from each pollination, and the number of hybrid
offspring from each successful cross.
[0010] Each breeding program should include a periodic, objective
evaluation of the efficiency of the breeding procedure. Evaluation
criteria vary depending on the goal and objectives, but should
include gain from selection per year (based on comparisons to an
appropriate standard), overall value of the advanced breeding
lines, and the number of successful cultivars produced per unit of
input (e.g., per year, per dollar expended, etc.). Promising
advanced breeding lines are then thoroughly tested and compared to
appropriate standards in environments representative of the
commercial target area(s) for three or more years. Candidates for
new commercial cultivars are selected from among the best lines;
those still deficient in a few traits may be used as parents to
produce new populations for further selection. These processes,
which lead to the final step of marketing and distribution, usually
take from 8 to 12 years from the time the first cross is made.
Therefore, development of new cultivars is a time-consuming process
that requires precise forward planning, efficient use of resources,
and a minimum of changes in direction.
[0011] A most difficult task in plant breeding is the
identification of individuals that are genetically superior. One
method of identifying a superior plant is to observe its
performance relative to other experimental plants and to a widely
grown standard cultivar. If a single observation is inconclusive,
replicated observations provide a better estimate of its genetic
worth. This task is so difficult, because (for most traits) the
true genotypic value is masked by other confounding plant traits or
environmental factors.
[0012] The goal of sunflower plant breeding is to develop new,
unique, and superior sunflower cultivars and hybrids. The breeder
initially selects and crosses two or more parental lines, followed
by repeated selfing and selection, producing many new genetic
combinations. The breeder can theoretically generate billions of
different genetic combinations via crossing, selfing, and
mutagenesis. Such a breeder has no direct control of the process at
the cellular level. Therefore, two breeders will never develop the
same line, or even very similar lines, having the same sunflower
traits.
[0013] Each year, the plant breeder selects the germplasm to
advance to the next generation. This germplasm is grown under
unique and different geographical, climatic, and soil conditions.
Further selections are then made, during and at the end of the
growing season. The cultivars that are developed are unpredictable.
This unpredictability is due to the breeder's selection, which
occurs in unique environments, and which allows no control at the
DNA level (using conventional breeding procedures), with millions
of different possible genetic combinations being generated. A
breeder of ordinary skill in the art cannot predict the final
resulting lines he develops, except possibly in a very gross and
general fashion. Similarly, the same breeder cannot produce the
same cultivar twice by using the exact same original parents and
the same selection techniques. This unpredictability results in the
expenditure of large amounts of resources, monetary and otherwise,
to develop superior new sunflower cultivars.
[0014] The development of new sunflower cultivars requires the
development and selection of sunflower varieties, crossing of these
varieties, and selection of superior hybrid crosses. Hybrid seed is
produced by manual crosses between selected male-fertile parents,
or by using male sterility systems. These hybrids are selected for
certain single gene traits (e.g., pod color, flower color,
pubescence color, and herbicide resistance) that indicate that the
seed is truly a hybrid. Data on parental lines, as well as the
phenotype of the hybrid, influence the breeder's decision regarding
whether to continue with the specific hybrid cross.
[0015] Pedigree breeding is used commonly for the improvement of
self-pollinating crops. In pedigree breeding, two parents that
possess favorable, complementary traits are crossed to produce
F.sub.1 progeny. An F.sub.2 population is produced by selfing one
or several plants from the F.sub.1 progeny generation. Selection of
the best individuals may begin in the F.sub.2 population; then,
beginning in the F.sub.3, the best individuals in the best families
are selected. To improve the effectiveness of selection for traits
with low heritability, replicated testing of families can begin in
the F.sub.4 generation. At an advanced stage of inbreeding (e.g.,
F.sub.6 or F.sub.7), the best lines or mixtures of lines with
similar phenotypes are tested for potential release as new
cultivars. Mass and recurrent selections can be used to improve
populations of either self- or cross-pollinating crops. A
genetically variable population of heterozygous individuals may be
either identified or created by intercrossing several different
parents. The best plants may be selected based on individual
superiority, outstanding progeny, or excellent combining ability.
The selected plants are intercrossed to produce a new population,
in which further cycles of selection may be continued.
[0016] Backcross breeding has been used to transfer genes for a
simply and highly heritable trait into a desirable homozygous
cultivar, or inbred line, which is the recurrent parent. The source
of the trait to be transferred is the "donor parent." The resulting
plant is expected to have the attributes of the recurrent parent
(e.g., cultivar), and the desirable trait transferred from the
donor parent. After the initial cross, individuals possessing the
phenotype of the donor parent are selected, and repeatedly crossed
(backcrossed) to the recurrent parent. The resulting plant is
expected to have the attributes of the recurrent parent and the
desirable trait transferred from the donor parent.
[0017] In sunflower breeding, the "single-seed descent procedure"
refers to the planting of a segregating population, followed by
harvesting a sample of one seed per resulting plant, and using the
harvested one-seed sample to plant the next generation. When the
population has been advanced from the F.sub.2 generation to the
desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2 individuals. The
number of plants in a population declines each generation, due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
[0018] In a multiple-seed procedure, sunflower breeders commonly
harvest seeds from each plant in a population and thresh them
together to form a bulk. Part of the bulk is used to plant the next
generation, and part is put in reserve. This procedure has been
referred to as modified single-seed descent. The multiple-seed
procedure has been used to save labor involved in the harvest. It
is considerably faster to remove seeds with a machine, than to
remove one seed from each by hand for the single-seed procedure.
The multiple-seed procedure also makes it possible to plant the
same number of seeds of a population for each generation of
inbreeding. Enough seeds are harvested to compensate for the number
of plants that did not germinate or produce seed.
[0019] Proper testing should detect any major faults and establish
the level of superiority or improvement of a new cultivar over
current cultivars. In addition to showing superior performance,
there should be a demand for a new cultivar that is compatible with
industry standards, or that creates a new market. The testing
preceding release of a new cultivar should take into consideration
research and development costs as well as technical superiority of
the final cultivar. The introduction of a new cultivar can incur
additional costs to the seed producer, the grower, the processor,
and the consumer due to special required advertising and marketing,
altered seed and commercial production practices, and new product
utilization. For seed-propagated cultivars, it must be feasible to
produce seed easily and economically.
[0020] It is the goal of the plant breeder to select plants and
enrich the plant population for individuals that have desired
traits, for example, decreased palmitic acid content, leading
ultimately to increased agricultural productivity. Consistent with
the foregoing, a continuing goal of sunflower breeders is to
develop stable, high-yielding cultivars that are agronomically
sound. Current goals include maximization of the amount of grain
produced on the land used, and the supply of food for both animals
and humans. To accomplish these goals, the sunflower breeder must
select and develop sunflower plants that have traits that result in
superior cultivars, and do so in the most cost-effective manner.
Molecular markers may be used in the process of marker-assisted
selection (MAS) to aid in the identification and selection of
individuals or families of individuals that possess inherited
attributes that are linked to the markers.
BRIEF SUMMARY OF THE DISCLOSURE
[0021] Molecular markers that are linked to low palmitic acid
content may be used to facilitate marker-assisted selection for the
low palmitic acid content trait in sunflower. Marker-assisted
selection provides significant advantages with respect to time,
cost, and labor, when compared to palmitic acid content
phenotyping. Disclosed herein are particular markers identified to
be within or near low palmitic acid content QTL regions in the
sunflower genome that are polymorphic in parent genotypes and
linked (e.g., tightly linked) to a low palmitic acid content
phenotype. These markers, offer superior utility in marker-assisted
selection of sunflower plants and cultivars having low palmitic
acid content.
[0022] Described herein are methods of identifying a first
sunflower plant that displays low palmitic acid content or
germplasm comprised within such a sunflower plant. A first
sunflower plant or germplasm that displays low palmitic acid
content may in some examples be a plant or germplasm comprising a
lower (i.e., decreased) palmitic acid content than is observed in a
parental plant or germplasm of the first plant or germplasm. A
first sunflower plant or germplasm that displays low palmitic acid
content may in some examples be a plant or germplasm comprising a
lower palmitic acid content than is observed in a particular
conventional plant or germplasm of the same species (e.g.,
sunflower) as the first plant or germplasm. Some embodiments of
such methods may comprise detecting in the first sunflower plant or
germplasm at least one marker linked to low palmitic acid content,
wherein the at least one marker is selected from the group
consisting of: HA0031B; HA0908; HA1665; HA0304A; HA0850; HA0743;
HA0870; HA0907; HA0612A; and markers linked (e.g., tightly linked)
to any of HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870,
HA0907, and HA0612A.
[0023] Also described are methods of producing an sunflower plant
or germplasm having low palmitic acid content. Some embodiments of
such methods may comprise introgressing at least one marker linked
to low palmitic acid content from a first sunflower plant or
germplasm into a second sunflower plant or germplasm to produce a
sunflower plant or germplasm that is likely to have low palmitic
acid content. In such examples, the at least one marker may be
selected from the group consisting of: HA0031B; HA0908; HA1665;
HA0304A; HA0850; HA0743; HA0870; HA0907; HA0612A; and markers
linked to any of HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743,
HA0870, HA0907, and HA0612A. A sunflower plant or germplasm
produced by the foregoing methods is also included in particular
embodiments.
[0024] Some embodiments include methods for producing a transgenic
sunflower plant. Examples of such methods may comprise introducing
one or more exogenous nucleic acid molecule(s) into a target
sunflower plant or progeny thereof, wherein at least one of the one
or more exogenous nucleic acid molecule(s) comprises a sunflower
genomic nucleotide sequence that is linked to at least one marker
that is linked to low palmitic acid content, or wherein at least
one of the one or more exogenous nucleic acid molecule(s) comprises
a nucleotide sequence that is specifically hybridizable to a
nucleotide sequence that is linked to at least one marker that is
linked to low palmitic acid content. A marker that is linked to low
palmitic acid content may be selected from the group consisting of:
HA0031B; HA0908; HA1665; HA0304A; HA0850; HA0743; HA0870; HA0907;
HA0612A; and markers linked to any of HA0031B, HA0908, HA1665,
HA0304A, HA0850, HA0743, HA0870, HA0907, and HA0612A. In certain
examples the foregoing methods for producing a transgenic sunflower
plant, a resulting transgenic sunflower plant may comprise low
palmitic acid content.
[0025] Some embodiments include systems and kits for identifying a
sunflower plant that is likely to comprise low palmitic acid
content. Particular examples of such systems and kits may comprise
a set of nucleic acid probes, each comprising a nucleotide sequence
that is specifically hybridizable to a nucleotide sequence that is
linked in sunflower to at least one marker that is linked to low
palmitic acid content. A marker that is linked in sunflower to low
palmitic acid content may be selected from the group consisting of:
HA0031B; HA0908; HA1665; HA0304A; HA0850; HA0743; HA0870; HA0907;
HA0612A; and markers linked to any of HA0031B, HA0908, HA1665,
HA0304A, HA0850, HA0743, HA0870, HA0907, and HA0612A. Particular
examples of systems and kits for identifying a sunflower plant that
is likely to comprise low palmitic acid content may also comprise a
detector that is configured to detect one or more signal outputs
from the set of nucleic acid probes, or an amplicon thereof,
thereby identifying the presence or absence of the at least one
marker that is linked to low palmitic acid content. Specific
examples include instructions that correlate the presence or
absence of the at least one marker with the likely decrease in
palmitic acid content.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 includes a GC-FID FAME chromatogram showing the
identification of palmitic acid methyl ester peaks by comparison
with the retention times of methyl ester reference standards.
Individual percent areas were calculated for all analytes in the
reference standard based upon the total integrated peak areas. A
heptane blank was also injected to identify any contamination on
the GC.
[0027] FIG. 2 includes a graphical display showing the distribution
of the palmitic acid content of 23,040 samples. Values describing
the distribution are set forth in Tables 1-2.
[0028] FIG. 3 includes a histogram of the palmitic acid content of
an F.sub.2 population of 384 individuals obtained from a cross of
an elite sunflower line with a line having low palmitic acid
content.
[0029] FIG. 4 includes a schematic representation of a major locus
on linkage group 5 (LG5) for low palmitic acid content in
sunflower. Several SSR markers have been identified to be tightly
linked or flanking the locus as depicted.
[0030] FIG. 5 includes a schematic representation of the linkage
between the locus for low palmitic acid content and several SSR
markers, showing the location of the major low palmitic acid QTL on
LG5. The LOD score is provided on the y-axis, and distance of the
marker from the locus in cM is provided on the x-axis. The LOD
score is determined using the multiple interval protocol
implemented by the Map QTL software program (J. W. Van Ooijen, M.
P. Boer, R. C. Jansen, C. Maliepaard (2002) Map QTL 4.0: software
for the calculation of QTL positions on genetic maps, Plant
Research International, Wageningen, The Netherlands).
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0031] It is desirable for a number of reasons to produce a
sunflower oil having low levels of palmitic and stearic acids and
high levels of oleic or linoleic acids. Embodiments of the
invention include, for example, compositions and methods for
identifying sunflower plants comprising a low palmitic acid content
and/or germplasm carrying a genotype that is predictive and
determinative of a low palmitic acid phenotype. Methods of making
such sunflower plants and germplasm are included in some
embodiments. Such methods may include, for example and without
limitation, introgression of desired low palmitic acid content
marker alleles and/or genetic transformation methods. Sunflower
plants and/or germplasm made by the methods such as the foregoing
are included in particular embodiments. Systems and kits for
selecting sunflower plants comprising a low palmitic acid content
and/or germplasm carrying a genotype that is predictive and
determinative of a low palmitic acid phenotype are also a feature
of certain embodiments.
[0032] The identification and selection of sunflower plants
comprising a low palmitic acid content using MAS are capable of
providing an effective and environmentally friendly approach for
generating plants with desirable oil content. Embodiments of the
present invention provide a number of sunflower marker loci and QTL
chromosome intervals that demonstrate statistically significant
co-segregation with (and therefore are predictive and determinative
of) low palmitic acid content. Detection of these markers, or
additional loci linked to the markers that are therefore equivalent
thereto, may be used in marker-assisted sunflower breeding programs
to produce low palmitic acid content plants and germplasm.
[0033] Some embodiments provide methods for identifying a first
sunflower plant or germplasm (e.g., a line or variety) that
displays low palmitic acid content. In some examples, at least one
allele of one or more marker locus (e.g., a plurality of marker
loci) that is linked (e.g., tightly linked) with a low palmitic
acid trait is/are detected in the first sunflower plant or
germplasm. The marker loci may be selected from the loci in FIG. 4,
including: HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743,
HA0870, HA0907, HA0612A, and other markers that are linked to at
least one of the foregoing QTL markers.
[0034] In some examples, a plurality of maker loci may be selected
or identified in the same plant or germplasm. All combinations of,
for example, HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743,
HA0870, HA0907, HA0612A, and other markers that are linked to at
least one of the foregoing QTL markers, may be included in a
plurality of marker loci to be selected or identified in a plant or
germplasm.
[0035] In aspects of some embodiments, the palmitic acid content of
a sunflower plant can be quantitated using any suitable means or
method known in the art.
II. Abbreviations
[0036] AFLP amplified fragment length polymorphism
[0037] ASH allele specific hybridization
[0038] CCD charge coupling device
[0039] EST expressed sequence tag
[0040] FAME fatty acid methyl ester
[0041] FID flame ionization detector
[0042] GC gas chromatography
[0043] LCR ligase chain reaction
[0044] LG linkage group
[0045] LNA locked nucleic acid
[0046] LOD logarithm (base 10) of odds
[0047] MAS marker-assisted selection
[0048] NASBA nucleic acid sequence based amplification
[0049] NIR near infrared (spectroscopy)
[0050] NMR nuclear magnetic resonance (spectroscopy)
[0051] ORF open reading frame
[0052] PCR polymerase chain reaction
[0053] PNA peptide nucleic acid
[0054] QTL quantitative trait locus
[0055] RAPD randomly amplified polymorphic DNA
[0056] RFLP restriction fragment length polymorphism
[0057] RT-PCR reverse transcriptase-PCR
[0058] SNP single nucleotide polymorphism
[0059] SSCP single-strand conformation polymorphism
[0060] SSR simple sequence repeat
III. Terms
[0061] As used in this application, including the claims, terms in
the singular and the singular forms, "a," "an," and "the," for
example, include plural referents, unless the content clearly
dictates otherwise. Thus, for example, a reference to "plant," "the
plant," or "a plant" also refers to a plurality of plants.
Furthermore, depending on the context, use of the term, "plant,"
may also refer to genetically similar or identical progeny of that
plant. Similarly, the term, "nucleic acid," may refer to many
copies of a nucleic acid molecule. Likewise, the term "probe" may
refer to many similar or identical probe molecules.
[0062] Numeric ranges are inclusive of the numbers defining the
range, and include each integer and non-integer fraction within the
defined range. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art.
[0063] In order to facilitate review of the various embodiments
described in this disclosure, the following explanation of specific
terms is provided:
[0064] Isolated: An "isolated" biological component (such as a
nucleic acid or protein) has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs
(i.e., other chromosomal and extra-chromosomal DNA and RNA, and
proteins), while effecting a chemical or functional change in the
component (e.g., a nucleic acid may be isolated from a chromosome
by breaking chemical bonds connecting the nucleic acid to the
remaining DNA in the chromosome). Nucleic acid molecules and
proteins that have been "isolated" include nucleic acid molecules
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell, as well as chemically synthesized
nucleic acid molecules, proteins, and peptides.
[0065] Mapping population: As used herein, the term "mapping
population" may refer to a plant population (e.g., a sunflower
plant population) used for gene mapping. Mapping populations are
typically obtained from controlled crosses of parent genotypes, as
may be provided by two inbred lines. Decisions on the selection of
parents, mating design for the development of a mapping population,
and the type of markers used depend upon the gene to be mapped, the
availability of markers, and the molecular map. The parents of
plants within a mapping population should have sufficient variation
for a trait(s) of interest at both the nucleic acid sequence and
phenotype level. Variation of the parents' nucleic acid sequence is
used to trace recombination events in the plants of the mapping
population. The availability of informative polymorphic markers is
dependent upon the amount of nucleic acid sequence variation. Thus,
informative markers may not be identified in particular crosses of
parent genotypes, though such markers may exist.
[0066] A "genetic map" is a description of genetic linkage
relationships among loci on one or more chromosomes (or linkage
groups) within a given species, as may be determined by analysis of
a mapping population. In some examples, a genetic map may be
depicted in a diagrammatic or tabular form. The term "genetic
mapping" may refer to the process of defining the linkage
relationships of loci through the use of genetic markers, mapping
populations segregating for the markers, and standard genetic
principles of recombination frequency. A "genetic map location"
refers to a location on a genetic map (relative to surrounding
genetic markers on the same linkage group or chromosome) where a
particular marker can be found within a given species. In contrast,
a "physical map of the genome" refers to absolute distances (for
example, measured in base pairs or isolated and overlapping
contiguous genetic fragments) between markers within a given
species. A physical map of the genome does not necessarily reflect
the actual recombination frequencies observed in a test cross of a
species between different points on the physical map.
[0067] Cross: As used herein, the term "cross" or "crossed" refers
to the fusion of gametes via pollination to produce progeny (e.g.,
cells, seeds, and plants). This term encompasses both sexual
crosses (i.e., the pollination of one plant by another) and selfing
(i.e., self-pollination, for example, using pollen and ovule from
the same plant).
[0068] Backcrossing: Backcrossing methods may be used to introduce
a nucleic acid sequence into plants. The backcrossing technique has
been widely used for decades to introduce new traits into plants.
N. Jensen, Ed. Plant Breeding Methodology, John Wiley & Sons,
Inc., 1988. In a typical backcross protocol, the original variety
of interest (recurrent parent) is crossed to a second variety
(non-recurrent parent) that carries a gene of interest to be
transferred. The resulting progeny from this cross are then crossed
again to the recurrent parent, and the process is repeated until a
plant is obtained wherein essentially all of the desired
morphological and physiological characteristics of the recurrent
plant are recovered in the converted plant, in addition to the
transferred gene from the non-recurrent parent.
[0069] Introgression: As used herein, the term "introgression"
refers to the transmission of an allele at a genetic locus into a
genetic background. In some embodiments, introgression of a
specific allele form at the locus may occur by transmitting the
allele form to at least one progeny via a sexual cross between two
parents of the same species, where at least one of the parents has
the specific allele form in its genome. Progeny comprising the
specific allele form may be repeatedly backcrossed to a line having
a desired genetic background. Backcross progeny may be selected for
the specific allele form, so as to produce a new variety wherein
the specific allele form has been fixed in the genetic background.
In some embodiments, introgression of a specific allele form may
occur by recombination between two donor genomes (e.g., in a fused
protoplast), where at least one of the donor genomes has the
specific allele form in its genome. Introgression may involve
transmission of a specific allele form that may be, for example and
without limitation, a selected allele form of a marker allele; a
QTL; and/or a transgene.
[0070] Germplasm: As used herein, the term "germplasm" refers to
genetic material of or from an individual plant, a group of plants
(e.g., a plant line, variety, and family), and a clone derived from
a plant or group of plants. A germplasm may be part of an organism
or cell, or it may be separate (e.g., isolated) from the organism
or cell. In general, germplasm provides genetic material with a
specific molecular makeup that is the basis for hereditary
qualities of the plant. As used herein, "germplasm" refers to cells
of a specific plant; seed; tissue of the specific plant (e.g.,
tissue from which new plants may be grown); and non-seed parts of
the specific plant (e.g., leaf, stem, pollen, and cells).
[0071] As used herein, the term "germplasm" is synonymous with
"genetic material," and it may be used to refer to seed (or other
plant material) from which a plant may be propagated. A "germplasm
bank" may refer to an organized collection of different seed or
other genetic material (wherein each genotype is uniquely
identified) from which a known cultivar may be cultivated, and from
which a new cultivar may be generated. In embodiments, a germplasm
utilized in a method or plant as described herein is from a
sunflower line or variety. In particular examples, a germplasm is
seed of the sunflower line or variety. In particular examples, a
germplasm is a nucleic acid sample from the sunflower line or
variety.
[0072] Gene: As used herein, the term "gene" (or "genetic element")
may refer to a heritable genomic DNA sequence with functional
significance. The term "gene" may also be used to refer to, for
example and without limitation, a cDNA and/or an mRNA encoded by a
heritable genomic DNA sequence.
[0073] Genotype: As used herein, the term "genotype" refers to the
genetic constitution of an individual (or group of individuals) at
one or more particular loci. The genotype of an individual or group
of individuals is defined and described by the allele forms at the
one or more loci that the individual has inherited from its
parents. The term genotype may also be used to refer to an
individual's genetic constitution at a single locus, at multiple
loci, or at all the loci in its genome. A "haplotype" is the
genotype of an individual at a plurality of genetic loci. In some
examples, the genetic loci described by a haplotype may be
physically and genetically linked; i.e., the loci may be positioned
on the same chromosome segment.
[0074] Quantitative trait locus: Specific chromosomal loci (or
intervals) may be mapped in an organism's genome that correlates
with particular quantitative phenotypes. Such loci are termed
quantitative trait loci, or QTL. As used herein, the term
"quantitative trait locus" (QTL) may refer to stretches of DNA that
have been identified as likely DNA sequences (e.g., genes,
non-coding sequences, and/or intergenic sequences) that underlie a
quantitative trait, or phenotype, that varies in degree, and can be
attributed to the interactions between two or more DNA sequences
(e.g., genes, non-coding sequences, and/or intergenic sequences) or
their expression products and their environment. Thus, the term
"quantitative trait locus" includes polymorphic genetic loci with
at least two alleles that differentially affect the expression of a
phenotypic trait in at least one genetic background (e.g., in at
least one breeding population or progeny). In practice, QTLs can be
molecularly identified to help map regions of the genome that
contain sequences involved in specifying a quantitative trait, such
as reduced palmitic acid content.
[0075] As used herein, the term "QTL interval" may refer to
stretches of DNA that are linked to the genes that underlie the QTL
trait. A QTL interval is typically, but not necessarily, larger
than the QTL itself. A QTL interval may contain stretches of DNA
that are 5' and/or 3' with respect to the QTL.
[0076] Multiple experimental paradigms have been developed to
identify and analyze QTLs. See, e.g., Jansen (1996) Trends Plant
Sci. 1:89. The majority of published reports on QTL mapping in crop
species have been based on the use of a bi-parental cross (Lynch
and Walsh (1997) Genetics and Analysis of Quantitative Traits,
Sinauer Associates, Sunderland). Typically, these paradigms involve
crossing one or more parental pairs that can be, for example, a
single pair derived from two inbred strains, or multiple related or
unrelated parents of different inbred strains or lines, which each
exhibit different characteristics relative to the phenotypic trait
of interest. Typically, this experimental protocol involves
deriving 100 to 300 segregating progeny from a single cross of two
divergent inbred lines that are, for example, selected to maximize
phenotypic and molecular marker differences between the lines. The
parents and segregating progeny are genotyped for multiple marker
loci, and evaluated for one to several quantitative traits (e.g.,
disease resistance). QTLs are then identified as significant
statistical associations between genotypic values and phenotypic
variability among the segregating progeny. The strength of this
experimental protocol comes from the utilization of the inbred
cross, because the resulting F.sub.1 parents all have the same
linkage phase (how the alleles were joined in the parental
generation). Thus, after selfing of F.sub.1 plants, all segregating
F.sub.2 progeny are informative and linkage disequilibrium is
maximized, the linkage phase is known, there are only two QTL
alleles, and (except for backcross progeny) the frequency of each
QTL allele is 0.5.
[0077] Numerous statistical methods for determining whether markers
are genetically linked to a QTL (or to another marker) are known to
those of skill in the art and include, for example and without
limitation, standard linear models, such as ANOVA or regression
mapping (Haley and Knott (1992) Heredity 69:315); and maximum
likelihood methods, such as expectation-maximization algorithms
(e.g., Lander and Botstein (1989) Genetics 121:185-99; Jansen
(1992) Theor. Appl. Genet. 85:252-60; Jansen (1993) Biometrics
49:227-31; Jansen (1994) "Mapping of quantitative trait loci by
using genetic markers: an overview of biometrical models," in J. W.
van Ooijen and J. Jansen (eds.), Biometrics in Plant breeding:
applications of molecular markers, pp. 116-24, CPRO-DLO
Netherlands; Jansen (1996) Genetics 142:305-11; and Jansen and Stam
(1994) Genetics 136:1447-55).
[0078] Exemplary statistical methods include single point marker
analysis; interval mapping (Lander and Botstein (1989) Genetics
121:185); composite interval mapping; penalized regression
analysis; complex pedigree analysis; MCMC analysis; MQM analysis
(Jansen (1994) Genetics 138:871); HAPLO-IM+ analysis, HAPLO-MQM
analysis, and HAPLO-MQM+ analysis; Bayesian MCMC; ridge regression;
identity-by-descent analysis; and Haseman-Elston regression, any of
which are suitable in the context of particular embodiments of the
invention. Alternative statistical methods applicable to complex
breeding populations that may be used to identify and localize QTLs
in particular examples are described in U.S. Pat. No. 6,399,855 and
PCT International Patent Publication No. WO0149104 A2. All of these
approaches are computationally intensive and are usually performed
with the assistance of a computer based system and specialized
software. Appropriate statistical packages are available from a
variety of public and commercial sources, and are known to those of
skill in the art.
[0079] Marker: Although specific DNA sequences that encode proteins
are generally well-conserved across a species, other regions of DNA
(e.g., non-coding DNA and introns) tend to develop and accumulate
polymorphism, and therefore, may be variable between individuals of
the same species. The genomic variability can be of any origin, for
example, the variability may be due to DNA insertions, deletions,
duplications, repetitive DNA elements, point mutations,
recombination events, and the presence and sequence of transposable
elements. Such regions may contain useful molecular genetic
markers. In general, any differentially inherited polymorphic trait
(including nucleic acid polymorphisms) that segregates among
progeny is a potential marker.
[0080] As used herein, the terms "marker" and "molecular marker"
refer to a nucleotide sequence or encoded product thereof (e.g., a
protein) used as a point of reference when identifying a linked
locus. Thus, a marker may refer to a gene or nucleotide sequence
that can be used to identify plants having a particular allele. A
marker may be described as a variation at a given genomic locus. A
genetic marker may be a short DNA sequence, such as a sequence
surrounding a single base-pair change (single nucleotide
polymorphism, or "SNP"), or a long one, for example, a
microsatellite/simple sequence repeat ("SSR"). A "marker allele" or
"marker allele form" refers to the version of the marker that is
present in a particular individual. The term "marker" as used
herein may refer to a cloned segment of chromosomal DNA, and may
also or alternatively refer to a DNA molecule that is complementary
to a cloned segment of chromosomal DNA. The term also refers to
nucleic acid sequences complementary to genomic marker sequences,
such as nucleic acid primers and probes.
[0081] A marker may be described, for example, as a specific
polymorphic genetic element at a specific location in the genetic
map of an organism. A genetic map may be a graphical representation
of a genome (or a portion of a genome, such as a single chromosome)
where the distances between landmarks on the chromosome are
measured by the recombination frequencies between the landmarks. A
genetic landmark can be any of a variety of known polymorphic
markers, for example and without limitation: simple sequence repeat
(SSR) markers; restriction fragment length polymorphism (RFLP)
markers; and single nucleotide polymorphism (SNP) markers. As one
example, SSR markers can be derived from genomic or expressed
nucleic acids (e.g., expressed sequence tags (ESTs)).
[0082] Additional markers include, for example and without
limitation, ESTs; amplified fragment length polymorphisms (AFLPs)
(Vos et al. (1995) Nucl. Acids Res. 23:4407; Becker et al. (1995)
Mol. Gen. Genet. 249:65; Meksem et al. (1995) Mol. Gen. Genet.
249:74); randomly amplified polymorphic DNA (RAPD), and isozyme
markers. Isozyme markers may be employed as genetic markers, for
example, to track isozyme markers or other types of markers that
are linked to a particular first marker. Isozymes are multiple
forms of enzymes that differ from one another with respect to amino
acid sequence (and therefore with respect to their encoding nucleic
acid sequences). Some isozymes are multimeric enzymes containing
slightly different subunits. Other isozymes are either multimeric
or monomeric, but have been cleaved from a pro-enzyme at different
sites in the pro-enzyme amino acid sequence. Isozymes may be
characterized and analyzed at the protein level or at the nucleic
acid level. Thus, any of the nucleic acid based methods described
herein can be used to analyze isozyme markers in particular
examples.
[0083] "Genetic markers" include alleles that are polymorphic in a
population, where the alleles of may be detected and distinguished
by one or more analytic methods (e.g., RFLP analysis, AFLP
analysis, isozyme marker analysis, SNP analysis, and SSR analysis).
The term "genetic marker" may also refer to a genetic locus (a
"marker locus") that may be used as a point of reference when
identifying a genetically linked locus (e.g., QTL). Such a marker
may also be referred to as a "QTL marker."
[0084] The nature of the foregoing physical landmarks (and the
methods used to detect them) vary, but all of these markers are
physically distinguishable from each other (as well as from the
plurality of alleles of any one particular marker) on the basis of
polynucleotide length and/or sequence. Numerous methods for
detecting molecular markers and identifying marker alleles are
well-established. A wide range of protocols are known to one of
skill in the art for detecting this variability, and these
protocols are frequently specific for the type of polymorphism they
are designed to detect. Such protocols include, for example and
without limitation, PCR amplification; detection of single-strand
conformation polymorphism (SSCP), e.g., via electrophoresis; and
self-sustained sequence replication (3SR) (see Chan and Fox (1999)
Reviews in Medical Microbiology 10:185-96).
[0085] The primary motivation for developing molecular marker
technologies from the perspective of plant breeders has been to
increase breeding efficiency through MAS. A molecular marker allele
that demonstrates linkage disequilibrium with a desired phenotypic
trait (e.g., a QTL) provides a useful tool for the selection of the
desired trait in a plant population. The key components to the
implementation of an MAS approach are the creation of a dense
(information rich) genetic map of molecular markers in the plant
germplasm; the detection of at least one QTL based on statistical
associations between marker and phenotypic variability; the
definition of a set of particular useful marker alleles based on
the results of the QTL analysis; and the use and/or extrapolation
of this information to the current set of breeding germplasm to
enable marker-based selection decisions to be made.
[0086] Genetic variability, for example as determined in a mapping
population, may be observed between different populations of the
same species (e.g., sunflower). In spite of the variability in the
genetic map that may occur between populations of the same species,
genetic map and marker information derived from one population
generally remains useful across multiple populations for the
purposes of identification and/or selection of plants and/or
germplasm comprising traits that are linked to the markers and
counter-selection of plants and/or germplasm comprising undesirable
traits.
[0087] Two types of markers used in particular MAS protocols
described herein are SSR markers, and SNP markers. SSR markers
include any type of molecular heterogeneity that results in nucleic
acid sequence length variability. Exemplary SSR markers are short
(up to several hundred base pairs) segments of DNA that consist of
multiple tandem repeats of a two or three base-pair sequence. These
repeated sequences result in highly polymorphic DNA regions of
variable length due to poor replication fidelity (e.g., by
polymerase slippage). SSRs appear to be randomly dispersed through
the genome, and are generally flanked by conserved regions. SSR
markers may also be derived from RNA sequences (in the form of a
cDNA, a partial cDNA, or an EST), as well as genomic material.
[0088] The heterogeneity of SSR markers make them well-suited for
use as molecular genetic markers. For example, SSR genomic
variability is inherited, and it is multi-allelic, co-dominant, and
reproducibly detectable. The proliferation of increasingly
sophisticated amplification-based detection techniques (e.g.,
PCR-based techniques) provides a variety of sensitive methods for
the detection of nucleotide sequence heterogeneity between samples.
Probes (e.g., nucleic acid primers) may be designed to hybridize to
conserved regions that flank the SSR, and the probes may be used to
amplify the variable SSR region. The differently sized amplicons
generated from an SSR region have characteristic and reproducible
sizes. Differently sized SSR amplicons observed from two homologous
chromosomes from an individual, or from different individuals, in
the plant population define SSR marker alleles. As long as there
exist at least two SSR marker alleles that produce PCR products
with different sizes, the SSR may be employed as a marker.
[0089] Linkage (dis)equilibrium: As used herein, the term "linkage
equilibrium" refers to the situation where two markers
independently segregate; i.e., the markers sort randomly among
progeny. Markers that show linkage equilibrium are considered
unlinked (whether or not they lie on the same chromosome). As used
herein, the term "linkage disequilibrium" refers to the situation
where two markers segregate in a non-random manner; i.e., the
markers have a recombination frequency of less than 50% (and thus
by definition, are separated by less than 50 cM on the same linkage
group). In some examples, markers that show linkage disequilibrium
are considered linked.
[0090] Linked, tightly linked, and extremely tightly linked: As
used herein, linkage between genes or markers may refer to the
phenomenon in which genes or markers on a chromosome show a
measurable probability of being passed on together to individuals
in the next generation. Thus, linkage of one marker to another
marker or gene may be measured and/or expressed as a recombination
frequency. The closer two genes or markers are to each other, the
closer to "1" this probability becomes. Thus, the term "linked" may
refer to one or more genes or markers that are passed together with
a gene with a probability greater than 0.5 (which is expected from
independent assortment where markers/genes are located on different
chromosomes). When the presence of a gene contributes to a
phenotype in an individual, markers that are linked to the gene may
be said to be linked to the phenotype. Thus, the term "linked" may
refer to a relationship between a marker and a gene, or between a
marker and a phenotype.
[0091] A relative genetic distance (determined by crossing over
frequencies and measured in centimorgans (cM)) is generally
proportional to the physical distance (measured in base pairs) that
two linked markers or genes are separated from each other on a
chromosome. One centimorgan is defined as the distance between two
genetic markers that show a 1% recombination frequency (i.e., a
crossing-over event occurs between the two markers once in every
100 cell divisions). In general, the closer one marker is to
another marker or gene (whether the distance between them is
measured in terms of genetic distance or physical distance), the
more tightly they are linked. Because chromosomal distance is
approximately proportional to the frequency of recombination events
between traits, there is an approximate physical distance that
correlates with recombination frequency. For example, in sunflower,
1 cM correlates, on average, to about 400 kb.
[0092] Thus, the term "linked" may refer herein to one or more
genes or markers that are physically located within about 4.0 Mb of
one another on the same sunflower chromosome (i.e., about 10 cM).
Thus, two "linked" genes or markers may be separated by 4.1 Mb;
about 4.0 Mb; about 3.0 Mb; about 2.5 Mb; 2.1 Mb; 2.00 Mb; about
1.95 Mb; about 1.90 Mb; about 1.85 Mb; about 1.80 Mb; about 1.75
Mb; about 1.70 Mb; about 1.65 Mb; about 1.60 Mb; about 1.55 Mb;
about 1.50 Mb; about 1.45 Mb; about 1.40 Mb; about 1.35 Mb; about
1.30 Mb; about 1.25 Mb; about 1.20 Mb; about 1.15 Mb; about 1.10
Mb; about 1.05 Mb; about 1.00 Mb; about 0.95 Mb; about 0.90 Mb;
about 0.85 Mb; about 0.80 Mb; about 0.75 Mb; about 0.70 Mb; about
0.65 Mb; about 0.60 Mb; about 0.55 Mb; about 0.50 Mb; about 0.45
Mb; about 0.40 Mb; about 0.35 Mb; about 0.30 Mb; about 0.25 Mb;
about 0.20 Mb; about 0.15 Mb; about 0.10 Mb; about 0.05 Mb; about
0.025 Mb; and about 0.01 Mb.
[0093] As used herein, the term "tightly linked" may refer to one
or more genes or markers that are located within about 2.0 Mb of
one another on the same chromosome. Thus, two "tightly linked"
genes or markers may be separated by 2.1 Mb; about 1.75 Mb; about
1.5 Mb; about 1.0 Mb; about 0.9 Mb; about 0.8 Mb; about 0.7 Mb;
about 0.6 Mb; about 0.55 Mb; 0.5 Mb; about 0.45 Mb; about 0.4 Mb;
about 0.35 Mb; about 0.3 Mb; about 0.25 Mb; about 0.2 Mb; about
0.15 Mb; about 0.1 Mb; and about 0.05 Mb.
[0094] As used herein, the term "extremely tightly linked" may
refer to one or more genes or markers that are located within about
500 kb of one another on the same chromosome. Thus, two "extremely
tightly linked" genes or markers may be separated by 600 kb; about
450 kb; about 400 kb; about 350 kb; about 300 kb; about 250 kb;
about 200 kb; about 175 kb; about 150 kb; about 125 kb; about 120
kb; about 115 kb; about 110 kb; about 105 kb; 100 kb; about 95 kb;
about 90 kb; about 85 kb; about 80 kb; about 75 kb; about 70 kb;
about 65 kb; about 60 kb; about 55 kb; about 50 kb; about 45 kb;
about 40 kb; about 35 kb; about 30 kb; about 25 kb; about 20 kb;
about 15 kb; about 10 kb; about 5 kb; and about 1 kb.
[0095] The closer a particular marker is to a gene that encodes a
polypeptide that contributes to a particular phenotype (whether
measured in terms of genetic or physical distance), the more
tightly linked is the particular marker to the phenotype. In view
of the foregoing, it will be appreciated that markers linked to a
particular gene or phenotype include those markers that are tightly
linked, and those markers that are extremely tightly linked, to the
gene or phenotype. In some embodiments, the closer a particular
marker is to a gene that encodes a polypeptide that contributes to
a low palmitic acid content phenotype (whether measured in terms of
genetic or physical distance), the more tightly linked is the
particular marker to the low palmitic acid content phenotype. Thus,
linked, tightly linked, and extremely tightly genetic markers of a
low palmitic acid content phenotype in sunflower may be useful in
MAS programs to identify sunflower varieties comprising a decreased
palmitic acid content (when compared to parental varieties and/or
at least one particular conventional variety), to identify
individual sunflower plants comprising a decreased palmitic acid
content, and to breed this trait into other sunflower varieties to
decrease palmitic acid content.
[0096] In some embodiments, the linkage relationship between a
molecular marker and a phenotype may be expressed as a
"probability" or "adjusted probability." Within this context, a
probability value is the statistical likelihood that a particular
combination of a phenotype and the presence or absence of a
particular marker allele form is random. Thus, the lower the
probability score, the greater the likelihood that the phenotype
and the particular marker allele form will co-segregate. In some
examples, the probability score may be described as "significant"
or "non-significant." In particular examples, a probability score
of 0.05 (p=0.05 (a 5% probability)) of random assortment is
considered a "significant" indication of co-segregation. However, a
significant probability may in other examples be any probability of
less than 50% (p=0.5). For instance, a significant probability may
be less than 0.25; less than 0.20; less than 0.15; or less than
0.1.
[0097] In some embodiments, a marker that is linked to a low
palmitic acid content phenotype may be selected from the QTL
markers of sunflower linkage group 5 that are illustrated in FIG.
4. In some embodiments, a marker that is linked to a low palmitic
acid content phenotype may be selected from those markers that are
located within about 10 cM of a QTL marker illustrated in FIG. 4.
Thus, marker that is linked to a low palmitic acid content
phenotype may be, for example, within 10 cM; 9 cM; 8 cM; 7 cM; 6
cM; 5 cM; 4 cM; 3 cM; 2 cM; 1 cM; 0.75 cM; 0.5 cM; 0.25 cM; or
less, from a QTL marker illustrated in FIG. 4.
[0098] A plant breeder can advantageously use molecular markers to
identify desired individuals by identifying marker alleles that
show a statistically significant probability of co-segregation with
a desired phenotype (e.g., low palmitic acid content), manifested
as linkage disequilibrium. By identifying a molecular marker or
clusters of molecular markers that co-segregate with a quantitative
trait, the breeder is thus identifying a QTL. By identifying and
selecting a marker allele (or desired alleles from multiple
markers) that associates with the desired phenotype, the plant
breeder is able to rapidly select the phenotype by selecting for
the proper molecular marker allele (i.e., MAS). The more molecular
markers that are placed on the genetic map, the more potentially
useful that map becomes for conducting MAS.
[0099] Marker set: As used herein, a "set" of markers or probes
refers to a specific collection of markers or probes (or data
derived therefrom) that may be used to identify individuals
comprising a trait of interest. In some embodiments, a set of
markers linked to a low palmitic acid phenotype may be used to
identify sunflower plants comprising low palmitic acid content.
Data corresponding to a marker set or probe set (or data derived
from the use of such markers or probes) may be stored in an
electronic medium. While each marker in a marker set may possess
utility with respect to trait identification, individual markers
selected from the set and subsets including some, but not all, of
the markers may also be effective in identifying individuals
comprising the trait of interest.
[0100] Allele: As used herein, the term "allele" refers to one of
two or more different nucleotide sequences that occur at a specific
locus. For example, a first allele may occur on one chromosome,
while a second allele may occur on a second homologous chromosome;
e.g., as occurs for different chromosomes of a heterozygous
individual, or between different homozygous or heterozygous
individuals in a population. In some embodiments, a particular
allele at a particular locus may be linked to an agronomically
desirable phenotype (e.g., low palmitic acid content). In some
embodiments, a particular allele at the locus may allow the
identification of plants that do not comprise the agronomically
desirable phenotype (e.g., high palmitic acid content plants), such
that those plants may be removed from a breeding program or
planting. A marker allele may segregate with a favorable phenotype,
therefore providing the benefit of identifying plants comprising
the phenotype. An "allelic form of a chromosome segment" may refer
to a chromosome segment that comprises a marker allele nucleotide
sequence that contributes to, or is linked to, a particular
phenotype at one or more genetic loci physically located on the
chromosome segment.
[0101] "Allele frequency" may refer to the frequency (expressed as
a proportion or percentage) at which an allele is present at a
locus within a plant, within a line, or within a population of
lines. Thus, for an allele "A," a diploid individual of genotype
"AA," "Aa," or "aa," has an allele frequency of 1.0, 0.5, or 0.0,
respectively. The allele frequency within a line may be estimated
by averaging the allele frequencies of a sample of individuals from
that line. Similarly, the allele frequency within a population of
lines may be calculated by averaging the allele frequencies of
lines that make up the population. For a population with a finite
number of individuals or lines, an allele frequency may be
expressed as a count of individuals or lines (or any other
specified grouping) containing the allele.
[0102] A marker allele "positively" correlates with a trait when
the marker is linked to the trait, and when presence of the marker
allele is an indicator that the desired trait or trait form will
occur in a plant comprising the allele. A marker allele
"negatively" correlates with a trait when the marker is linked to
the trait, and when presence of the marker allele is an indicator
that the desired trait or trait form will not occur in a plant
comprising the allele.
[0103] A "homozygous" individual has only one form of allele at a
given locus (e.g., a diploid plant has a copy of the same allele
form at a particular locus for each of two homologous chromosomes).
An individual is "heterozygous" if more than one allele form is
present at the locus (e.g., a diploid individual has one copy of a
first allele form and one copy of a second allele form at the
locus). The term "homogeneity" refers to members of a group that
have the same genotype (i.e., the same allele frequency) at one or
more specific loci of interest. In contrast, the term
"heterogeneity" refers to individuals within a group that differ in
genotype at one or more specific loci of interest.
[0104] Any technique that may be used to characterize the
nucleotide sequence at a locus may be used to identify a marker
allele. Methods for marker allele detection include, for example
and without limitation, molecular identification methods (e.g.,
amplification and detection of a marker amplicon). For example, an
allelic form of an SSR marker, or of a SNP marker, may be detected
by an amplification based technology. In a typical
amplification-based detection method, a marker locus or a portion
of the marker locus is amplified (using, e.g., PCR, LCR, and
transcription using a nucleic acid isolated from a sunflower plant
of interest as an amplification template), and the resulting
amplified marker amplicon is detected. In some embodiments, plant
RNA may be utilized as the template for an amplification reaction.
In some embodiments, plant genomic DNA may be utilized as the
template for the amplification reaction. In some examples, the QTL
marker is an SNP marker, and the detected allele is a SNP marker
allele, and the method of detection is allele specific
hybridization (ASH). In some examples, the QTL marker is an SSR
marker, and the detected allele is an SSR marker allele.
[0105] ASH technology is based on the stable annealing of a short,
single-stranded, oligonucleotide probe to a completely
complementary single-strand target nucleic acid. Detection may be
accomplished via detection of an isotopic or non-isotopic label
attached to the probe. For each polymorphism, two or more different
ASH probes may be designed to have identical DNA sequences, except
at site of a polymorphism. Each probe may be perfectly homologous
with one allele sequence, so that the range of probes can
distinguish all the known alternative allele sequences. When each
probe is hybridized to target DNA under appropriate probe design
and hybridization conditions, a single-base mismatch between the
probe and target DNA prevents hybridization. In this manner, only
one of the alternative probes will hybridize to a target sample
that is homozygous for an allele. Samples that are heterozygous or
heterogeneous for two alleles will hybridize to both of two
alternative probes.
[0106] ASH markers may be used as dominant markers, where the
presence or absence of only one allele is determined from
hybridization or lack of hybridization by only one probe. The
alternative allele may be inferred from a lack of hybridization. In
examples, ASH probe and target molecules may be RNA or DNA
molecules; a target molecule may comprise any length of nucleotides
beyond the sequence that is complementary to the probe; the probe
may be designed to hybridize with either strand of a DNA target;
and the size of the probe may be varied to conform with the
requirements of different hybridization conditions.
[0107] Amplified variable sequences refer to amplified sequences of
the plant genome that exhibit high nucleic acid residue variability
between members of the same species. All organisms have variable
genomic sequences, and each organism (with the exception of a
clone) has a different set of variable sequences. Once identified,
the presence of specific variable sequence can be used to predict
phenotypic traits. DNA from a plant may in some examples be used as
a template for amplification with primers that flank a variable
sequence of DNA. The variable sequence may be amplified and then
sequenced.
[0108] Self-sustained sequence replication may also and
alternatively be used to identify genetic markers. Self-sustained
sequence replication refers to a method of nucleic acid
amplification using target nucleic acid sequences that are
replicated exponentially in vitro under substantially isothermal
conditions, using three enzymatic activities involved in retroviral
replication: reverse transcriptase; Rnase H; and a DNA-dependent
RNA polymerase. Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA
87:1874. By mimicking the retroviral strategy of RNA replication by
means of cDNA intermediates, this reaction accumulates cDNA and RNA
copies of the original target.
[0109] Data representing detected marker allele(s) may be
transmitted (for example, electronically; and via infrared,
wireless, or optical transmission) to a computer or
computer-readable medium for analysis or storage.
[0110] For example, an amplification primer or amplification primer
pair may be admixed with a genomic nucleic acid isolated from a
first sunflower plant or germplasm, wherein the primer or primer
pair is complementary or partially complementary to at least a
portion of a marker locus, and the primer or primer pair is capable
of initiating DNA polymerization by a DNA polymerase using the
sunflower genomic nucleic acid as a template. The primer or primer
pair (e.g., a primer pair provided in Table 6) is extended in a DNA
polymerization reaction utilizing a DNA polymerase and a template
genomic nucleic acid to generate at least one amplicon.
[0111] "Positional cloning" refers to a particular cloning
procedure in which a target nucleic acid is identified and isolated
by its genomic proximity to a marker. For example, a genomic
nucleic acid clone may include all or part of two more chromosomal
regions that are proximal to one another. If a marker can be used
to identify the genomic nucleic acid clone from a genomic library,
standard methods such as sub-cloning and/or sequencing may be used
to identify and or isolate sub-sequences of the clone that are
located near the marker.
[0112] Locus: As used herein, the term "locus" refers to a position
on the genome that corresponds to a measurable characteristic
(e.g., a trait) or polymorphism. An SNP locus is defined by a probe
that hybridizes to DNA contained within the locus.
[0113] Marker-assisted breeding: As used herein, the term
"marker-assisted breeding" may refer to an approach to breeding
directly utilizing MAS for one or more traits (e.g., reduced
palmitic acid content). In current practice, plant breeders attempt
to identify easily detectable traits, such as flower color, seed
coat appearance, or isozyme variants that are linked to an
agronomically desired trait. The plant breeders then follow the
agronomic trait in the segregating, breeding populations by
following the segregation of the easily detectable trait. However,
there are very few of these linkage relationships available for use
in plant breeding.
[0114] Marker-assisted breeding provides a time- and cost-efficient
process for improvement of plant varieties. Several examples of the
application of marker-assisted breeding involve the use of isozyme
markers. See, e.g., Tanksley and Orton, eds. (1983) Isozymes in
Plant Breeding and Genetics, Amsterdam: Elsevier. One example is an
isozyme marker associated with a gene for resistance to a nematode
pest in tomato. The resistance, controlled by a gene designated Mi,
is located on chromosome 6 of tomato and is very tightly linked to
Aps1, an acid phosphatase isozyme. Use of the Aps1 isozyme marker
to indirectly select for the Mi gene provided the advantages that
segregation in a population can be determined unequivocally with
standard electrophoretic techniques; the isozyme marker can be
scored in seedling tissue, obviating the need to maintain plants to
maturity; and co-dominance of the isozyme marker alleles allows
discrimination between homozygotes and heterozygotes. See Rick
(1983) in Tanksley and Orton, supra.
[0115] Probe: In some embodiments, the presence of a marker in a
plant may be detected through the use of a nucleic acid probe. A
probe may be a DNA molecule or an RNA molecule. RNA probes can be
synthesized by means known in the art, for example, using a DNA
molecule template. A probe may contain all or a portion of the
nucleotide sequence of the marker and additional, contiguous
nucleotide sequence from the plant genome. This is referred to
herein as a "contiguous probe." The additional, contiguous
nucleotide sequence is referred to as "upstream" or "downstream" of
the original marker, depending on whether the contiguous nucleotide
sequence from the plant chromosome is on the 5' or the 3' side of
the original marker, as conventionally understood. As is recognized
by those of ordinary skill in the art, the process of obtaining
additional, contiguous nucleotide sequence for inclusion in a
marker may be repeated nearly indefinitely (limited only by the
length of the chromosome), thereby identifying additional markers
along the chromosome.
[0116] An oligonucleotide probe sequence may be prepared
synthetically or by cloning. Suitable cloning vectors are
well-known to those of skill in the art. An oligonucleotide probe
may be labeled or unlabeled. A wide variety of techniques exist for
labeling nucleic acid molecules, including, for example and without
limitation: radiolabeling by nick translation; random priming;
tailing with terminal deoxytransferase, or the like, where the
nucleotides employed are labeled, for example, with radioactive
.sup.32P. Other labels which may be used include, for example and
without limitation: Fluorophores (e.g., FAM and VIC); enzymes;
enzyme substrates; enzyme cofactors; enzyme inhibitors; and the
like. Alternatively, the use of a label that provides a detectable
signal, by itself or in conjunction with other reactive agents, may
be replaced by ligands to which receptors bind, where the receptors
are labeled (for example, by the above-indicated labels) to provide
detectable signals, either by themselves, or in conjunction with
other reagents. See, e.g., Leary et al. (1983) Proc. Natl. Acad.
Sci. USA 80:4045-9.
[0117] A probe may contain a nucleotide sequence that is not
contiguous to that of the original marker; this probe is referred
to herein as a "noncontiguous probe." The sequence of the
noncontiguous probe is located sufficiently close to the sequence
of the original marker on the genome so that the noncontiguous
probe is genetically linked to the same gene or trait (e.g., low
palmitic acid content). For example, in some embodiments, a
noncontiguous probe is located within about 10 cM; 9 cM; 8 cM; 7
cM; 6 cM; 5 cM; 4 cM; 3 cM; 2 cM; 1 cM; 0.75 cM; 0.5 cM; 0.25 cM;
or less, from a QTL marker illustrated in FIG. 4.
[0118] A probe may be an exact copy of a marker to be detected. A
probe may also be a nucleic acid molecule comprising, or consisting
of, a nucleotide sequence which is substantially identical to a
cloned segment of the subject organism's (e.g., sunflower)
chromosomal DNA. As used herein, the term "substantially identical"
may refer to nucleotide sequences that are more than 85% identical.
For example, a substantially identical nucleotide sequence may be
85.5%; 86%; 87%; 88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%;
98%; 99% or 99.5% identical to a reference sequence.
[0119] A probe may also be a nucleic acid molecule that is
"specifically hybridizable" or "specifically complementary" to an
exact copy of the marker to be detected ("DNA target").
"Specifically hybridizable" and "specifically complementary" are
terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the nucleic acid
molecule and the DNA target. A nucleic acid molecule need not be
100% complementary to its target sequence to be specifically
hybridizable. A nucleic acid molecule is specifically hybridizable
when there is a sufficient degree of complementarity to avoid
non-specific binding of the nucleic acid to non-target sequences
under conditions where specific binding is desired, for example,
under stringent hybridization conditions.
[0120] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are known
to those of ordinary skill in the art, and are discussed, for
example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames
and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford,
1985. Further detailed instruction and guidance with regard to the
hybridization of nucleic acids may be found, for example, in
Tijssen, "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," in Laboratory Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Acid
Probes, Part I, Chapter 2, Elsevier, N Y, 1993; and Ausubel et al.,
Eds., Current Protocols in Molecular Biology, Chapter 2, Greene
Publishing and Wiley-Interscience, N Y, 1995.
[0121] As used herein, "stringent conditions" encompass conditions
under which hybridization will only occur if there is less than 50%
mismatch between the hybridization molecule and the DNA target.
"Stringent conditions" include further particular levels of
stringency. Thus, as used herein, "moderate stringency" conditions
are those under which molecules with more than 50% sequence
mismatch will not hybridize; conditions of "high stringency" are
those under which sequences with more than 20% mismatch will not
hybridize; and conditions of "very high stringency" are those under
which sequences with more than 10% mismatch will not hybridize.
[0122] The following are representative, non-limiting hybridization
conditions.
[0123] Very High Stringency (detects sequences that share at least
90% sequence identity): Hybridization in 5.times.SSC buffer at
65.degree. C. for 16 hours; wash twice in 2.times.SSC buffer at
room temperature for 15 minutes each; and wash twice in
0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.
[0124] High Stringency (detects sequences that share at least 80%
sequence identity): Hybridization in 5.times.-6.times.SSC buffer at
65-70.degree. C. for 16-20 hours; wash twice in 2.times.SSC buffer
at room temperature for 5-20 minutes each; and wash twice in
1.times.SSC buffer at 55-70.degree. C. for 30 minutes each.
[0125] Moderate Stringency (detects sequences that share at least
50% sequence identity): Hybridization in 6.times.SSC buffer at room
temperature to 55.degree. C. for 16-20 hours; wash at least twice
in 2.times.-3.times.SSC buffer at room temperature to 55.degree. C.
for 20-30 minutes each.
[0126] With respect to all probes discussed, supra, the probe may
comprise additional nucleic acid sequences, for example, promoters;
transcription signals; and/or vector sequences. Any of the probes
discussed, supra, may be used to define additional markers that are
linked to a gene involved in reduced palmitic acid content in
sunflower, and markers thus identified may be equivalent to
exemplary markers named in the present disclosure, and thus are
within the scope of the invention.
[0127] Sequence identity: The term "sequence identity" or
"identity," as used herein in the context of two nucleic acid or
polypeptide sequences, may refer to the residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window.
[0128] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences) over a comparison
window, wherein the portion of the sequence in the comparison
window may comprise additions or deletions (i.e., gaps) as compared
to the reference sequence (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The
percentage is calculated by determining the number of positions at
which the identical nucleotide or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
comparison window, and multiplying the result by 100 to yield the
percentage of sequence identity.
[0129] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482;
Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and
Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS
5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang
et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994)
Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol.
Lett. 174:247-50. A detailed consideration of sequence alignment
methods and homology calculations can be found in, e.g., Altschul
et al. (1990) J. Mol. Biol. 215:403-10.
[0130] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default BLOSUM62 matrix set to default
parameters. Nucleic acid sequences with even greater similarity to
the reference sequences will show increasing percentage identity
when assessed by this method.
[0131] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" may refer to a polymeric form of nucleotides, which
may include both sense and anti-sense strands of RNA, cDNA, genomic
DNA, and synthetic forms and mixed polymers of the above. A
nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a
modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." The term includes single- and double-stranded
forms of DNA. A nucleic acid molecule can include either or both
naturally occurring and modified nucleotides linked together by
naturally occurring and/or non-naturally occurring nucleotide
linkages.
[0132] An "exogenous" molecule is a molecule that is not native to
a specified system (e.g., a germplasm, variety, elite variety,
and/or plant) with respect to nucleotide sequence and/or genomic
location for a polynucleotide, and with respect to amino acid
sequence and/or cellular localization for a polypeptide. In
embodiments, exogenous or heterologous polynucleotides or
polypeptides may be molecules that have been artificially supplied
to a biological system (e.g., a plant cell, a plant gene, a
particular plant species or variety, and/or a plant chromosome) and
are not native to that particular biological system. Thus, the
designation of a nucleic acid as "exogenous" may indicate that the
nucleic acid originated from a source other than a naturally
occurring source, or it may indicate that the nucleic acid has a
non-natural configuration, genetic location, or arrangement of
elements.
[0133] In contrast, for example, a "native" or "endogenous" nucleic
acid is a nucleic acid (e.g., a gene) that does not contain a
nucleic acid element other than those normally present in the
chromosome or other genetic material on which the nucleic acid is
normally found in nature. An endogenous gene transcript is encoded
by a nucleotide sequence at its natural chromosomal locus, and is
not artificially supplied to the cell.
[0134] The term "recombinant" refers to a material (e.g.,
recombinant nucleic acid, recombinant gene, recombinant
polynucleotide, and/or recombinant polypeptide) that has been
altered by human intervention. For example, the arrangement of the
parts or elements of a recombinant molecule may not be a native
arrangement, and/or the primary sequence of the recombinant
molecule may been changed from its native sequence in some way. A
material may be altered to produce a recombinant material within or
removed from its natural environment or state. An open reading
frame of a nucleic acid is recombinant if the nucleotide sequence
of the open reading frame has been removed from it natural context
and cloned into any type of artificial nucleic acid (e.g., a
vector). Protocols and reagents to produce recombinant molecules,
especially recombinant nucleic acids, are common and routine in the
art. The term "recombinant" may also herein refer to a cell or
organism that comprises recombinant material (e.g., a plant and/or
plant cell that comprises a recombinant nucleic acid). In some
examples, a recombinant organism is a transgenic organism.
[0135] As used herein, the term "introduced," when referring to
translocation of a heterologous or exogenous nucleic acid into a
cell, refers to the incorporation of the nucleic acid into the cell
using any methodology available in the art. This term encompasses
nucleic acid introduction methods including, for example and
without limitation, transfection; transformation; and
transduction.
[0136] As used herein, the term "vector" refers to a polynucleotide
or other molecules that is capable of transferring at least one
nucleic acid segment(s) into a cell. A vector may optionally
comprise components/elements that mediate vector maintenance and
enable its intended use (e.g., sequences necessary for replication,
genes imparting drug or antibiotic resistance, a multiple cloning
site, and/or operably linked promoter/enhancer elements that enable
the expression of a cloned gene). Vectors may be derived, for
example, from plasmids, bacteriophages, or plant or animal viruses.
A "cloning vector," "shuttle vector," or "subcloning vector"
generally comprises operably linked elements to facilitate cloning
or subcloning steps (e.g., a multiple cloning site containing
multiple restriction endonuclease sites).
[0137] The term "expression vector," as used herein, refers to a
vector comprising operably linked polynucleotide sequences that may
facilitate expression of a coding sequence in a particular host
organism. For example, a bacterial expression vector may facilitate
expression of a coding sequence in a bacterium. A plant expression
vector may facilitate expression of a coding sequence in a plant
cell. Polynucleotide sequences that facilitate expression in
prokaryotes may include, for example and without limitation, a
promoter; an operator; and a ribosome binding site. Eukaryotic
expression vectors (e.g., a plant expression vector) comprise
promoters, enhancers, termination, and polyadenylation signals (and
other sequences) that are generally different from those used in
prokaryotic expression vectors.
[0138] Single-nucleotide polymorphism: As used herein, the term
"single-nucleotide polymorphism" (SNP) may refer to a DNA sequence
variation occurring when a single nucleotide in the genome (or
other shared sequence) differs between members of a species or
paired chromosomes in an individual. Within a population, SNPs can
be assigned a minor allele frequency that is the lowest allele
frequency at a locus that is observed in a particular population.
This is simply the lesser of the two allele frequencies for
single-nucleotide polymorphisms. Different populations are expected
to exhibit at least slightly different allele frequencies.
Particular populations may exhibit significantly different allele
frequencies. In some examples, markers linked to SCN resistance are
SNP markers.
[0139] SNPs may fall within coding sequences of genes, non-coding
regions of genes, or in the intergenic regions between genes. SNPs
within a coding sequence will not necessarily change the amino acid
sequence of the protein that is produced, due to degeneracy of the
genetic code. An SNP in which both forms lead to the same
polypeptide sequence is termed "synonymous" (sometimes called a
silent mutation). If a different polypeptide sequence is produced,
they are termed "non-synonymous." A non-synonymous change may
either be missense or nonsense, where a missense change results in
a different amino acid, and a nonsense change results in a
premature stop codon. SNPs that are not in protein-coding regions
may still have consequences for gene splicing, transcription factor
binding, or the sequence of non-coding RNA. SNPs are usually
biallelic and thus easily assayed in plants and animals.
Sachidanandam (2001) Nature 409:928-33.
[0140] Plant: As used herein, the term "plant" may refer to a whole
plant, a cell or tissue culture derived from a plant, and/or any
part of any of the foregoing. Thus, the term "plant" encompasses,
for example and without limitation, whole plants; plant components
and/or organs (e.g., leaves, stems, and roots); plant tissue; seed;
and a plant cell. A plant cell may be, for example and without
limitation, a cell in and/or of a plant, a cell isolated from a
plant, and a cell obtained through culturing of a cell isolated
from a plant. Thus, the term "sunflower plant" may refer to, for
example and without limitation, a whole sunflower plant; multiple
sunflower plants; sunflower plant cell(s); sunflower plant
protoplast; sunflower tissue culture (e.g., from which a sunflower
plant can be regenerated); sunflower plant callus; sunflower plant
parts (e.g., sunflower seed, sunflower flower, sunflower cotyledon,
sunflower leaf, sunflower stem, sunflower bud, sunflower root, and
sunflower root tip); and sunflower plant cells that are intact in
sunflower plants or in parts of sunflower plants.
[0141] A "transgenic plant" is a plant comprising within at least
one of its cells an exogenous polynucleotide. In examples, the
exogenous polynucleotide is stably integrated within the genome of
the cell, such that the polynucleotide may be inherited in
successive generations. In some examples, the heterologous
polynucleotide may be integrated into the genome as part of a
recombinant expression cassette. The term "transgenic" is used
herein to refer to any cell, cell line, callus, tissue, plant part,
or plant, the genotype of which has been altered by the presence of
a exogenous nucleic acid. Thus, this term encompasses transgenic
organisms and cells that have been initially altered to comprise
the exogenous polynucleotide, and those organisms and cells created
by crosses or asexual propagation of the initial transgenic
organism or cell. The term "transgenic," as used herein, does not
encompass genome (chromosomal or extra-chromosomal) alternations
introduced by conventional plant breeding methods (e.g., crosses of
only non-transgenic organisms) or by naturally occurring events
(e.g., random cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, and spontaneous mutation).
[0142] A plant "line," "variety," or "strain" is a group of
individual plants having the same parentage. Plants of a line
generally are inbred to some degree, and are generally homozygous
and homogeneous at most genetic loci. A "subline" may refer to an
inbred subset of descendents from a common progenitor that are
genetically distinct from other similarly inbred subsets descended
from the same progenitor. In some embodiments, a "subline" may be
produced by inbreeding seed from an individual sunflower plant
selected at the F.sub.3 to F.sub.5 generation until the residual
segregating loci are homozygous across most or all loci.
[0143] Commercial sunflower varieties are typically produced by
aggregating the self-pollinated progeny ("bulking") of a single
F.sub.3 to F.sub.5 plant from a controlled cross between 2
genetically different parents. While such a variety typically
appears uniform, a self-pollinating variety derived from the
selected plant eventually (for example, by the F.sub.8 generation)
becomes a mixture of homozygous plants that may vary in genotype at
any locus that was heterozygous in the originally selected F.sub.3
to F.sub.5 plant. In embodiments described herein, marker-based
sublines that differ from each other based on qualitative marker
polymorphism at the DNA level at one or more specific loci, are
produced by genotyping a sample of seed derived from individual
self-pollinated progeny derived from a selected F.sub.3 to F.sub.5
plant. Such a seed sample may be genotyped directly as seed, or as
plant tissue grown from seed. In some examples, seed sharing a
common genotype at one or more specified marker locus are bulked to
produce a subline that is genetically homogenous at one or more
locus that is linked to a trait of interest (e.g., low palmitic
acid content).
[0144] An "ancestral line" refers to a parent line that is or has
been used as a source of genetic material, for example, for the
development of elite lines. An "ancestral population" refers to a
group of ancestors that have contributed the bulk of the genetic
variation that was used to develop an elite line. "Descendants" are
progeny of ancestors, and descendents may be separated from their
ancestors by many generations of breeding. For example, elite lines
are the descendants of their ancestors. A pedigree may be used to
describe the relationship between a descendant and each of its
ancestors. A pedigree may span one or more generations, and thus
may describe relationships between a descendant and its ancestors
removed by 1, 2, 3, 4, etc., generations.
[0145] An "elite line" or "elite strain" refers to an agronomically
superior line that has been bred and selected (often through many
cycles) for superior agronomic performance. Numerous elite
sunflower lines are available and known to those of skill in the
art. An elite population is an assortment of elite lines or
individuals from elite lines that may be used to represent the
state of the art in terms of the available agronomically superior
genotypes of a given crop species (e.g., sunflower). Similarly, an
elite germplasm or elite strain of germplasm is an agronomically
superior germplasm. An elite germplasm may be obtained from a plant
with superior agronomic performance, and may capable of being used
to generate a plant with superior agronomic performance, such as a
sunflower of an existing or newly developed elite line.
[0146] In contrast to elite lines, an "exotic line" or "exotic
strain" (or an "exotic germplasm") refers to a line or germplasm
obtained from a sunflower not belonging to an available elite
sunflower line or strain of germplasm. In the context of a cross
between two sunflower plants or germplasms, an exotic germplasm is
not closely related by descent to the elite germplasm with which it
is crossed. Most commonly, exotic germplasm has been selected to
introduce a novel genetic element (e.g., an allele form of
interest) into a breeding program.
[0147] Trait or phenotype: The terms "trait" and "phenotype" are
used interchangeably herein to refer to a measurable or observable
heritable characteristic. A phenotype may in some examples be
directly controlled by a single gene or genetic locus (i.e., a
single gene trait). In other examples, a phenotype may be the
result of an interaction between several genes (a complex trait).
Thus, a QTL can act through a single gene mechanism or by a
polygenic mechanism. In some examples, a trait or phenotype can be
assigned a "phenotypic value," which corresponds to a quantitative
value measured for the phenotypic trait.
[0148] The term "molecular phenotype" may refer to a phenotype that
is detectable at the level of a population of (one or more)
molecules. In some examples, the molecular phenotype may only be
detectable at the molecular level. The detectable molecules of the
phenotype may be nucleic acids (e.g., genomic DNA or RNA);
proteins; and/or metabolites. For example, a molecular phenotype
may be an expression profile for one or more gene products (e.g.,
at a specific stage of plant development, or in response to an
environmental condition or stress).
[0149] Low palmitic acid content: For the purposes of the present
disclosure, a trait of particular interest is "low palmitic acid
content." Although the fatty acid composition of sunflower plants
may be affected to an extent by environmental factors, those in the
art understand that palmitic acid content (as well as other oil
traits) are predominantly determined by heritable genetic factors.
Thus, for example, the selection of a particular sunflower variety
for cultivation may be based at least in part on the characteristic
palmitic acid content of that particular variety under normal field
growing conditions (e.g., conditions without drought, disease, and
adequate soil nutrients). In examples, a sunflower plant having a
low palmitic acid content may comprise a palmitic acid (16:0)
content that is about 3% or less of the total oil content in seed
of the plant. In some examples, such a sunflower plant having a low
palmitic acid content comprises a palmitic acid content that is
about 2.5% or less of the total oil content in seed of the plant,
for example and without limitation, the palmitic acid content may
be 2.6%; 2.5%; 2.4%; 2.3%; 2.2%; 2.1%; 2.0%; 1.9%; 1.8%; about
1.7%; and lower.
[0150] In some embodiments, "low palmitic acid content" is
determined by comparison with the characteristic palmitic acid
content of a wild-type or parental variety. Thus, a first sunflower
comprising a low palmitic acid content phenotype may have
"decreased" or "lowered" levels of palmitic acid relative to a
wild-type sunflower, or relative to a parental sunflower variety
from which the first sunflower was derived. Decreased and lowered
are relative terms, indicating that the plant produces or contains
less palmitic acid than a similar wild-type plant.
[0151] Sunflower plant palmitic acid content varies widely, and the
characteristic palmitic acid contents measured in particular
varieties represent a spectrum of higher and lower palmitic acid
content phenotypes. However, by simple observation, the relative
palmitic acid content of different plants, plant lines, or plant
families may be determined. Furthermore, sunflower varieties
represent genetically determinable phenotypic gradations of
"palmitic acid content." One of skill in the art is familiar with
assays for quantitating and scoring sunflower plant palmitic acid
content. The palmitic acid content of a plant may be quantitated by
using various analytical techniques standard in the art including,
for example and without limitation, NMR; NIR; and Soxhlet
extraction.
[0152] Verification of low palmitic acid content may be
accomplished by using or adapting available palmitic acid content
protocols. For example, NMR, NIR, and/or Soxhlet extraction may be
utilized to verify that a low palmitic acid content trait still
segregates with a particular marker in any particular plant or
population. These and other protocols may also be used in some
embodiments to measure the degree of palmitic acid content
reduction achieved by introgressing or recombinantly introducing a
marker linked to low palmitic acid content into a desired genetic
background.
IV. Markers for Low Palmitic Acid Content in Sunflower
[0153] Embodiments of the invention include markers that are linked
to low palmitic acid content in sunflower. Such markers may be
used, for example and without limitation, to identify sunflower
plants and germplasm having an increased likelihood of comprising a
low palmitic acid phenotype; to select such sunflower plants and
germplasm (e.g., in a marker-assisted selection program); and to
identify and select sunflower plants and germplasm that do not have
an increased likelihood of comprising a low palmitic acid
phenotype. Use of one or more of the markers describe herein may
provide advantages to plant breeders with respect to the time,
cost, and labor involved in sunflower breeding, when compared to
currently available compositions and methods in the art.
[0154] Disclosed herein are particular markers identified to be
within or near a low palmitic acid content QTL region in linkage
group 5 (LG5) in the sunflower genome that are polymorphic in
parent genotypes. Among such QTL markers are particular marker
alleles that are linked to a low palmitic acid content phenotype in
sunflower. In some embodiments, a QTL marker that is linked to a
low palmitic acid content phenotype in sunflower is selected from
the subset of markers provided in FIG. 1. For example and without
limitation, a QTL marker that is linked to a low palmitic acid
content phenotype in sunflower may be selected from HA0031B;
HA0908; HA1665; HA0304A; HA0850; HA0743; HA0870; HA0907; and
HA0612A.
[0155] Mapping populations may be used to determine a marker that
is linked to a low palmitic acid content. In some embodiments, such
a mapping population may be derived from the cross, H757B/H280R,
though other populations may also and alternatively be used. Any of
many suitable software platforms may be used to determine a linked
marker locus. For example and without limitation, TASSEL.RTM.;
GeneFlow.RTM.; and MapManager-QTX.RTM. may be used in particular
examples. In some embodiments, such as when software is used in a
linkage analysis, data reflecting detected allele information may
be electronically transmitted or electronically stored during use
or prior to use, for example, in a computer readable medium.
[0156] In some embodiments, a first sunflower plant or germplasm
that is likely to comprise a low palmitic acid content phenotype is
identified by detecting a plurality of marker alleles in the first
sunflower plant or germplasm. For example and without limitation,
particular embodiments include methods for identifying plants or
germplasm that is likely to comprise a low palmitic acid content
phenotype, where a marker allele linked to low palmitic acid is
detected from among the molecular markers, HA0031B; HA0908; HA1665;
HA0304A; HA0850; HA0743; HA0870; HA0907; and HA0612A. Methods for
identifying plants or germplasm that is likely to comprise a low
palmitic acid content phenotype according to some embodiments
comprise detecting more than one marker allele linked to low
palmitic acid from among the molecular markers, HA0031B; HA0908;
HA1665; HA0304A; HA0850; HA0743; HA0870; HA0907; and HA0612A.
Particular embodiments include methods for identifying plants or
germplasm that is likely to comprise a low palmitic acid content
phenotype, where a marker allele is detected from among molecular
markers that are linked to at least one marker linked to low
palmitic acid selected from HA0031B; HA0908; HA1665; HA0304A;
HA0850; HA0743; HA0870; HA0907; and HA0612A.
[0157] In some embodiments, a detected allele is an allele form
that positively correlates with low palmitic acid content.
Alternatively, an allele that is detected may be an allele form
that negatively correlates with low palmitic acid content, in which
case the allele may be counter-selected. In the case where more
than one marker allele is selected for detection, an allele is
selected for each of the markers; thus, two or more alleles are
detected. In some examples, a marker may comprise more than one
advantageous (e.g., positively correlated) allele form; in such an
example, any of such advantageous allele forms can be detected.
[0158] Thus, a plurality of marker alleles may be simultaneously
detected in a single plant, germplasm, or population of plants. In
examples of such methods, a plant or germplasm may be selected that
contains positively correlated alleles from more than one marker
linked to low palmitic acid content. In particular examples,
positively correlated alleles from more than one marker linked to
low palmitic acid content may be introgressed into a target (e.g.,
recipient) sunflower germplasm. It will be appreciated by those of
skill in the art that the simultaneous selection (and/or
introgression) of positively correlated alleles from more than one
low palmitic acid content marker in the same plant or germplasm may
result in an additive (e.g., synergistic) phenotype in the plant or
germplasm.
[0159] Although particular marker alleles may co-segregate with a
low palmitic acid content phenotype, such marker loci are not
necessarily part of a QTL locus contributing to (e.g., responsible
for) the low palmitic acid content. For example, it is not a
requirement that a co-segregating marker be comprised within a gene
(e.g., as part of the gene open reading frame) that contributes to
or imparts low palmitic acid content. The association between a
specific marker allele with a low palmitic acid content phenotype
may be due to the original "coupling" linkage phase between the
co-segregating marker allele and a QTL low palmitic acid content
allele in the ancestral sunflower line from which the low palmitic
acid content allele originated. Eventually, with repeated
recombination, crossing-over events between the co-segregating
marker and QTL locus may change this orientation. Thus, a
positively correlated marker allele may change through successive
generations, depending on the linkage phase that exists within the
low palmitic acid content parent used to create segregating
populations. This fact does not reduce the utility of the marker
for monitoring segregation of the phenotype; it only changes which
marker allele form is positively (vs. negatively) correlated in a
given segregating population.
[0160] When referring to the relationship between two genetic
elements (e.g., a genetic element contributing to low palmitic acid
content and a proximal marker), "coupling" phase linkage refers to
the circumstance where the positively correlated allele at a low
palmitic acid content QTL is physically associated on the same
chromosome strand as the positively correlated allele of the
respective linked marker locus. In "coupling phase," both alleles
are inherited together by progeny that inherit that chromosome
strand. In "repulsion" phase linkage, the positively correlated
allele at a locus of interest (e.g., a QTL for low palmitic acid
content) is physically linked with a normally negatively correlated
allele at the proximal marker locus, and thus the two alleles that
are normally positively correlated are not inherited together
(i.e., the two loci are "out of phase" with each other).
[0161] As used herein, a "positively correlated" allele of a marker
is that allele of the marker that co-segregates with a desired
phenotype (e.g., low palmitic acid content) in the mapping
populations described herein. However, in view of the foregoing, it
will be understood that due to the possibility of repulsion phase
linkage, other allele forms of the marker may be used equivalently
in other embodiments involving different populations.
[0162] Similarly, a linked marker allele form that does not
co-segregate with low palmitic acid content may also and
alternatively be used in some embodiments, since such an allele
form may be used to identify a plant that is not likely to comprise
a low palmitic acid phenotype. For example, such an allele may be
used for exclusionary purposes (e.g., counter-selection) during
breeding to identify alleles that negatively correlate with low
palmitic acid content, and/or to eliminate increased palmitic acid
content plants or germplasm from subsequent rounds of breeding.
[0163] A QTL marker has a minimum of one positively correlated
allele, although in some examples the QTL marker may have two or
more positively correlated alleles found in the population. Any
positively correlated allele of such a marker may be used, for
example, for the identification and construction of low (e.g.,
decreased) palmitic acid content sunflower lines. In some examples,
one, two, three, or more positively correlated allele(s) of
different markers linked to low palmitic acid content are
identified in (or introgressed into) a plant, and all or a subset
of the positively correlated markers may be selected for or against
during MAS. In some embodiments, at least one plant or germplasm is
identified that has at least one such allele that positively
correlates with a low palmitic acid content phenotype.
[0164] Marker loci are themselves traits, and may thus be analyzed
according to standard linkage analysis, e.g., by tracking the
marker loci during segregation. Therefore, in some embodiments,
linkage between markers is determined, for example, where one cM is
equal to a 1% chance that a first marker locus will be separated by
crossing-over in a single generation from a second locus (which may
be any other trait, (e.g., a second marker locus), or another trait
locus that comprises or is comprised within a QTL).
[0165] Genetic markers that are linked to QTL markers (e.g., QTL
markers provided in FIG. 1 and their equivalents) are particularly
useful when they are sufficiently proximal (i.e., sufficiently
tightly linked) to a given QTL marker, so that the genetic marker
and the QTL marker display a low recombination frequency. In some
embodiments, a linked marker and a QTL marker display a
recombination frequency of about 10% or less (i.e., the given
marker is within about 10 cM of the QTL). By definition, these
linked loci will co-segregate at least 90% of the time. Indeed, the
closer a marker is to a QTL marker, the more effective and
advantageous that marker becomes as an indicator for the desired
trait. Nonetheless, markers that are, for example, more than about
10 cM from a QTL may be useful, particularly when combined with
other linked markers.
[0166] Thus, in some embodiments, linked loci such as a QTL marker
locus and a second marker locus display an inter-locus
recombination frequency of about 10% or less; for example and
without limitation, about 9% or less, about 8% or less, about 7% or
less, about 6% or less, about 5% or less, about 4% or less, about
3% or less, and about 2% or less. In some examples, the relevant
loci (e.g., a marker locus and a target locus, such as a QTL)
display a recombination a frequency of about 1% or less; for
example and without limitation, about 0.75% or less, about 0.5% or
less, and about 0.25% or less. Thus, loci may in particular
embodiments be separated by about 10 cM; about 9 cM; about 8 cM;
about 7 cM; about 6 cM; about 5 cM; about 4 cM; about 3 cM; about 2
cM; about 1 cM; about 0.75 cM; about 0.5 cM; about 0.25 cM; or
less. In some examples, specific linked markers may be determined
by review of a genetic map of the sunflower genome including, for
example, LG5.
[0167] In some aspects, linkage may be expressed as a recombination
frequency limit, or as a genetic or physical distance range. For
example, in some embodiments, two linked loci are two loci that are
separated by less than 50 cM map units. In some examples, linked
loci are two loci that are separated by less than 40 cM. In some
examples, two linked loci are two loci that are separated by less
than 30 cM. In some examples, two linked loci are two loci that are
separated by less than 25 cM. In some examples, two linked loci are
two loci that are separated by less than 20 cM. In some examples,
two linked loci are two loci that are separated by less than 15 cM.
In some examples, linkage may be expressed as a range with an upper
and a lower limit; for example and without limitation, between
about 10 and 20 cM; between about 10 and 30 cM; between about 10
and 40 cM; between about 0.5 and about 10 cM; between about 0.1 and
about 9 cM; between about 0.1 and about 8 cM; between about 0.1 and
about 7 cM; between about 0.1 and about 6 cM; between about 0.1 and
about 5 cM; between about 0.1 and about 4 cM; between about 0.1 and
about 3 cM; between about 0.1 and about 2 cM; between about 0.1 and
about 1 cM; and between about 0.1 and about 0.5 cM.
[0168] Markers described herein (e.g., those markers set forth in
FIG. 1, HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870,
HA0907, HA0612A, and markers linked to at least one of the
foregoing) are positively correlated with low sunflower palmitic
acid content in some embodiments. Thus, these markers may be
sufficiently proximal to a low palmitic acid content QTL and/or
trait that one or more of the markers may be used as a predictor
for a low palmitic acid content trait. This predictive ability is
extremely useful in the context of MAS, as discussed in more detail
herein.
[0169] Use of particular markers described herein that are linked
to a low palmitic acid content phenotype and/or QTL marker is not
restricted to any particular sunflower genetic map or methodology.
It is noted that lists of linked markers may vary between maps and
methodologies due to various factors. For example, the markers that
are placed on any two maps may not be identical, and a first map
may have a greater marker density than another, second map. Also,
the mapping populations, methodologies, and algorithms used to
construct genetic maps may differ. One of skill in the art
recognizes that one genetic map is not necessarily more or less
accurate than another, and the skilled person furthermore
recognizes that any sunflower genetic map may be used to determine
markers that are linked to a particular marker. For example,
particular linked markers can be determined from any genetic map
known in the art (e.g., an experimental map or an integrated map),
and can be determined from any new mapping dataset.
[0170] Embodiments of the present invention are not limited to any
particular sunflower population or use of any particular
methodology (e.g., any particular software or any particular set of
software parameters) to identify or determine linkage of a
particular marker with a low palmitic acid content phenotype. In
view of the present disclosure, one of ordinary skill in the art
will be able to extrapolate features of the markers described
herein to any sunflower gene pool or population of interest, and
use any particular software and software parameters in so
doing.
V. Detection of Markers for Low Palmitic Acid Content in
Sunflower
[0171] Methods for detecting (identifying) sunflower plants or
germplasm that carry particular alleles of low palmitic acid
content marker loci are a feature of some embodiments. In some
embodiments, any of a variety of marker detection protocols
available in the art may be used to detect a marker allele,
depending on the type of marker being detected. In examples,
suitable methods for marker detection may include amplification and
identification of the resulting amplified marker by, for example
and without limitation, PCR; LCR; and transcription-based
amplification methods (e.g., ASH, SSR detection, RFLP analysis, and
many others).
[0172] In general, a genetic marker relies on one or more property
of nucleic acids for its detection. For example, some techniques
for detecting genetic markers utilize hybridization of a probe
nucleic acid to a nucleic acid corresponding to the genetic marker
(e.g., an amplified nucleic acid produced using a genomic sunflower
DNA molecule as a template). Hybridization formats including, for
example and without limitation, solution phase; solid phase; mixed
phase; and in situ hybridization assays may be useful for allele
detection in particular embodiments. An extensive guide to the
hybridization of nucleic acids may be found, for example, in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes Elsevier, N.Y.
[0173] Markers corresponding to genetic polymorphisms between
members of a population may be detected by any of numerous methods
including, for example and without limitation, nucleic acid
amplification-based methods; and nucleotide sequencing of a
polymorphic marker region. Many detection methods (including
amplification-based and sequencing-based methods) may be readily
adapted to high throughput analysis in some examples, for example,
by using available high throughput sequencing methods, such as
sequencing by hybridization.
[0174] Amplification primers for amplifying SSR-type marker loci
are included in particular examples of some embodiments. Table 6
provides specific primers for amplification of particular markers
described herein. However, one of skill will immediately recognize
that other sequences on either side of the given primers may be
used in place of the given primers, so long as the primers are
capable of amplifying a nucleotide sequence comprising the allele
to be detected. Further, the precise probe used for allele
detection may vary. For example, any probe capable of identifying
the region of a marker amplicon to be detected may be substituted
for the exemplary probes listed herein. Further, the configuration
of amplification primers and detection probes may also vary. Thus,
embodiments are not limited to the primers and probes specifically
recited herein. Although many specific examples of primers are
provided herein (see Table 6), suitable primers to be used with the
invention may be designed using any suitable method. For example,
equivalent primers may be designed using any suitable software
program, such as for example and without limitation,
LASERGENE.RTM..
[0175] Molecular markers may be detected by established methods
available in the art including, for example and without limitation:
ASH, or other methods for detecting SNPs; AFLP detection; amplified
variable sequence detection; RAPD detection; RFLP detection;
self-sustained sequence replication detection; SSR detection; SSCP
detection; and isozyme markers detection. While the exemplary
markers provided in FIG. 1 and Table 6 are SSR markers, any of the
aforementioned marker types may be employed in particular
embodiments to identify chromosome segments encompassing a genetic
element that contributes to a low palmitic acid content phenotype
in sunflower.
[0176] For example, markers that comprise RFLPs may be detected,
for example, by hybridizing a probe (which is typically a
sub-fragment or synthetic oligonucleotide corresponding to a
sub-fragment) of the nucleic acid to be detected to
restriction-digested genomic DNA. The restriction enzyme is
selected so as to provide restriction fragments of at least two
alternative (or polymorphic) lengths in different individuals or
populations. Determining one or more restriction enzyme(s) that
produces informative fragments for each cross is a simple procedure
that is easily accomplished by those of skill in the art after
provision of the target DNA sequence. After separation by length in
an appropriate matrix (e.g., agarose or polyacrylamide) and
transfer to a membrane (e.g., nitrocellulose or nylon), a labeled
probe may be hybridized under conditions that result in equilibrium
binding of the probe to the target, followed by removal of excess
probe by washing, and detection of the labeled probe.
[0177] In some embodiments, an amplification step is utilized as
part of a method to detect/genotype a marker locus. However, an
amplification step is not in all cases a requirement for marker
detection. For example, an unamplified genomic DNA may be detected
simply by performing a Southern blot on a sample of genomic DNA.
Separate detection probes may also be omitted in
amplification/detection methods, for example and without
limitation, by performing a real time amplification reaction that
detects product formation by modification of an amplification
primer upon incorporation into a product; incorporation of labeled
nucleotides into an amplicon; and by monitoring changes in
molecular rotation properties of amplicons as compared to
unamplified precursors (e.g., by fluorescence polarization).
[0178] PCR, RT-PCR, real-time PCR, and LCR are in particularly
broad use as amplification and amplification-detection methods for
amplifying and detecting nucleic acids (e.g., those comprising
marker loci). Details regarding the use of these and other
amplification methods can be found in any of a variety of standard
texts including, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual (2000) 3rd Ed., Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.; Current Protocols in
Molecular Biology (supplemented through 2002) F. M. Ausubel et al.,
eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc.; and PCR Protocols
A Guide to Methods and Applications (1990) Innis et al. eds)
Academic Press Inc., San Diego, Calif. Additional details regarding
detection of nucleic acids in plants can also be found, for
example, in Plant Molecular Biology (1993) Croy (ed.) BIOS
Scientific Publishers, Inc.
[0179] Additional details regarding techniques sufficient to direct
persons of skill through particular in vitro amplification and
detection methods, including the polymerase chain reaction (PCR),
the ligase chain reaction (LCR), Q.beta.-replicase amplification,
and other RNA polymerase-mediated techniques (e.g., NASBA), and
examples thereof, may also be found in, for example: U.S. Pat. No.
4,683,202; Arnheim and Levinson (1991) J. NIH Res. 3:81-94; Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al.
(1990), supra; Lomell et al. (1989) J. Clin. Chem. 35:1826;
Landegren et al. (1988) Science 241:1077-80; Van Brunt (1990)
Biotechnology 8:291-4; Wu and Wallace (1989) Gene 4:560; Barringer
et al. (1990) Gene 89:117; and Sooknanan and Malek (1995)
Biotechnology 13:563-4. Improved methods of amplifying large
nucleic acids by PCR, which may be useful in some applications of
positional cloning, are further described in Cheng et al. (1994)
Nature 369:684, and the references cited therein, in which PCR
amplicons of up to 40 kb are generated.
[0180] Many available biology texts also have extended discussions
regarding PCR and related amplification methods. One of skill will
appreciate that essentially any RNA can be converted into a
double-stranded DNA that is suitable for restriction digestion, PCR
amplification, and sequencing using reverse transcriptase and a
polymerase (e.g., by RT-PCR).
[0181] In some embodiments, a nucleic acid probe may be used to
detect a nucleic acid that comprises a marker allele nucleotide
sequence. Such probes can be used, for example, in positional
cloning to isolate nucleotide sequences that are linked to a marker
allele sequence. Nucleic acid probes that are useful in particular
embodiments are not limited by any particular size constraint. In
some embodiments, a nucleic acid probe may be, for example and
without limitation, at least 20 nucleotides in length; at least 50
nucleotides in length; at least 100 nucleotides in length; and at
least 200 nucleotides in length. Nucleic acid probes to a marker
locus may be cloned and/or synthesized.
[0182] Any suitable label may be used with a probe in particular
examples. Detectable labels suitable for use with nucleic acid
probes include any composition detectable by spectroscopic,
radioisotopic, photochemical, biochemical, immunochemical,
electrical, optical, or chemical means. Thus, a hybridized probe
may be detected using, for example, autoradiography, fluorography,
or other similar detection techniques, depending on the particular
label to be detected. Useful labels include biotin (for staining
with labeled streptavidin conjugate), magnetic beads, fluorescent
dyes, radiolabels, enzymes, and colorimetric labels. Other labels
include ligands that bind to antibodies or specific binding targets
labeled with fluorophores, chemiluminescent agents, and enzymes. A
probe may also comprise radiolabeled PCR primers that are used to
generate a radiolabeled amplicon. Additional information regarding
labeling strategies for labeling nucleic acids, and corresponding
detection strategies may be found, for example, in Haugland (1996)
Handbook of Fluorescent Probes and Research Chemicals, Sixth
Edition, Molecular Probes, Inc., Eugene Oreg.; and Haugland (2001)
Handbook of Fluorescent Probes and Research Chemicals, Eighth
Edition, Molecular Probes, Inc., Eugene, Oreg. (Available on CD
ROM). In particular examples, PCR detection and quantification is
carried out using dual-labeled fluorogenic oligonucleotide probes,
for example, TaqMan.RTM. probes (Applied Biosystems).
[0183] In some embodiments, primers are not labeled, and marker PCR
amplicons may be visualized, for example, following their size
resolution (e.g., following agarose gel electrophoresis). In
particular examples, ethidium bromide staining of PCR amplicons
following size resolution allows visualization of differently size
amplicons corresponding to different marker alleles.
[0184] Primers for use in embodiments are not limited to those
capable of generating an amplicon of any particular size. For
example, primers used to amplify particular marker loci and alleles
are not limited to those amplifying the entire region of the
relevant locus. The primers may generate an amplicon of any
suitable length that is longer or shorter than those given in the
allele definitions. In examples, marker amplification may produce
an amplicon that is, for example and without limitation, at least
20 nucleotides in length; at least 50 nucleotides in length; at
least 100 nucleotides in length; and at least 200 nucleotides in
length.
[0185] Synthetic methods for making oligonucleotides and useful
compositions comprising oligonucleotides (e.g., probes, primers,
molecular beacons, PNAs, and LNAs) are generally well-known by
those of skill in the art. For example, oligonucleotides may be
synthesized chemically according to the solid phase phosphoramidite
triester method described in, for example, Beaucage and Caruthers
(1981) Tetrahedron Letts. 22(20):1859-62. Such methods may employ
an automated synthesizer, for example and without limitation, as
described in Needham-VanDevanter et al. (1984) Nucleic Acids Res.
12:6159-68. Oligonucleotides (including modified oligonucleotides)
may also be ordered from a variety of commercial sources including,
for example and without limitation, The Midland Certified Reagent
Company; The Great American Gene Company; ExpressGen Inc.; and
Operon Technologies Inc. Similarly, PNAs may be custom ordered from
any of a variety of sources including, for example and without
limitation, PeptidoGenic; HTI Bio-Products, Inc.; BMA Biomedicals
Ltd (U.K.); and Bio. Synthesis, Inc.
[0186] In some embodiments, an in silico method may be used to
detect a marker allele. For example, the sequence of a nucleic acid
comprising a marker sequence may be stored in a computer. The
desired marker locus sequence (or its homolog) may be identified
using an appropriate nucleic acid search algorithm, as provided by,
for example and without limitation, BLAST.TM., or even simple word
processors.
[0187] In some embodiments, a marker allele is detected using a
PCR-based detection method, where the size or sequence of a PCR
amplicon comprising the marker is indicative of the absence or
presence of a particular marker allele. In some examples, PCR
primers are hybridized to conserved regions flanking the
polymorphic marker region. PCR primers so used to amplify a
molecular marker are sometimes referred to in the art as "PCR
markers," or simply "markers."
[0188] A primary motivation for development of molecular markers in
crop species is the potential for increased efficiency in plant
breeding through marker assisted selection (MAS). Genetic markers
that are linked to a trait or gene of interest may be used to
identify plants that contain a desired marker allele at one or more
loci, which plants are thus expected to transfer the desired marker
allele, along with the trait or gene of interest, to their progeny.
Genetic markers may be used to identify plants that contain a
particular genotype at one locus, or at several unlinked or linked
loci (e.g., a haplotype). Similarly, marker alleles described
herein may be introgressed into any desired sunflower genetic
background, germplasm, plant, line, variety, etc., as part of an
overall MAS breeding program designed to enhance sunflower
yield.
[0189] According to some embodiments, markers described herein
provide the means to identify sunflower plants and germplasm that
comprise a low or reduced palmitic acid content (or high or
increased palmitic acid content) by identifying plants and
germplasm comprising a specific allele at a locus such as HA0031B,
HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A,
and a marker locus linked to at least one of the foregoing. By
identifying plants lacking a marker allele that co-segregates with
low palmitic acid, high palmitic acid plants and germplasm (or
plants with a lesser decrease of palmitic acid content) may be
identified, for example, for elimination from subsequent crosses
and breeding.
[0190] According to the foregoing, embodiments of the invention
include molecular markers that have a significant probability of
co-segregation with a QTL that contributes to or imparts a low
(e.g., decreased) palmitic acid content phenotype. These QTL
markers find use in marker assisted selection for desired traits
(decreased palmitic acid content), and also have other uses.
Embodiments of the invention are not limited to any particular
method for the detection or analysis of these markers.
VI. Introgression of Markers for Low Palmitic Acid Content into
Sunflower Lines
[0191] As set forth, supra, identification of sunflower plants or
germplasm that includes a marker allele or alleles that is/are
linked to a low (e.g., decreased) palmitic acid content phenotype
provides a basis for performing marker assisted selection of
sunflower. In some embodiments, at least one sunflower plant that
comprises at least one marker allele that is positively correlated
with low palmitic acid is selected, while sunflower plants that
comprise marker alleles that are negatively correlated with low
palmitic acid content may be selected against.
[0192] Desired marker alleles that are positively correlated with
low palmitic acid may be introgressed into sunflower having a
particular (e.g., elite or exotic) genetic background, so as to
produce an introgressed low palmitic acid content sunflower plant
or germplasm. In some embodiments, a plurality of low palmitic acid
content markers may be sequentially or simultaneous selected and/or
introgressed into sunflower. The particular combinations of low
palmitic acid content markers that may be selected for in a single
plant or germplasm is not limited, and can include a combination of
markers such as those set forth in FIG. 1, any markers linked to
the markers recited in FIG. 1, or any markers located within the
QTL intervals defined herein.
[0193] In embodiments, the ability to identify QTL marker alleles
that are positively correlated with low palmitic acid content of a
sunflower plant provides a method for selecting plants that have
favorable marker loci as well. For example, any plant that is
identified as comprising a desired marker allele (e.g., a marker
allele that positively correlates with low palmitic acid content)
may be selected for, while plants that lack the allele (or that
comprise an allele that negatively correlates with low palmitic
acid content) may be selected against. Thus, in particular
embodiments, subsequent to identification of a marker allele in a
first plant or germplasm, an introgression method includes
selecting the first sunflower plant or germplasm, or selecting a
progeny of the first plant or germplasm. In some examples, the
resulting selected sunflower plant or germplasm may be crossed with
a second sunflower plant or germplasm (e.g., an elite sunflower or
an exotic sunflower), so as to produce progeny comprising the
marker allele and desirable characteristics and/or alleles of the
second plant or germplasm.
[0194] In some embodiments, a method of introgressing a low
palmitic acid QTL may include, for example, providing at least one
marker linked to low palmitic acid (e.g., a marker that
co-segregates with low palmitic acid); determining the marker
allele in a first plant or germplasm comprising a low palmitic acid
QTL; and introgressing the marker allele into a second sunflower
plant or germplasm, so as to produce an introgressed sunflower
plant or germplasm. In particular embodiments, the second sunflower
plant or germplasm may comprise increased palmitic acid content as
compared to the first sunflower plant or germplasm, while the
introgressed sunflower plant or germplasm will comprise a decreased
palmitic acid content as compared to the second plant or germplasm.
As discussed in more detail below, an introgressed sunflower plant
or germplasm produced by these and other embodiments are also
included in embodiments of the invention.
[0195] In some embodiments, where an introgressed sunflower plant
or germplasm is produced by any of the methods provided herein, the
introgressed sunflower plant or germplasm may be characterized by
the fatty acid composition of the oil in seed from the plant. An
introgressed plant or germplasm may comprise, for example and
without limitation, about 3% or less palmitic acid in seed oil from
the plant. In some examples, such an introgressed sunflower plant
or germplasm comprises about 2.5% or less palmitic acid in seed oil
from the plant, such as for example and without limitation, 2.6%;
2.5%; 2.4%; 2.3%; 2.2%; 2.1%; 2.0%; 1.9%; 1.8%; about 1.7%; and
lower.
[0196] In addition to introgressing selected marker alleles (e.g.,
through standard breeding methods) into desired genetic
backgrounds, so as to introgress a low palmitic acid QTL into the
background, transgenic approaches may be used in some embodiments
to produce low palmitic acid content sunflower plants and/or
germplasm. In some embodiments, an exogenous nucleic acid (e.g., a
gene or open reading frame) that is linked to at least one marker
described herein in sunflower may be introduced into a target plant
or germplasm. For example, a nucleic acid coding sequence linked to
at least one marker described herein may be cloned from sunflower
genomic DNA (e.g., via positional cloning) and introduced into a
target plant or germplasm.
[0197] Thus, particular embodiments include methods for producing a
sunflower plant or germplasm comprising a low palmitic acid content
phenotype, wherein the method comprises introducing an exogenous
nucleic acid into a target sunflower plant or progeny thereof,
wherein the exogenous nucleic acid is substantially identical to a
nucleotide sequence that is linked to at least one positively
correlated marker allele at one or more marker locus that is linked
to low palmitic acid content. In some examples, the marker locus
may be selected from: HA0031B; HA0908; HA1665; HA0304A; HA0850;
HA0743; HA0870; HA0907; HA0612A; and a marker that is linked (e.g.,
demonstrating not more than 10% recombination frequency) to at
least one of the foregoing. In some embodiments, a plurality of
linked markers may be used to construct a transgenic plant. Which
of the markers described herein that are used in such a plurality
is within the discretion of the practitioner.
[0198] Any of a variety of methods can be used to provide an
exogenous nucleic acid to a sunflower plant or germplasm. In some
embodiments, a nucleotide sequence is isolated by positional
cloning, and is identified by linkage to a marker allele that is
positively correlated with low palmitic acid content. For example,
the nucleotide sequence may correspond to an open reading frame
(ORF) that encodes a polypeptide that, when expressed in a
sunflower plant, results in or contributes to the sunflower plant
having low palmitic acid content. The nucleotide sequence may then
be incorporated into an exogenous nucleic acid molecule. The
precise composition of the exogenous nucleic acid may vary. For
example, an exogenous nucleic acid may comprise an expression
vector to provide for expression of the nucleotide sequence in the
plant wherein the exogenous nucleic acid is introduced.
[0199] Markers linked to low palmitic acid content may be
introgressed (for example, thereby introgressing a low palmitic
acid content phenotype) into a sunflower plant or germplasm
utilizing a method comprising marker assisted selection. In
embodiments, MAS is performed using polymorphic markers that have
been identified as having a significant likelihood of
co-segregation with a low palmitic acid content trait. Such markers
(e.g., those set forth in FIG. 1) are presumed to map within or
near a gene or genes that contribute to the decreased palmitic acid
content of the plant (compared to a plant comprising the wild-type
gene or genes). Such markers may be considered indicators for the
trait, and may be referred to as QTL markers. In embodiments, a
plant or germplasm is tested for the presence of a positively
correlated allele in at least one QTL marker.
[0200] In embodiments, linkage analysis is used to determine which
polymorphic marker allele demonstrates a statistical likelihood of
co-segregation with a low palmitic acid content phenotype.
Following identification of such a positively correlated marker
allele for the low palmitic acid content phenotype, the marker may
then be used for rapid, accurate screening of plant lines for the
low palmitic acid content allele without the need to grow the
plants through their life cycle and await phenotypic evaluations.
Furthermore, the identification of the marker permits genetic
selection for the particular low palmitic acid content allele, even
when the molecular identity of the actual low palmitic acid content
QTL is unknown. A small tissue sample (for example, from the first
leaf of the plant) may be taken from a progeny sunflower plant
produced by a cross and screened with the appropriate molecular
marker. Thereby, it may be rapidly determined whether the progeny
should be advanced for further breeding. Linked markers also remove
the impact of environmental factors that may influence phenotypic
expression, thereby allowing the selection for low palmitic acid
content sunflower in an environmental neutral manner. Therefore,
while the contributions of various environmental factors to the oil
traits of plants may appear at first glance to confound the use of
the markers described herein, in fact a particular advantage of
these markers is that they do not depend on environment for their
utility.
[0201] In some embodiments comprising MAS, a polymorphic QTL marker
locus may be used to select a plant that contains a marker allele
(or alleles) that is positively correlated with a low palmitic acid
content phenotype. For example, a nucleic acid corresponding to the
marker nucleic acid allele may be detected in a biological sample
from the plant to be selected. This detection may take the form of
hybridization of a probe nucleic acid to a marker allele or
amplicon thereof (e.g., using allele-specific hybridization,
Southern analysis, northern analysis, in situ hybridization, and
hybridization of primers followed by PCR amplification of a region
of the marker). After the presence (or absence) of the particular
marker allele in the biological sample is verified, the plant is
selected, and may in some examples be used to make progeny plants
by selective breeding.
[0202] Sunflower plant breeders desire combinations of low palmitic
acid content marker loci with markers/genes other desirable traits
(e.g., high yield) to develop improved sunflower varieties.
Screening large numbers of samples by non-molecular methods (e.g.,
trait evaluation in sunflower plants) is generally expensive, time
consuming, and unreliable. Use of the polymorphic markers described
herein, which are linked to low palmitic acid content QTL, provides
an effective method for selecting desirable varieties in breeding
programs. Advantages of marker-assisted selection over field
evaluations for low palmitic acid content include, for example,
that MAS can be done at any time of year, regardless of the growing
season. Moreover, as set forth, supra, environmental effects are
largely irrelevant to marker-assisted selection.
[0203] When a population is segregating for multiple marker loci
linked to one or more traits (e.g., multiple markers linked to low
palmitic acid content), the efficiency of MAS compared to
phenotypic screening becomes even greater, because all of the
marker loci may be evaluated in the lab together from a single
sample of DNA. In particular embodiments of the invention, the
HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907,
and HA0612A markers, as well as markers linked to at least one of
the foregoing, may be assayed simultaneously or sequentially from a
single sample, or from a plurality of parallel samples.
[0204] Another use of MAS in plant breeding is to assist the
recovery of the recurrent parent genotype by backcross breeding.
Backcrossing is usually performed for the purpose of introgressing
one or a few markers or QTL loci from a donor parent (e.g., a
parent comprising desirable low palmitic acid content marker loci)
into an otherwise desirable genetic background from a recurrent
parent (e.g., an otherwise high yielding sunflower line). The more
cycles of backcrossing that are done, the greater the genetic
contribution of the recurrent parent to the resulting introgressed
variety. In some examples, many cycles of backcrossing may be
carried out, for example, because low palmitic acid content plants
may be otherwise undesirable, e.g., due to low yield, low
fecundity, etc. In contrast, strains which are the result of
intensive breeding programs may have excellent yield, fecundity,
etc., merely being deficient in one desirable respect, such as
palmitic acid content. In marker assisted backcrossing of specific
markers from a donor source, which may or may not constitute an
elite genetic background to an elite variety that will serve as the
recurrent line, the practitioner may select among backcross progeny
for the donor marker, and then use repeated backcrossing to the
recurrent line to reconstitute as much of the recurrent line's
genome as possible.
[0205] According to the foregoing, markers and methods described
herein may be utilized to guide marker assisted selection or
breeding of sunflower varieties with the desired complement (set)
of allelic forms of chromosome segments associated with superior
agronomic performance (e.g., low palmitic acid content, along with
any other available markers for yield, disease resistance, etc.).
Any of the described marker alleles may be introduced into a
sunflower line via introgression (e.g., by traditional breeding,
via transformation, or both) to yield a sunflower plant with
superior agronomic performance. If nucleic acids from a plant are
positive for a desired genetic marker allele, the plant may be
self-fertilized in some embodiments to create a true breeding line
with the same genotype, or it may be crossed with a plant
comprising the same marker allele, or other desired markers and/or
characteristics to create a sexually crossed hybrid generation.
[0206] Often, a method of the present invention is applied to at
least one related sunflower plant such as from progenitor or
descendant lines in the subject sunflower plants pedigree such that
inheritance of the desired decreased palmitic acid content allele
can be traced. The number of generations separating the sunflower
plants being subject to the methods of the present invention will
generally be from 1 to 20, commonly 1 to 5, and typically 1, 2, or
3 generations of separation, and quite often a direct descendant or
parent of the sunflower plant will be subject to the method (i.e.,
one generation of separation).
[0207] Genetic diversity is important in breeding programs. With
limited diversity, the genetic gain achieved in a breeding program
will eventually plateau when all of the favorable alleles have been
fixed within the elite population. Therefore, one objective of
plant breeding is to incorporate diversity into an elite pool
without losing the genetic gain that has already been made, and
with the minimum possible investment. MAS provide an indication of
which genomic regions, and which favorable alleles from the
original ancestors, have been selected for and conserved over time,
facilitating efforts to incorporate favorable variation from exotic
germplasm sources (parents that are unrelated to the elite gene
pool) in the hopes of finding favorable alleles that do not
currently exist in the elite gene pool. Thus, in some embodiments,
markers described herein may be used for MAS in crosses involving
(elite.times.exotic) sunflower lines by subjecting segregating
progeny to MAS to maintain major yield alleles, along with the
decreased palmitic acid content marker alleles herein.
[0208] The molecular marker loci and alleles described herein
(e.g., HA0031B, HA0908, HA1665, HA0304A, HA0850, HA0743, HA0870,
HA0907, HA0612A, and markers linked to at least one of the
foregoing) may be used in some embodiments, as indicated
previously, to identify a low palmitic acid content QTL, which may
then be cloned by familiar procedures. Such decreased low acid
content clones may be first identified by their genetic linkage to
markers described herein. For example, "positional gene cloning"
takes advantage of the physical proximity of a low palmitic acid
content marker to define an isolated chromosomal fragment
containing a low palmitic acid content QTL gene. The isolated
chromosomal fragment may be produced by such well-known methods as,
for example and without limitation, digesting chromosomal DNA with
one or more restriction enzymes, by amplifying a chromosomal region
using PCR, and any suitable alternative amplification reaction. The
digested or amplified fragment may subsequently be ligated into a
vector suitable for replication and/or expression of the inserted
fragment. Markers that are adjacent to an ORF associated with a
phenotypic trait may be specifically hybridized to a DNA clone
(e.g., a clone from a genomic DNA library), thereby identifying a
clone on which the ORF (or a fragment of the ORF) is located. If a
marker is more distant from the low palmitic acid content QTL gene,
a fragment containing the ORF may be identified by successive
rounds of screening and isolation of clones, which together
comprise a contiguous sequence of DNA. This process is commonly
referred to as "chromosome walking," and it may be used to produce
a "contig" or "contig map."
[0209] Protocols sufficient to guide one of skill through the
isolation of clones associated with linked markers are found in,
for example, Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al., Eds.,
Current Protocols in Molecular Biology, Chapter 2, Greene
Publishing and Wiley-Interscience, N Y, 1995.
VII. Plants Comprising Markers for Low Palmitic Acid Content
[0210] Some embodiments include methods for making a sunflower
plant, and further include these sunflower plants, per se. In
particular embodiments, such a method may comprise crossing a first
parent sunflower plant comprising at least one marker allele that
is positively correlated with low palmitic acid with a second
sunflower plant at a marker linked to low palmitic acid described
herein, and growing the female sunflower plant under plant growth
conditions to yield sunflower plant progeny. Such sunflower plant
progeny may be assayed for marker alleles linked to low palmitic
acid content, and desired progeny may be selected. Such progeny
plants, or seed thereof, may be subject to a variety of uses
including, for example and without limitation, they may be sold
commercially for sunflower production; used for food; processed to
obtain a desired sunflower product (e.g., sunflower oil); and/or
further utilized in subsequent rounds of breeding. Sunflower plants
according to some embodiments include progeny plants that comprise
at least one of the allelic forms of the markers described herein,
such that further progeny are capable of inheriting the marker
allele.
[0211] Some embodiments include methods for producing a sunflower
plant comprising low palmitic acid content (e.g., decreased
palmitic acid content). In particular embodiments, such methods may
include production of such a plant by conventional plant breeding
or by introducing an exogenous DNA (e.g., a transgene) into a
sunflower variety or plant.
[0212] Thus, some embodiments include host cells and organisms that
are transformed with nucleic acids corresponding to a low palmitic
acid content QTL identified using at least one marker linked to low
palmitic acid content described herein. In some examples, such
nucleic acids may include chromosome intervals (e.g., genomic
fragments), ORFs, and/or cDNAs that encode expression products that
contribute to a low palmitic acid content phenotype.
[0213] Host cells may be genetically engineered (e.g., transduced,
transfected, transformed, etc.) with a vector (e.g., a cloning
vector, shuttle vector, or expression vector) that comprises an ORF
linked to a marker of low palmitic acid content. Vectors include,
for example and without limitation, plasmids; phagemids;
Agrobacterium; viruses; naked polynucleotides (linear or circular);
and conjugated polynucleotides. Many vectors may be introduced into
bacteria, especially for the purpose of propagation and
expansion.
[0214] Vectors may be introduced into plant tissues, cultured plant
cells, and plant protoplasts by any of a variety of standard
methods known in the art including, for example and without
limitation: electroporation (From et al. (1985) Proc. Natl. Acad.
Sci. USA 82:5824); infection by viral vectors such as cauliflower
mosaic virus (CaMV) (see, e.g., U.S. Pat. No. 4,407,956); ballistic
penetration by small particles comprising the nucleic acid (Klein
et al. (1987) Nature 327:70); use of pollen as vector (PCT
International Patent Publication No. WO 85/01856); and use of
Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid
in which DNA fragments are cloned (Fraley et al. (1983) Proc. Natl.
Acad. Sci. USA 80:4803). Any suitable method, including without
limitation the specific methods explicitly identified herein, which
provides for effective introduction of a nucleic acid into a cell
or protoplast, may be employed in certain embodiments of the
invention.
[0215] Engineered host cells can be cultured in conventional
nutrient media or media modified for, for example, activating
promoters or selecting transformants. In some embodiments, host
plant cells may be cultured into transgenic plants. Plant
regeneration from cultured protoplasts is described in, for
example, Evans et al. (1983) "Protoplast Isolation and Culture," In
Handbook of Plant Cell Cultures 1, MacMillan Publishing Co., NY,
pp. 124-176; Davey (1983) "Recent Developments in the Culture and
Regeneration of Plant Protoplasts," In Protoplasts, Birkhauser,
Basel, pp. 12-29; Dale (1983) "Protoplast Culture and Plant
Regeneration of Cereals and Other Recalcitrant Crops," In
Protoplasts, supra, pp. 31-41; and Binding (1985) "Regeneration of
Plants," In Plant Protoplasts, CRC Press, Boca Raton, Fla., pp.
21-73. Additional resources providing useful details regarding
plant cell culture and regeneration include Payne et al. (1992)
Plant Cell and Tissue Culture in Liquid Systems, John Wiley &
Sons, Inc., NY; Gamborg and Phillips (eds.) (1995) Plant Cell,
Tissue and Organ Culture; Fundamental Methods, Springer Lab Manual,
Springer-Verlag (Berlin Heidelberg NY); and R. R. D. Croy (Ed.)
Plant Molecular Biology (1993) Bios Scientific Publishers, Oxford,
UK (ISBN 0 12 198370 6).
[0216] Transformed plant cells that are produced using any of the
above transformation techniques may be cultured to regenerate a
whole plant that possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques generally rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced into the cell together with the desired
nucleotide sequences. Regeneration and growth processes used to
produce a whole plant generally include the steps of selection of
transformant cells and shoots; rooting the transformant shoots; and
growth of the plantlets in soil.
[0217] Plant transformation with nucleic acids that lower palmitic
acid content (e.g., that comprise markers described herein) may be
used to transform species other than sunflower. For example, it is
contemplated that expression products from QTLs that contribute to
or provide a low palmitic acid content phenotype in sunflower can
also decrease palmitic acid content when transformed and expressed
in other agronomically and horticulturally important plant species.
Such species include dicots, for example and without limitation, of
the families: Leguminosae (including pea, beans, lentil, peanut,
yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine,
vetch, lotus, sweet clover, wisteria, and sweetpea) and Compositae
(the largest family of vascular plants, including at least 1,000
genera, including important commercial crops such as sunflower).
Additional plants comprising nucleic acids that lower palmitic acid
content (e.g., that comprise markers described herein) may be
plants from among the genera: Allium, Apium, Arachis, Brassica,
Capsicum, Cicer, Cucumis, Curcubita, Daucus, Fagopyrum, Glycine,
Helianthus, Lactuca, Lens, Lycopersicon, Medicago, Pisum,
Phaseolus, Solanum, Trifolium, Vigna, and many others. Common crop
plants which may be used in particular examples include, for
example and without limitation: soybean, sunflower, canola, peas,
beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover,
alfalfa, lupine, vetch, sweet clover, sweetpea, field pea, fava
bean, broccoli, brussel sprouts, cabbage, cauliflower, kale,
kohlrabi, celery, lettuce, carrot, onion, pepper, potato, eggplant
and tomato.
VIII. Systems for Detecting and/or Correlating Low Palmitic Acid
Content Markers
[0218] Systems, including automated systems, for identifying plants
that comprise at least one marker linked to a low palmitic acid
phenotype in sunflower, and/or for correlating presence of a
specific linked marker allele with low palmitic acid content, are
also included in some embodiments. Exemplary systems may include
probes useful for allele detection at a marker locus described
herein; a detector for detecting labels on the probes; appropriate
fluid handling elements and temperature controllers, for example,
that mix probes and templates and/or amplify templates; and/or
system instructions that correlate label detection to the presence
of a particular marker locus or allele.
[0219] In particular embodiments, a system for identifying a
sunflower plant predicted to have low palmitic acid content is
provided. Such a system may include, for example and without
limitation: a set of marker primers and/or probes configured to
detect at least one allele of at least one marker linked to low
palmitic acid content (e.g., HA0031B, HA0908, HA1665, HA0304A,
HA0850, HA0743, HA0870, HA0907, HA0612A, and a marker linked to at
least one of the foregoing); a detector that is configured to
detect one or more signal outputs from the set of marker probes or
primers, or amplicon thereof, thereby identifying the presence or
absence of the allele; and system instructions that correlate the
presence or absence of the allele with low (e.g., decreased) or
higher palmitic acid content.
[0220] A system that performs marker detection and/or correlation
may include a detector that is configured to detect one or more
signal outputs from the set of marker probes or primers, or
amplicon thereof. The precise configuration of the detector may
depend on the type of label used to detect a marker allele.
Particular examples may include light detectors and/or
radioactivity detectors. For example, detection of light emission
or other property of a labeled probe may be indicative of the
presence or absence of a marker allele interacting with the probe
(e.g., via specific hybridization). The detector(s) optionally
monitors one or a plurality of signals from an amplification
reaction. For example, a detector may monitor optical signals which
correspond to "real time" amplification assay results.
[0221] A wide variety of signal detection devices are available
including, for example and without limitation, photo multiplier
tubes; spectrophotometers; CCD arrays; arrays and array scanners;
scanning detectors; phototubes and photodiodes; microscope
stations; galvo-scanns; and microfluidic nucleic acid amplification
detection appliances. In addition to the type of label used to
detect a marker allele, the precise configuration of a detector may
depend, in part, on the instrumentation that is most conveniently
obtained for the user. Detectors that detect fluorescence,
phosphorescence, radioactivity, pH, charge, absorbance,
luminescence, temperature, or magnetism may be used in some
examples.
[0222] The precise form of instructions provided in a system
according to some embodiments may similarly vary, depending on the
components of the system. For example, instructions may be present
as system software in one or more integrated unit(s) of the system,
or they may be present in one or more computers or computer
readable media operably coupled to a detector. In some examples,
system instructions include at least one reference table that
includes a correlation between the presence or absence of a
particular marker allele in a plant or germplasm and a predicted
palmitic acid content. Instructions may also include directions for
establishing a user interface with the system; e.g., to permit a
user to view results of a sample analysis and to input parameters
into the system.
[0223] A system may include in particular embodiments components
for storing or transmitting computer readable data representing or
designating detected marker alleles, for example, in an automated
(e.g., fully automated) system. For example, a computer readable
media may be provided that includes cache, main, and storage
memory, and/or other electronic data storage components (e.g., hard
drives, floppy drives, and storage drives) for storage of computer
code. Data representing alleles detected by the method of the
present invention can also be electronically, optically, or
magnetically transmitted in a computer data signal embodied in a
transmission medium over a network, such as an intranet or internet
or combinations thereof. A system may also or alternatively
transmit data via wireless, infrared, or other available
transmission alternatives.
[0224] During operation, the system typically comprises a sample
that is to be analyzed, such as a plant tissue, or material
isolated from the tissue such as genomic DNA, amplified genomic
DNA, cDNA, amplified cDNA, RNA, amplified RNA, or the like.
[0225] In some embodiments, a system may be comprised of separate
elements, or may alternatively be integrated into a single unit for
convenient detection of markers alleles, and optionally for
additionally performing marker-phenotype correlations. In
particular embodiments, the system may also include a sample, for
example and without limitation, genomic DNA; amplified genomic DNA;
cDNA; amplified cDNA; RNA; and amplified RNA, from sunflower or
from a selected sunflower plant tissue.
[0226] Automated systems provided in some embodiments optionally
include components for sample manipulation; e.g., robotic devices.
For example, an automated system may include a robotic liquid
control armature for transferring solutions (e.g., plant cell
extracts) from a source to a destination (e.g., from a microtiter
plate to an array substrate) that may be operably linked to a
digital computer (e.g., in an integrated computer system). An input
device for entering data to the digital computer to control high
throughput liquid transfer by the robotic liquid control armature
(and, optionally, to control transfer by the armature to the solid
support) may also be a feature of an automated system. Many
automated robotic fluid handling systems are commercially
available. For example, a variety of automated systems that utilize
various Zymate.TM. systems, and typically include, robotics and
fluid handling modules, are available from Caliper Technologies
Corp. (Hopkinton, Mass.). Similarly, the common ORCA.RTM. robot,
which is used in a variety of laboratory systems (e.g., for
microtiter tray manipulation) is also commercially available from,
for example, Beckman Coulter, Inc. (Fullerton, Calif.). As an
alternative to conventional robotics, microfluidic systems for
performing fluid handling and detection are now widely available
from Caliper Technologies and Agilent technologies (Palo Alto,
Calif.).
[0227] In particular embodiments, a system for molecular marker
analysis may include, for example and without limitation, a digital
computer comprising high-throughput liquid control software; a
digital computer comprising image analysis software for analyzing
data from marker labels; a digital computer comprising data
interpretation software; a robotic liquid control armature for
transferring solutions from a source to a destination; an input
device (e.g., a computer keyboard) for entering data into the
system (e.g., to control high throughput liquid transfer by the
robotic liquid control armature); and an image scanner for
digitizing label signals from labeled probes.
[0228] Optical images (e.g., hybridization patterns) viewed and/or
recorded by a camera or other device (e.g., a photodiode and data
storage device) may be further processed in any of the embodiments
herein. For example and without limitation, such images may be
processed by digitizing the image and/or storing and analyzing the
image on a computer. A variety of commercially available peripheral
equipment and software is available for digitizing, storing and
analyzing a digitized video or digitized optical image, for
example, using various computer and programming platforms.
[0229] Some embodiments also include kits useful for identifying
plants that comprise at least one marker linked to a low palmitic
acid phenotype in sunflower, and/or for correlating presence of a
specific linked marker allele with low palmitic acid content. In
some examples, such a kit may include appropriate primers or probes
for detecting at least one marker linked to low palmitic acid
content and particular marker alleles; and instructions for using
the primers or probes to detect the at least one marker and
correlate the marker allele with a predicted palmitic acid content.
A kit may in some examples include packaging materials for
packaging probes, primers, and/or instructions; and controls (e.g.,
control amplification reactions that include probes, primers or
template nucleic acids for amplifications, and molecular size
markers).
[0230] In some embodiments, a kit or system for identifying plants
that comprise at least one marker linked to a low palmitic acid
phenotype in sunflower, and/or for correlating presence of a
specific linked marker allele with low palmitic acid content may
include nucleic acids that detect particular SSR QTL markers
described herein. For example, a system or kit may comprise an
amplification primer pair capable of initiating DNA polymerization
by a DNA polymerase on a sunflower nucleic acid template to
generate a sunflower marker amplicon, where the marker amplicon
corresponds to a sunflower marker selected from HA0031B, HA0908,
HA1665, HA0304A, HA0850, HA0743, HA0870, HA0907, HA0612A, and a
marker linked to at least one of the foregoing. For example, the
primer pair that is specific for the marker can be selected from
the primer pairs set forth in Table 6, or their equivalents.
EXAMPLES
[0231] The following examples are offered to illustrate, but not to
limit, certain embodiments of the invention. It is understood that
the examples and embodiments described herein are for illustrative
purposes only, and persons skilled in the art will recognize
various reagents, techniques, systems, and parameters that can be
altered without departing from the spirit or scope of the
invention.
Example 1: Natural Variation for Palmitic Acid Content in
Sunflower
[0232] Natural variation of palmitic acid content in sunflower was
measured based upon the AOCS.TM. method Ce 2-66(97) (AOCS.TM.
product code MC-CE266).
[0233] Five sunflower seeds from each sample to be tested were
placed into a labeled 96-well extraction plate (Corning Inc.
catalog no. 4411) containing one 1/8-inch steel ball (Small Parts
Inc. catalog no. BS-0125-C). 200 .mu.L heptanes was added to each
well, which were then capped. The capped samples were placed in a
GenoGrinder.TM. for 2.0 minutes at 1300 strokes/minute. Samples
were removed, and any unground samples were crushed by hand with a
spatula and re-ground.
[0234] After the first grind, 400 .mu.L heptanes was added to each
well, and the material was re-ground at 1300 strokes/minute. The
samples were then centrifuged for 10 minutes at 3700 rpm at
6.degree. C. Then, using a Beckman Coulter MC robot, the
supernatant was transferred to a 96-well plate with glass inserts
(MicroLiter Analytical Supplies Inc. catalog no. 07-8045 MB-1200)
containing 400 .mu.L heptanes. 40 .mu.L 1% sodium methoxide was
then added to each well. Sodium methoxide was diluted from a stock
30% solution with methanol (Sigma-Aldrich Fluka catalog no. 71748).
The plates were capped with a Teflon mat cap, and incubated at room
temperature for 60 minutes prior to analysis.
[0235] Samples were analyzed to determine their fatty acid
compositions on an Agilent 6890 GC-FID (Agilent Technologies)
equipped with a J&W Scientific DB-23 15 m.times.0.25 mm ID
column and 0.25 .mu.m film thickness (Agilent Technologies, catalog
no. 122-2312). The initial oven temperature was 200.degree. C.,
which temperature was maintained for the duration of the run. The
inlet was set to split ratio of 1:50 and a temperature of
280.degree. C. A ramped flow rate of 0.8 mL/minute helium was
maintained for the initial two minutes. The flow was then increased
at a rate of 1.0 mL/minute to 2.5 mL/minute, where it was held for
1.5 minutes. The detector was set to 300.degree. C. with a constant
carrier gas make up and column flow of 30 mL/minute, fuel hydrogen
flow of 30 mL/minute, and oxidizer flow of 400 mL/minute. An
injection volume of 2 .mu.L was used for all samples.
[0236] Palmitic acid methyl ester peaks were identified by
comparison with the retention times of methyl ester reference
standards (Nu-Chek-Prep, Inc., GLC#428). See FIG. 1 and Table 1.
Individual percent areas were calculated for all analytes in the
reference standard based upon the total integrated chromatographic
peak areas. A heptane blank was also injected to identify any
contamination on the GC.
TABLE-US-00001 TABLE 1 Statistics of palmitic acid content
distribution Distribution Quantile % palmitic acid 100.0% maximum
15.05 99.5 5.87 97.5 5.23 90.0 4.63 75.0 quartile 4.20 50.0 median
3.72 25.0 quartile 3.21 10.0 2.92 2.5 2.66 0.5 2.41 0.0 minimum
0.00
[0237] Using this method, the palmitic acid content of field grown
samples was assessed on sunflower varieties that were developed as
part of a seven-year sunflower breeding program. The distribution
of the palmitic acid contents measured is presented in FIG. 2 and
Table 2. Typically observed values for palmitic acid in
conventional sunflower germplasm ranged from approximately 2.5% to
6% of total fatty acids, with a mean of 3.75%.
TABLE-US-00002 TABLE 2 Statistics of palmitic acid content
distribution Mean 3.74945 Std. Dev. 0.70766 Std. Err. Mean 0.00466
Upper 95% Mean 3.75858 Lower 95% Mean 3.74031
Example 2: Identification of Germplasm with Low Palmitic Acid
Content
[0238] A low palmitic acid profile was discovered during a program
designed to improve an elite black-hulled, high linoleic acid
sunflower line (687R) by breeding it with a line having an elevated
oleic acid profile. This was accomplished by means of backcross
breeding using as the high oleic acid donor a striped-hull, high
oleic acid confection parent (H280R). H280R has, in general, a
lower palmitic acid content, but the observed level is not normally
below about 2.5%. To achieve the targeted oleic acid levels, FAME
analysis as described in Example 1 was conducted at each generation
during this back-cross breeding program. During the routine
screening of fatty acid levels, a segregant was observed that had
substantially reduced levels of palmitic acid. In Table 3, the
palmitic acid content values for four individuals from the first
back-cross generation of the back-cross breeding program of 687R
with H280R is shown.
TABLE-US-00003 TABLE 3 Palmitic acid values determined using
protocol described in Example 1 of a bulked sample of 8-10 seeds
from four heads selected from the first back-cross breeding
generation of 687R/H280R Head Palmitic acid content (%) 1 2.18 2
2.13 3 2.06 4 2.04 H280R 3.18
Example 3: Variation for Palmitic Acid Content in a Sunflower
Population made between a Low Palmitic Acid Parent and a
Conventional Sunflower Elite Parent
[0239] Variation in the palmitic acid content when an elite
sunflower inbred is crossed to a source of the reduced palmitic
acid content was demonstrated by crossing a high oleic acid
restorer (line-R) with a low palmitic acid source derived from the
discovery described in Example 2. The low palmitic acid source had
been converted to a cytoplasmic male sterile background (line-A).
An F.sub.2 population from the cross of line-A by line-R was
generated, and 384 seeds were collected. The seeds were cut in
half, with half of the seed being analyzed according to the
protocol described in Example 1. The other half of the seed was
planted for subsequent analysis. The summary statistics for the
palmitic acid content in the F.sub.2 population line-A/line-R
(N=384) are presented in Tables 4-5.
TABLE-US-00004 TABLE 4 Statistics of palmitic acid content
distribution in an F.sub.2 population between an elite inbred
having typical palmitic acid content with a line having low
palmitic acid Distribution Quantile % palmitic acid 100.0% Maximum
4.216 99.5 4.216 97.5 3.612 90.0 3.342 75.0 Quartile 3.1995 50.0
Median 2.989 25.0 Quartile 2.5995 10.0 1.976 2.5 1.734 0.5 1.648
0.0 Minimum 1.648
TABLE-US-00005 TABLE 5 Statistics of palmitic acid content
distribution in an F.sub.2 population between an elite inbred
having typical palmitic acid content with a line having low
palmitic acid Mean 2.83964 Std. Dev. 0.51878 Std. Err. Mean 0.02647
Upper 95% Mean 2.89169 Lower 95% Mean 2.78759
Example 4: Demonstration of Bimodal Distribution of Palmitic Acid
Content
[0240] The distribution of palmitic acid content in the population
described in Example 3 is presented in FIG. 3. The distribution is
bimodal: part of the population is centered around about 3.15%
palmitic acid, with a lower tail ending at about 2.5% and an upper
tail extending to about 4%; and a second part of the population is
centered at about 2.1% palmitic acid, with a lower tail reaching
about 1.75% and an upper tail extending to about 2.5%. From the
quantiles presented in Example 3 it was observed that 25% of the
population has a palmitic acid content below 2.6%, with the
remainder of the population having a higher palmitic acid content.
The value of the first quartile (2.6%) corresponds closely with the
inflection point where the bimodal distribution transitions from
the lower cluster to the upper cluster. Noting that there is a 3:1
ratio between individuals with a higher palmitic acid content and
those with a lower value, it was concluded that there is a single,
major genetic element that is responsible for low palmitic acid
content in this population, with the recessive allele conferring
the low palmitic acid phenotype.
Example 5: QTL Mapping of a Genetic Determinant of Palmitic Acid
Content
[0241] A major locus for palmitic acid content was mapped on to
sunflower linkage group 5 (LG5) using microsatellite or SSR markers
and the palmitic acid content data presented in Examples 3 and
4.
[0242] The maps in sunflower are usually referred to by linkage
group. Maps of linkage group 5 are available. See Yu et al. (2003)
Crop Sci. 43:367-87; see also Tang et al. (2002) Theor. Appl.
Genet. 105:1124-36. It should be noted that sunflower linkage group
numbers of maps developed by European scientists are different
from, for example, the ones set forth in the above-cited
references. The chromosome numbers corresponding to linkage groups
in sunflower have not yet been defined.
TABLE-US-00006 TABLE 6 Primer sequences and map locations of SSR
markers mapped on LG5 for identifying the palmitic acid locus Map
Position Marker (cm) Forward Primer Sequence Reverse Primer
Sequence HA0357_H757B 0.0 GTTCCTGTCGGGTAACTGTAGC
CATTGATGGAGATGGCTGG (SEQ ID NO: 1) (SEQ ID NO: 2) HA0694B_H280R
17.8 GCCGTGAATAATGGGATTGA GATTGGGTCAGCTTGTGTGA (SEQ ID NO: 3) (SEQ
ID NO: 4) HA1485 19.7 GGGAAGTGGGCTTGTCTATGTAT
AACACACCGAAATCACCTATGAA (SEQ ID NO: 5) (SEQ ID NO: 6) HA1838 20.1
AGAGGAATGAGATCGGGTTGAT GTGGGACAACTCAGCAACGTC (SEQ ID NO: 7) (SEQ ID
NO: 8) HA1489_H280R 20.5 CTTATTCCAAGGACGCATAGTCG
CGATGGTATGATTCTCGACGTTA (SEQ ID NO: 9) (SEQ ID NO: 10) HA1146B 21.6
ACACCAACCAGACGCAGAAT GTGCAAGAACGAGGAAGAGG (SEQ ID NO: 11) (SEQ ID
NO: 12) HA0037 21.8 GAACATGGCCATAACTCATAGACG CCTTCGACCCAACATC (SEQ
ID NO: 13) (SEQ ID NO: 14) HA0654 21.8 ACGCACATGAGAGAGAAAGAG
ACCTTCGACCCAACATCAAG (SEQ ID NO: 15) (SEQ ID NO: 16) HA1620_H280R
22.6 TTTCGTGATGGTGATTGATGATT CAGCAACTCTGACCGTTTCATTA (SEQ ID NO:
17) (SEQ ID NO: 18) HA0031B 23.0 CTCACGAAACTCTTCATGCTG
CTCTCACACTTACTGAAC (SEQ ID NO: 19) (SEQ ID NO: 20) HA0908 23.1
TTGTCTTCATCTGCGTGTGA TTGCTGTTGTTGATCGGTGT (SEQ ID NO: 21) (SEQ ID
NO: 22) HA1665 23.2 CCTAAGGGGATGAATTCTCTTTC AACTTCCAATGTTCTCCAACCAT
(SEQ ID NO: 23) (SEQ ID NO: 24) HA0304A_H757B 23.6
GTGCCCTAACACTGTTCCGT AGCGAAAGGATCGAGAATC (SEQ ID NO: 25) (SEQ ID
NO: 26) HA0850_H757B 23.8 CCCTGGAGTGTATGTCCGTTA
ATCCGTCTGCTGCCTAATCC (SEQ ID NO: 27) (SEQ ID NO: 28) HA0743_H757B
24.2 ACGGAAAGCTCTTGAAAGCA GCGGGCATTCCAACTAGTAA (SEQ ID NO: 29) (SEQ
ID NO: 30) HA0870 24.3 GTGCGTTGGCTCTTATGGAT AGTGATGGCATTCCCAATTT
(SEQ ID NO: 31) (SEQ ID NO: 32) HA0907 24.6 CATGAACATCGCCAATTCAG
TGCAAGGAACCATCAGAATC (SEQ ID NO: 33) (SEQ ID NO: 34) HA0612A_H757B
25.8 CTTGGGTTCTTCATAACTC CATGTAATCACCTTTCAAG (SEQ ID NO: 35) (SEQ
ID NO: 36) HA1923 27.1 AACCAAAGATTCAAGGCAATCA CAGACATTAGACGCGAAGCAG
(SEQ ID NO: 37) (SEQ ID NO: 38) HA1357A 28.2
CACAAAACAATCGCTAAAAGAACA AATGATGATGGTCACGAAGAAGA (SEQ ID NO: 39)
(SEQ ID NO: 40) HA1357B_H757B 29.5 CACAAAACAATCGCTAAAAGAACA
AATGATGATGGTCACGAAGAAGA (SEQ ID NO: 39) (SEQ ID NO: 40)
HA1819_H280R 30.9 GTTTCGGGTGGGGGATTACGG ATGGTCGACAACAAGCGCAAAC (SEQ
ID NO: 41) (SEQ ID NO: 42) HA0894 33.2 TGGTGGAGGTCACTATTGGA
AGGAAAGAAGGAAGCCGAGA (SEQ ID NO: 43) (SEQ ID NO: 44) HA1790 37.6
TCCCCAAACTTGCGTGTAGGT CATTACAAACCACAGCTCCTTCC (SEQ ID NO: 45) (SEQ
ID NO: 46) HA0041 47.1 CTAGCAACCAACCTCATTG GTCTCCTTCTCTTTCTCGGC
(SEQ ID NO: 47) (SEQ ID NO: 48) HA1313 52.3 CGACCCACCTAGTAAAAGCAAAC
TGCCATAAAAAGATTTGGTCTCC (SEQ ID NO: 49) (SEQ ID NO: 50)
HA1776_H757B 59.6 TCACAGGAGAATGCAAAGAGTG GCATAATAGGAGTAACTGCCAAAAC
(SEQ ID NO: 51) (SEQ ID NO: 52)
[0243] PCR Procedures for SSR Markers.
[0244] PCR reactions were performed in a GeneAmp.TM. PCR System
9700 (Applied Biosystems) with a dual-384 well block. Each PCR
reaction was carried out in a volume of 8 .mu.L containing 10 ng of
genomic DNA with a final concentration of 1.times. Qiagen.TM. PCR
buffer (Qiagen, Valencia, Calif.), 0.25 .mu.M of each primer
(forward and reverse), 1 mM MgCl2, 0.1 mM of each dNTP, 0.4% PVP,
and 0.04 units of HotStart.TM. Taq DNA polymerase (Qiagen,
Valencia, Calif.).
[0245] PCR conditions were set up as follows: 12 minutes at
95.degree. C. for template DNA denaturing; 40 cycles for DNA
amplifications (each cycle: 5 seconds at 94.degree. C. for
denaturing, 15 seconds at 55.degree. C. for annealing, and 30
seconds at 72.degree. C. for extension); and 30 minutes at
72.degree. C. for final extensions.
[0246] Fragment Analysis.
[0247] PCR products of different primer pairs were multiplexed in a
final volume of 100 .mu.L (using autoclaved water to bring the
volume to 100 .mu.L). 0.5 .mu.L multiplexed PCR products were mixed
with 5 .mu.L of loading buffer. Gels were run on an AB3730XL DNA
Analyzer (Applied Biosystems) with G5-RCT spectral matrix using
standard conditions. Data were then imported into GeneMapper.RTM.
version 4.0 (Applied Biosystems). All dye colors were imported, and
the 2 highest peaks, with minimum intensity of 100 relative
fluorescent units (rfu), were labeled. Alleles were assigned a
numeric value according to PCR fragment size. Numeric allele scores
were imported into Excel.TM. (Microsoft), where they were converted
into formats appropriate for JoinMap.TM. 3.0 and MapQTL.TM.
4.0.
[0248] Statistical Analysis
[0249] Linkage Mapping.
[0250] Join Map.TM. 3.0 was used to create a genetic linkage map of
the line-A/line-R F.sub.2 population. JoinMap.TM. 3.0 requires one
input file, referred to as a locus genotype file. In the locus
genotype file, elite parent alleles were called as "A," donor
parent alleles were called as "B," while heterozygous alleles were
called as "H." Missing data were represented with a dash in the
locus genotype file. Results were calculated in Kosambi
centimorgans. The map generated from this analysis was compared to
the public map (see S. Tang, J. K. Yu, M. B. Slabaugh, D. K.
Shintani, S. J. Knapp (2002) Simple sequence repeat map of the
sunflower genome. Theor. Appl. Genet. 105:1124-1136) for final data
interpretation.
[0251] QTL Analysis.
[0252] Interval mapping for palmitic acid content was conducted
using MapQTL.TM. 4.0 to locate a potential QTL. MapQTL.TM. 4.0
requires three input files, including a locus genotype file, a map
file, and a quantitative data file. The locus genotype file
contained the genotype codes for all loci of the segregating
population as described above. The map file, generated from
JoinMap.TM. 3.0, contained the estimated map positions on LG5 of
the 27 loci listed in Example 5. The quantitative data file
contained the palmitic acid content as determined using the
analytical chemistry methods described in Example 1.
[0253] Interval mapping analysis evaluates the likelihood of a QTL
located along an interval between two markers. Jansen (1993)
Genetics 135:205-11. Interval mapping analysis was performed, and
the likelihood that a QTL was within an interval was calculated.
When a LOD score exceeded the predefined significance threshold of
P<0.05 or P<0.01, as calculated from a 1,000 iteration
experiment-wise permutation test (Churchill and Doerge (1994)
Genetics 138(3):963-71), a QTL determination was made. The position
with the largest LOD on the linkage group was used as the estimated
position of the QTL on the map. As this was an F.sub.2 population,
it was possible to perform interval mapping using a statistical
model to detect a QTL associated with additive genetic variance
alone, and using a statistical model that accounted for (and
therefore detected) a QTL associated with both additive and
dominant genetic variation. The previously defined data were
analyzed using both models.
Example 6: Selecting Backcross Progeny According to Palmitic Acid
Content
[0254] The markers described herein were used to select progeny
obtained by means of backcross breeding using a donor line having
the allele associated with the low palmitic acid phenotype. An
elite line with a palmitic acid content of approximately 3.5% was
crossed to a donor line having the alleles associated with the low
palmitic acid phenotype at loci HA0907, HA0041, HA1790, HA1665,
HA0908, and HA1620. A selection from the resulting progeny was
backcrossed to the elite line to produce the first backcross
generation. A selection from the first backcross generation was
backcrossed again to the elite line to produce the second backcross
generation. The genotype of an individual from the second backcross
generation is shown in Table 7.
TABLE-US-00007 TABLE 7 Genotypes of an elite line having an
elevated palmitic acid phenotype, a donor with the alleles
associated with the reduced palmitic acid phenotype, and a
selection from the second backcross generation with these two lines
as the recurrent parent and donor, respectively. Chromosome 5
HA0907 HA0041 HA1790 HA1665 HA0908 HA1620 End Sample 18 20 25 31 35
39 59 ON6725R A, A A, A A, A A, A A, A A, A NS1982.8 B, B B, B B, B
B, B B, B B, B ON6725R[2]/NS1982.8#1 = 1 = 3 Plant #19 A, A A, B A,
B A, B A, B A, B
[0255] Since the low palmitic acid content phenotype is recessive,
the individual from the second backcross generation shown in Table
7 would not display the low palmitic acid phenotype itself. To
verify that the alleles associated with reduced palmitic acid will
confer the low palmitic acid phenotype in the elite background, a
progeny test was performed. The individual in Table 7 was
self-pollinated, and eight seeds representing the progeny of the
self pollination were subjected to FAME analysis to determine the
palmitic acid content. The results, presented in Table 8, show that
three of the eight progeny have the low palmitic acid content
phenotype, consistent with the expected ratio of one to three for a
recessive trait controlled by a single locus. This result
demonstrates that the low palmitic acid content phenotype is being
inherited in progeny.
TABLE-US-00008 TABLE 8 Palmitic acid phenotypes of eight
individuals from the self pollination of plant number 19 from the
second backcross generation of ON6725R[2]/NS1982.8#1=1=3 Individual
Palmitic acid content (%) 1 2.68 2 2.73 3 1.71 4 2.23 5 1.98 6 2.85
7 3.31 8 1.92
Example 7: Selecting Cytoplasmic Male-sterile Maintainer Line
Progeny with Elevated Palmitic Acid Content
[0256] In commercial sunflower hybrid seed production, a
cytoplasmic male sterility system is used to produce the required
quantities of seed. The sunflower line in Example 6 was a restorer
line which restores normal fertility when used as a pollinator with
a female having a male sterile cytoplasm. Hybrids having the low
palmitic acid content phenotype may be produced from male and
female inbreds carrying the low palmitic acid QTL allele(s) linked
to the markers described herein, since the low palmitic acid
content phenotype is recessive. In a cytoplasmic male sterile
hybrid production system, the female inbred consists of two
near-isogenic lines: the A-line that carries the cytoplasm
conferring male sterility; and the B-line that has a normal
cytoplasm, but does not carry the restorer gene. The B-line is male
fertile, and it can be used to pollinate the A-line, with the
resulting progeny being male sterile, since they inherit the
cytoplasm from the female A-line. These progeny are also
essentially identical to the A-line parent, since the A and B lines
are near-isogenic. The B-line is thus known as the maintainer line.
The A-line is derived from the B-line using a cytoplasmic male
sterile line as the donor and the B-line as the recurrent parent.
Following repeated back-crossing with the B-line as the recurrent
(male) parent, the B-line genotype can be recovered while retaining
the male sterile cytoplasm of the donor. The resulting line is
known as the A-line. The first step in creating a new A-line,
B-line pair is to create a new B-line. The A-line is then derived
from the B-line.
[0257] To demonstrate the utility of the markers described herein
for the purpose of creating cytoplasmic male sterile maintainer
lines having a low palmitic acid content phenotype, an elite B-line
with a palmitic acid content of approximately 3.5% was crossed to a
donor line having the alleles associated with the low palmitic acid
phenotype at loci HA0850, HA0907, and HA0908. The remaining loci
were monomorphic between the donor and recurrent parent. A
selection from the resulting progeny was backcrossed to the elite
line to produce a first backcross generation. The genotype of an
individual from the first backcross generation is shown in Table
9.
TABLE-US-00009 TABLE 9 Genotypes of an elite B line having an
elevated palmitic acid phenotype, a donor with the alleles
associated with the reduced palmitic acid phenotype, and a
selection from the first backcross generation with these two lines
as the recurrent parent and donor, respectively. Chromosome 5
HA0850 HA0907 HA0908 End Sample 13 18 35 59 ON1919B A, A A, A A, A
NS1982.8 B, B B, B B, B ON1919B[1]// Plant # 11 A, A A, B A, B
CN1919B/ NS1982.8#3= 1-17=5
[0258] To verify that the low palmitic acid alleles carried by the
individual in Table 9 will confer the low palmitic acid phenotype
when in the homozygous state, a progeny test was performed. The
individual in Table 9 was self-pollinated, and eight seeds
representing the progeny of the self-pollination were subjected to
FAME analysis to determine the palmitic acid content. The results,
presented in Table 10, show that two of the eight progeny had the
low palmitic acid content phenotype, which is consistent with the
expected ratio of one-to-three for a recessive trait controlled by
a single locus. This result demonstrates that the low palmitic acid
content phenotype can be introgressed into a B-line using backcross
breeding.
TABLE-US-00010 TABLE 10 Palmitic acid phenotypes of eight
individuals from the self pollination of plant number 11 from the
first backcross generation of
ON1919B[1]//CN1919B/NS1982.8#3=1-17=5. Individual Palmitic acid
content (%) 1 2.74 2 3.16 3 3.01 4 1.77 5 2.97 6 3.47 7 2.73 8
1.87
Example 8: Development of Finished Cytoplasmic Male-sterile Elite
Maintainer and Restorer Lines with Elevated Palmitic Acid
Content
[0259] Following two generations of backcrossing, selected
individuals were self-pollinated for 3 generations, and selections
with desirable agronomic traits were subjected to FAME analysis.
The results, shown in Table 11, demonstrate that finished elite
B-lines with the low palmitic acid content phenotype can be
developed using the backcross breeding method.
TABLE-US-00011 TABLE 11 Palmitic acid content of elite B-lines
developed using the backcross breeding method Name Palmitic acid
content (%) H251B[3]/NS1982.12-20=1=4-1-20-07 1.93
ON7479B[2]/NS1982-8#2=1=5-12-10-01 1.96
[0260] Following two generations of backcrossing, selected
individuals were self-pollinated for 3 generations and selections
desirable agronomic traits was subjected to FAME analysis. The
results, shown in Table 12, demonstrate that finished elite
restorer lines with the low palmitic acid phenotype can be
developed using the backcross breeding method.
TABLE-US-00012 TABLE 12 Palmitic acid content of elite restorer
lines developed using the backcross breeding method Name Palmitic
acid content (%) OND163R[4]/NS1982-16=20=2=3=13-7-2-2 1.79
ON7385R[3]/NS1982.8=7=2=5-4-2-01 1.79
[0261] While the foregoing embodiments have been described in
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations.
[0262] All publications, patents, patent applications, and/or other
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated by
reference for all purposes.
Sequence CWU 1
1
52122DNAArtificialForward primer- marker HA0357_H757B 1gttcctgtcg
ggtaactgta gc 22219DNAArtificialReverse primer- marker HA0357_H757B
2cattgatgga gatggctgg 19320DNAArtificialForward primer- marker
HA0694B_H280R 3gccgtgaata atgggattga 20420DNAArtificialReverse
primer- marker HA0694B_H280R 4gattgggtca gcttgtgtga
20523DNAArtificialForward primer- marker HA1485 5gggaagtggg
cttgtctatg tat 23623DNAArtificialReverse primer- marker HA1485
6aacacaccga aatcacctat gaa 23722DNAArtificialForward primer- marker
HA1838 7agaggaatga gatcgggttg at 22821DNAArtificialReverse primer-
marker HA1838 8gtgggacaac tcagcaacgt c 21923DNAArtificialForward
primer- marker HA1489_H280R 9cttattccaa ggacgcatag tcg
231023DNAArtificialReverse primer- marker HA1489_H280R 10cgatggtatg
attctcgacg tta 231120DNAArtificialForward primer- marker HA1146B
11acaccaacca gacgcagaat 201220DNAArtificialReverse primer- marker
HA1146B 12gtgcaagaac gaggaagagg 201324DNAArtificialForward primer-
marker HA0037 13gaacatggcc ataactcata gacg
241416DNAArtificialReverse primer- marker HA0037 14ccttcgaccc
aacatc 161521DNAArtificialForward primer- marker HA0654
15acgcacatga gagagaaaga g 211620DNAArtificialReverse primer- marker
HA0654 16accttcgacc caacatcaag 201723DNAArtificialForward primer-
marker HA1620_H280R 17tttcgtgatg gtgattgatg att
231823DNAArtificialReverse primer- marker HA1620_H280R 18cagcaactct
gaccgtttca tta 231921DNAArtificialForward primer- marker HA0031B
19ctcacgaaac tcttcatgct g 212018DNAArtificialReverse primer- marker
HA0031B 20ctctcacact tactgaac 182120DNAArtificialForward primer-
marker HA0908 21ttgtcttcat ctgcgtgtga 202220DNAArtificialReverse
primer- marker HA0908 22ttgctgttgt tgatcggtgt
202323DNAArtificialForward primer- marker HA1665 23cctaagggga
tgaattctct ttc 232423DNAArtificialReverse primer- marker HA1665
24aacttccaat gttctccaac cat 232520DNAArtificialForward primer-
marker HA0304A_H757B 25gtgccctaac actgttccgt
202619DNAArtificialReverse primer- marker HA0304A_H757B
26agcgaaagga tcgagaatc 192721DNAArtificialForward primer- marker
HA0850_H757B 27ccctggagtg tatgtccgtt a 212820DNAArtificialReverse
primer- marker HA0850_H757B 28atccgtctgc tgcctaatcc
202920DNAArtificialForward primer- marker HA0743_H757B 29acggaaagct
cttgaaagca 203020DNAArtificialReverse primer- marker HA0743_H757B
30gcgggcattc caactagtaa 203120DNAArtificialForward primer- marker
HA0870 31gtgcgttggc tcttatggat 203220DNAArtificialReverse primer-
marker HA0870 32agtgatggca ttcccaattt 203320DNAArtificialForward
primer- marker HA0907 33catgaacatc gccaattcag
203420DNAArtificialReverse primer- marker HA0907 34tgcaaggaac
catcagaatc 203519DNAArtificialForward primer- marker HA0612A_H757B
35cttgggttct tcataactc 193619DNAArtificialReverse primer- marker
HA0612A_H757B 36catgtaatca cctttcaag 193722DNAArtificialForward
primer- marker HA1923 37aaccaaagat tcaaggcaat ca
223821DNAArtificialReverse primer- marker HA1923 38cagacattag
acgcgaagca g 213924DNAArtificialForward primer- markers HA1357A and
HA1357B_H757B 39cacaaaacaa tcgctaaaag aaca
244023DNAArtificialReverse primer- markers HA1357A and
HA1357B_H757B 40aatgatgatg gtcacgaaga aga
234121DNAArtificialForward primer- marker HA1819_H280R 41gtttcgggtg
ggggattacg g 214222DNAArtificialReverse primer- marker HA1819_H280R
42atggtcgaca acaagcgcaa ac 224320DNAArtificialForward primer-
marker HA0894 43tggtggaggt cactattgga 204420DNAArtificialReverse
primer- marker HA0894 44aggaaagaag gaagccgaga
204521DNAArtificialForward primer- marker HA1790 45tccccaaact
tgcgtgtagg t 214623DNAArtificialReverse primer- marker HA1790
46cattacaaac cacagctcct tcc 234719DNAArtificialForward primer-
marker HA0041 47ctagcaacca acctcattg 194820DNAArtificialReverse
primer- marker HA0041 48gtctccttct ctttctcggc
204923DNAArtificialForward primer- marker HA1313 49cgacccacct
agtaaaagca aac 235023DNAArtificialReverse primer- marker HA1313
50tgccataaaa agatttggtc tcc 235122DNAArtificialForward primer-
marker HA1776_H757B 51tcacaggaga atgcaaagag tg
225225DNAArtificialReverse primer- marker HA1776_H757B 52gcataatagg
agtaactgcc aaaac 25
* * * * *