U.S. patent application number 14/133807 was filed with the patent office on 2014-05-01 for genetic loci associated with iron deficiency tolerance in soybean.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. The applicant listed for this patent is Pioneer Hi-Bred International, Inc.. Invention is credited to Martin Fabrizius, Feng Han, HONG LU, SCOTT SEBASTIAN, Leon Streit.
Application Number | 20140123346 14/133807 |
Document ID | / |
Family ID | 35839980 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140123346 |
Kind Code |
A1 |
SEBASTIAN; SCOTT ; et
al. |
May 1, 2014 |
GENETIC LOCI ASSOCIATED WITH IRON DEFICIENCY TOLERANCE IN
SOYBEAN
Abstract
The invention relates to methods and compositions for
identifying soybean plants that are tolerant, have improved
tolerance or are susceptible to iron deficient growth conditions.
The methods use molecular genetic markers to identify, select
and/or construct disease-tolerant plants or identify and
counterselect disease-susceptible plants. Soybean plants that
display tolerance or improved tolerance to Phytophthora root rot
infection that are generated by the methods of the invention are
also a feature of the invention.
Inventors: |
SEBASTIAN; SCOTT; (Polk
City, IA) ; LU; HONG; (Des Moines, IA) ; Han;
Feng; (Hockessin, DE) ; Fabrizius; Martin;
(Willmar, MN) ; Streit; Leon; (Johnston,
IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pioneer Hi-Bred International, Inc. |
Johnsonton |
IA |
US |
|
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
Johnsonton
IA
|
Family ID: |
35839980 |
Appl. No.: |
14/133807 |
Filed: |
December 19, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13162634 |
Jun 17, 2011 |
|
|
|
14133807 |
|
|
|
|
12346920 |
Dec 31, 2008 |
7977533 |
|
|
13162634 |
|
|
|
|
11200539 |
Aug 8, 2005 |
7582806 |
|
|
12346920 |
|
|
|
|
60599497 |
Aug 6, 2004 |
|
|
|
60599379 |
Aug 6, 2004 |
|
|
|
Current U.S.
Class: |
800/312 ;
435/6.11 |
Current CPC
Class: |
A01H 1/04 20130101; C12N
15/8282 20130101; C12Q 1/6895 20130101; C12N 15/8271 20130101; C12Q
2600/156 20130101; A01H 5/10 20130101; C12Q 2600/13 20130101 |
Class at
Publication: |
800/312 ;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of identifying a first soybean plant or germplasm that
displays tolerance, improved tolerance or susceptibility to iron
deficiency, the method comprising detecting in the first soybean
plant or germplasm at least one allele of one or more marker locus
that is associated with the tolerance, improved tolerance or
susceptibility, wherein the one or more marker locus is selected
from the group consisting of: (a) S60210-TB, SAC1006, SATT391,
SAC1724, SATT307, P13073A-1, P10598A-1, SATT334, SATT510, SATT335,
P5219A-1, P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451,
SATT367, SATT495, P10649C-3, SATT613, SATT257, SATT581 and SATT153;
(b) a marker locus closely linked to a marker locus of (a); (c) a
marker locus localizing within a chromosome interval flanked by and
including S60210-TB and SATT391; (d) a marker locus localizing
within a chromosome interval flanked by and including P10598A-1 and
SATT334; (e) a marker locus localizing within a chromosome interval
flanked by and including SATT510 and SATT335; (f) a marker locus
localizing within a chromosome interval flanked by and including
P5219A-1 and P7659A-2; (g) a marker locus localizing within a
chromosome interval flanked by and including SAT.sub.--117 and
S60143-TB; (h) a marker locus localizing within a chromosome
interval flanked by and including SATT451 and STT367; (i) a marker
locus localizing within a chromosome interval flanked by and
including SATT495 and P10649C-3; and a marker locus localizing
within a chromosome interval flanked by and including SATT250 and
SATT346.
2. The method of claim 1, wherein the closely linked marker locus
of (b) displays a genetic recombination frequency of less than
about 10% with the marker locus of (a).
3. The method of claim 1, wherein the one or more marker locus of
(a) is selected from the group consisting of SAC1724, SATT307,
P13073A-1, P10598A-1, SATT334, SATT495, P10649C-3, SATT613 and
SATT257.
4. The method of claim 1, wherein the one or more marker locus
associated with tolerance, improved tolerance or susceptibility is
selected from the marker loci of (a) and (b).
5. The method of claim 1, wherein the one or more marker locus
associated with tolerance, improved tolerance or susceptibility is
a plurality of loci selected from the marker loci of (a) and
(b).
6. The method of claim 1, wherein the one or more marker locus
associated with tolerance, improved tolerance or susceptibility is
selected from marker loci localizing within the chromosome
intervals of (c), (d), (e), (f), (g), (h), (i) and (j).
7. The method of claim 1, wherein the one or more marker locus
associated with tolerance, improved tolerance or susceptibility is
a plurality of loci selected from marker loci localizing within the
chromosome intervals of (c), (d), (e), (f), (g), (h), (i) and
(j).
8. The method of claim 1, wherein the germplasm is a soybean line
or soybean variety.
9. The method of claim 1, wherein the tolerance, improved tolerance
or susceptibility is assayed in a population of soybean in a stand
that is known to produce chlorotic soybean plants.
10. The method of claim 1, wherein the detecting comprises
detecting at least one allelic form of a polymorphic simple
sequence repeat (SSR) or a single nucleotide polymorphism
(SNP).
11. The method of claim 1, wherein the detecting comprises
amplifying the marker locus or a portion of the marker locus and
detecting the resulting amplified marker amplicon.
12. A plant comprising in its genome one or more locus related to
tolerance or susceptibility to iron deficiency, wherein the one or
more locus is within a chromosome interval flanked by and including
SATT334 and SATT510 or a chromosome interval flanked by and
including SATT277 and SATT433.
13. A plant comprising in its genome one or more locus related to
tolerance or susceptibility to iron deficiency, wherein the one or
more locus is closely linked to a marker selected from the group
consisting of SATT334, SCT.sub.--033, SAT.sub.--120, SATT510,
SAC1724, SATT319, SAT.sub.--142-DB, SATT708-TB, SATT460, P13073A-1,
and SATT307.
14. The plant of claim 12, wherein the plant is a soybean line or
soybean variety.
15. The plant of claim 13, wherein the plant is a soybean line or
soybean variety.
16. The plant of claim 12, wherein the tolerance or susceptibility
is assayed in a population of soybean in a stand that is known to
produce chlorotic soybean plants.
17. The plant of claim 13, wherein the tolerance or susceptibility
is assayed in a population of soybean in a stand that is known to
produce chlorotic soybean plants.
18. The plant of claim 12, wherein the plant comprises an elite
soybean strain or an exotic soybean strain.
19. The plant of claim 13, wherein the plant comprises an elite
soybean strain or an exotic soybean strain.
20. A field comprising a plurality of plants of claim 12.
21. A field comprising a plurality of plants of claim 13.
22. A seed of the plant of claim 12.
23. A seed of the plant of claim 13.
24. A method of producing a plant with tolerance or susceptibility
to iron deficiency, the method comprising planting the seed of
claim 22 in soil and growing a plant therefrom.
25. A method of producing a plant with tolerance or susceptibility
to iron deficiency, the method comprising planting the seed of
claim 23 in soil and growing a plant therefrom.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. Ser.
No. 11/200,539 filed Aug. 8, 2005 which claims priority to and
benefit of U.S. Provisional Patent Application Ser. No. 60/599,497,
filed on Aug. 6, 2004, and U.S. Provisional Patent Application Ser.
No. 60/599,379, filed on Aug. 6, 2004, which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] Iron deficiency pathology in soybean is manifested as iron
deficiency chlorosis (FEC or IDC). The invention relates to
compositions and methods for identifying soybean plants that are
tolerant, have improved tolerance or are susceptible to
iron-deficient growth conditions, where the methods use molecular
genetic markers to identify, select and/or construct low
iron-tolerant plants. The invention also relates to the soybean
plants that display tolerance or improved tolerance to low iron
growth conditions that are generated by the methods of the
invention.
BACKGROUND OF THE INVENTION
[0003] Soybean, a legume, has become the world's primary source of
seed oil and seed protein. In addition, its utilization is being
expanded to the industrial, manufacturing and pharmaceutical
sectors. Soybean productivity is a vital agricultural and economic
consideration. Improving soybean tolerance to diverse and/or
adverse growth conditions is crucial for maximizing yields.
Iron Deficiency Chlorosis
[0004] Iron-deficiency chlorosis (IDC; alternatively, FEC), reduces
soybean yields, particularly on calcareous or other high pH soils.
IDC develops in soybean due to a lack of chlorophyll in the leaves
of affected plants, manifesting as yellowing on the leaves. Iron is
required for the synthesis of chlorophyll and, although iron is
sufficiently present in most soils, it is often in an insoluble
form that cannot be used by the plant. Iron deficiency occurs in
soils due to high pH, high salt content, cool temperatures or other
environmental factors that decrease iron solubility. Studies have
shown that even mild IDC symptoms are an indication that yield is
being negatively affected (Fehr (1982) Journal of Plant Nutrition,
611-621.)
[0005] Iron is found in soil mainly as insoluble oxyhydroxide
polymers (FeOOH) that are extremely insoluble (10.sup.-17 M) at
neutral pH. Since the optimal concentration of soluble Fe for plant
growth is approximately 10.sup.-6 M, plants have evolved two
different strategies to mine the iron they need from soil (Fox and
Guerinot 1998 "Molecular biology of cation transport in plants,"
Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:669-96).
[0006] So-called "Strategy I" is used by all plants except grasses.
This strategy involves a two step process. In the first step, the
oxidized iron Fe(III) is reduced to the more soluble Fe(II) by a
membrane-bound ferric chelate reductase located in root epidermal
cells. This reductase activity is inducible and necessary for iron
uptake under iron deficient conditions (Yi and Guerinot (1996)
Plant Journal 10:835-844). A gene FRO2 that encodes such a ferric
chelate reductase enzyme has been identified and sequenced in
Arabidopsis (Robinson et al, Nature 397:694-697, 1999). Following
the reduction step, a separate transport protein is required to
move the reduced iron across the root plasma membrane. A gene IRT1
(iron regulated transporter) which codes for the transport protein
has also been found in Arabidopsis (Eide et al, PNAC 93:5624-5628).
This same transport protein has been shown to transport manganese,
zinc, and cobalt as well (Korshunova et al, Plant Mol. Biology
40:37-44, 1999). In addition to this two step process, Strategy I
plants also acidify the soil by exuding protons from the roots via
the conversion of ATP to ADP within the roots. This lowers the pH
in the rhizosphere and makes the iron oxides more soluble.
[0007] While iron availability can, to an extent, be modulated
environmentally (e.g., by modifying soil pH or adding soluble iron,
applying foliar iron treatments, or applying iron to seed), these
approaches can cause unwanted side effects in the soybean or the
environment and also add to soybean production costs. Some
treatments, such as iron treatment of seed, display inconsistent
results in different cultivars or field environments. Despite these
difficulties, most producers currently rely on the use of seed,
foliar, or soil treatments to reduce IDC (Weirsma (2002) "Iron
Deficiency Chlorosis (IDC) In Soybean," Cropping Issues in
Northwest Minnesota 1(7): 1-2); Goos and Germain (2001) "Solubility
of Twelve Iron Fertilizer Products in Alkaline Soils"
Communications in Soil Science and Plant Analysis 32:2317-2323.
[0008] For some time, soybean producers have sought to develop IDC
tolerant plants as a cost-effective alternative or supplement to
standard foliar, soil and/or seed treatments (e.g., Hintz et al.
(1987) "Population development for the selection of high-yielding
soybean cultivars with resistance to iron deficiency chlorosis,"
Crop Sci. 28:369-370). Recent studies also suggest that cultivar
selection is more reliable and universally applicable than foliar
sprays or iron seed treatment methods, though environmental and
cultivar selection methods can also be used effectively in
combination. See also, Goos and Johnson (2000) "A Comparison of
Three Methods for Reducing Iron-Deficiency Chlorosis in Soybean"
Agronomy Journal 92:1135-1139; and Goos and Johnson "Seed
Treatment, Seeding Rate, and Cultivar Effects on Iron Deficiency
Chlorosis of Soybean" Journal of Plant Nutrition 24 (8)
1255-1268.
[0009] The advent of molecular genetic markers has facilitated
mapping and selection of agriculturally important traits in
soybean. Markers tightly linked to disease tolerance genes are an
asset in the rapid identification of tolerant soybean lines on the
basis of genotype by the use of marker assisted selection (MAS).
Introgressing disease tolerance genes into a desired cultivar would
also be facilitated by using suitable DNA markers.
[0010] Soybean cultivar improvement for IDS tolerance can be
performed using classical breeding methods, or, more preferably,
using marker assisted selection (MAS). Genetic markers for IDC
tolerance/susceptibility have been identified (e.g., Lin et al.
(2000) "Molecular characterization of iron deficiency chlorosis in
soybean" Journal of Plant Nutrition 23:1929-1939). Recent work
suggests that marker assisted selection is particularly beneficial
when selecting plants for IDC tolerance, because the strength of
environmental effects on chlorosis expression impedes progress in
improving IDC resistance. See also, Charlson et al., "Associating
SSR Markers with Soybean Resistance to Iron Chlorosis," Journal of
Plant Nutrition, vol. 26, nos. 10 & 11; 2267-2276 (2003).
Molecular Markers and Marker Assisted Selection
[0011] A genetic map is 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
but not limited to, molecular markers such as SSR markers, RFLP
markers, or SNP markers. Furthermore, SSR markers can be derived
from genomic or expressed nucleic acids (e.g., ESTs). The nature of
these 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.
[0012] Although specific DNA sequences which encode proteins are
generally well-conserved across a species, other regions of DNA
(typically non-coding) tend to accumulate polymorphism, and
therefore, can be variable between individuals of the same species.
Such regions provide the basis for numerous molecular genetic
markers. In general, any differentially inherited polymorphic trait
(including nucleic acid polymorphism) that segregates among progeny
is a potential marker. The genomic variability can be of any
origin, for example, insertions, deletions, duplications,
repetitive elements, point mutations, recombination events, or the
presence and sequence of transposable elements. A large number of
soybean molecular markers are known in the art, and are published
or available from various sources, such as the SOYBASE internet
resource. Similarly, numerous methods for detecting molecular
markers are also well-established.
[0013] The primary motivation for developing molecular marker
technologies from the point of view of plant breeders has been the
possibility to increase breeding efficiency through marker assisted
selection (MAS). A molecular marker allele that demonstrates
linkage disequilibrium with a desired phenotypic trait (e.g., a
quantitative trait locus, or QTL, such as resistance to a
particular disease) provides a useful tool for the selection of a
desired trait in a plant population. The key components to the
implementation of this approach are: (i) the creation of a dense
genetic map of molecular markers, (ii) the detection of QTL based
on statistical associations between marker and phenotypic
variability, (iii) the definition of a set of desirable marker
alleles based on the results of the QTL analysis, and (iv) the use
and/or extrapolation of this information to the current set of
breeding germplasm to enable marker-based selection decisions to be
made.
[0014] The availability of integrated linkage maps of the soybean
genome containing increasing densities of public soybean markers
has facilitated soybean genetic mapping and MAS. See, e.g., Cregan
et al. (1999) "An Integrated Genetic Linkage Map of the Soybean
Genome" Crop Sci. 39:1464-1490; Song et al., "A New Integrated
Genetic Linkage Map of the Soybean," Theor. Appl. Genet.,
109:122-128 (2004); Diwan and Cregan (1997) "Automated sizing of
fluorescent-labeled simple sequence repeat (SSR) markers to assay
genetic variation in Soybean," Theor. Appl. Genet., 95:220-225; the
SOYBASE resources on the world wide web, including the Shoemaker
Lab Home Page and other resources that can be accessed through
SOYBASE; and see the Soybean Genomics and Improvements Laboratory
(SGIL) on the world wide web.
[0015] Two types of markers are frequently used in marker assisted
selection protocols, namely simple sequence repeat (SSR, also known
as microsatellite) markers, and single nucleotide polymorphism
(SNP) markers. The term SSR refers generally to any type of
molecular heterogeneity that results in length variability, and
most typically is a short (up to several hundred base pairs)
segment of DNA that consists 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., caused by polymerase slippage. SSRs appear to be
randomly dispersed through the genome and are generally flanked by
conserved regions. SSR markers can also be derived from RNA
sequences (in the form of a cDNA, a partial cDNA or an EST) as well
as genomic material.
[0016] The characteristics of SSR heterogeneity make them well
suited for use as molecular genetic markers; namely, SSR genomic
variability is inherited, is multiallelic, codominant and is
reproducibly detectable. The proliferation of increasingly
sophisticated amplification-based detection techniques (e.g.,
PCR-based) provides a variety of sensitive methods for the
detection of nucleotide sequence heterogeneity. Primers (or other
types of probes) are designed to hybridize to conserved regions
that flank the SSR domain, resulting in the amplification of the
variable SSR region. The different sized amplicons generated from
an SSR region have characteristic and reproducible sizes. The
different sized SSR amplicons observed from two homologous
chromosomes in an individual, or from different individuals in the
plant population are generally termed "marker alleles." As long as
there exists at least two SSR alleles that produce PCR products
with at least two different sizes, the SSRs can be employed as a
marker.
[0017] Soybean markers that rely on single nucleotide polymorphisms
(SNPs) are also well known in the art. Various techniques have been
developed for the detection of SNPs, including allele specific
hybridization (ASH; see, e.g., Coryell et al., (1999) "Allele
specific hybridization markers for soybean," Theor. Appl. Genet.,
98:690-696). Additional types of molecular markers are also widely
used, including but not limited to expressed sequence tags (ESTs)
and SSR markers derived from EST sequences, restriction fragment
length polymorphism (RFLP), amplified fragment length polymorphism
(AFLP), randomly amplified polymorphic DNA (RAPD) and isozyme
markers. 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. For example, PCR amplification, single-strand
conformation polymorphisms (SSCP) and self-sustained sequence
replication (3 SR; see Chan and Fox, "NASBA and other
transcription-based amplification methods for research and
diagnostic microbiology," Reviews in Medical Microbiology
10:185-196 [1999]).
[0018] Linkage of one molecular marker to another molecular marker
is measured as a recombination frequency. In general, the closer
two loci (e.g., two SSR markers) are on the genetic map, the closer
they lie to each other on the physical map. A relative genetic
distance (determined by crossing over frequencies, measured in
centimorgans; cM) is generally proportional to the physical
distance (measured in base pairs, e.g., kilobase pairs [kb] or
megabasepairs [Mbp]) that two linked loci are separated from each
other on a chromosome. A lack of precise proportionality between cM
and physical distance can result from variation in recombination
frequencies for different chromosomal regions, e.g., some
chromosomal regions are recombinational "hot spots," while others
regions do not show any recombination, or only demonstrate rare
recombination events. In general, the closer one marker is to
another marker, whether measured in terms of recombination or
physical distance, the more strongly they are linked. In some
aspects, the closer a molecular marker is to a gene that encodes a
polypeptide that imparts a particular phenotype (disease
tolerance), whether measured in terms of recombination or physical
distance, the better that marker serves to tag the desired
phenotypic trait.
[0019] Genetic mapping variability can also be observed between
different populations of the same crop species, including soybean.
In spite of this variability in the genetic map that may occur
between populations, genetic map and marker information derived
from one population generally remains useful across multiple
populations in identification of plants with desired traits,
counter-selection of plants with undesirable traits and in guiding
MAS.
QTL Mapping
[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, pathogen tolerance, leading ultimately to
increased agricultural productivity. It has been recognized for
quite some time that specific chromosomal loci (or intervals) can
be mapped in an organism's genome that correlate with particular
quantitative phenotypes. Such loci are termed quantitative trait
loci, or QTL. The 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., pathogenic infection
tolerance), 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 a desired phenotype by
selecting for the proper molecular marker allele (a process called
marker-assisted selection, or MAS). The more molecular markers that
are placed on the genetic map, the more potentially useful that map
becomes for conducting MAS.
[0021] Multiple experimental paradigms have been developed to
identify and analyze QTL (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 the 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, which 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 (e.g., 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). QTL 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 F1 parents all have the same linkage phase. Thus,
after selfing of the F1 plants, all segregating progeny (F2) 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.
[0022] 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, e.g., standard linear
models, such as ANOVA or regression mapping (Haley and Knott (1992)
Heredity 69:315), maximum likelihood methods such as
expectation-maximization algorithms, (e.g., Lander and Botstein
(1989) "Mapping Mendelian factors underlying quantitative traits
using RFLP linkage maps," Genetics 121:185-199; Jansen (1992) "A
general mixture model for mapping quantitative trait loci by using
molecular markers," Theor. Appl. Genet., 85:252-260; Jansen (1993)
"Maximum likelihood in a generalized linear finite mixture model by
using the EM algorithm," Biometrics 49:227-231; 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. 116-124, CPRO-DLO Metherlands; Jansen (1996) "A
general Monte Carlo method for mapping multiple quantitative trait
loci," Genetics 142:305-311; and Jansen and Stam (1994) "High
Resolution of quantitative trait into multiple loci via interval
mapping," Genetics 136:1447-1455). 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,
Haseman-Elston regression, any of which are suitable in the context
of the present invention. In addition, additional details regarding
alternative statistical methods applicable to complex breeding
populations which can be used to identify and localize QTLs are
described in: U.S. Ser. No. 09/216,089 by Beavis et al. "QTL
MAPPING IN PLANT BREEDING POPULATIONS" and PCT/US00/34971 by Jansen
et al. "MQM MAPPING USING HAPLOTYPED PUTATIVE QTLS ALLELES: A
SIMPLE APPROACH FOR MAPPING QTLS IN PLANT BREEDING POPULATIONS."
Any 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.
[0023] There is a need in the art for improved soybean strains that
are tolerant to iron-deficient growth conditions. There is a need
in the art for methods that identify soybean plants or populations
(germplasm) that display tolerance to iron-deficient growth
conditions. What is needed in the art is to identify molecular
genetic markers that co-segregate with to low-iron tolerance loci
(e.g., tolerance QTL) in order to facilitate MAS, and also to
facilitate gene discovery and cloning of gene alleles that impart
tolerance to low iron growth conditions. Such markers can be used
to select individual plants and plant populations that show
favorable marker alleles in soybean populations and then employed
to select the tolerant phenotype, or alternatively, be used to
counterselect plants or plant populations that show a low-iron
susceptibility phenotype. The present invention provides these and
other advantages.
SUMMARY OF THE INVENTION
[0024] Compositions and methods for identifying soybean plants or
germplasm with tolerance to low iron growth conditions are
provided. Methods of making soybean plants or germplasm that are
tolerant to low iron growth conditions, e.g., through introgression
of desired tolerance marker alleles and/or by transgenic production
methods, as well as plants and germplasm made by these methods, are
also provided. Systems and kits for selecting tolerant plants and
germplasm are also a feature of the invention.
[0025] Low iron growth conditions can produce plant pathology
termed iron deficiency chlorosis (IDC) or iron chlorosis (FEC). The
identification and selection of soybean plants that show tolerance
to low iron growth conditions using MAS can provide an effective
and environmentally friendly approach to overcoming losses caused
by this disease. The present invention provides a number of soybean
marker loci and QTL chromosome intervals that demonstrate
statistically significant co-segregation with tolerance to low iron
growth conditions. Detection of these QTL markers or additional
loci linked to the QTL markers can be used in marker-assisted
soybean breeding programs to produce tolerant plants, or plants
with improved tolerance.
[0026] In some aspects, the invention provides methods for
identifying a first soybean plant or germplasm (e.g., a line or
variety) that has tolerance, improved tolerance or susceptibility
to low iron growth conditions. In the methods, at least one allele
of one or more marker locus (e.g., a plurality of marker loci) that
is associated with the tolerance, improved tolerance or
susceptibility are detected in the first soybean plant or
germplasm. The marker loci can be selected from the loci provided
in FIG. 1, including: S60210-TB, SAC1006, SATT391, SAC1724,
SATT307, P13073A-1, P10598A-1, SATT334, SATT510, SATT335, P5219A-1,
P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367,
SATT495, P10649C-3, SATT613, SATT257, SATT581 and SATT153, as well
as any other marker that is closely linked to these QTL markers
(e.g., within about 10 cM of these loci). The invention also
provides chromosomal QTL intervals that correlate with low iron
tolerance. These intervals are located on linkage groups Cl, F, G,
I, L and M. Any marker located within these intervals also finds
use as a marker for low iron tolerance. These intervals include any
marker locus localizing within a chromosome interval flanked by and
including:
[0027] (a) S60210-TB and SATT391 (LG-C1);
[0028] (b) P10598A-1 and SATT334 (LG-F);
[0029] (c) SATT510 and SATT335 (LG-F);
[0030] (d) P5219A-1 and P7659A-2 (LG-G);
[0031] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0032] (f) SATT451 and SATT367 (LG-I);
[0033] (g) SATT495 and P10649C-3 (LG-L); and
[0034] (h) SATT250 and SATT346 (LG-M).
[0035] A plurality of maker loci can be selected in the same plant.
Which QTL markers are selected in combination is not particularly
limited. The QTL markers used in combinations can be any of the
makers listed in FIG. 1, any other marker that is closely linked to
the markers in FIG. 1 (e.g., the closely linked markers as
determined from FIG. 4 and FIG. 5, or determined from the SOYBASE
resource), or any marker within the QTL intervals described
herein.
[0036] The markers that are linked to the QTL markers of the
invention (e.g., those markers provided in FIG. 1) are closely
linked, for example, within about 10 cM from the QTL markers. In
desirable embodiments, the linked locus displays a genetic
recombination distance of 9 centiMorgans, 8, 7, 6, 5, 4, 3, 2, 1,
0.75, 0.5 or 0.25, or less from the QTL marker. In some
embodiments, the closely linked locus is selected from the list of
marker loci determined from FIG. 6 or FIG. 6.
[0037] In some embodiments, preferred QTL markers are selected from
SAC 1724, SATT307, P13073A-1, P10598A-1, SATT334, SATT495,
P10649C-3, SATT613 and SATT257.
[0038] In some embodiments, the germplasm is a soybean line or
variety. In some aspects, the tolerance or improved tolerance is a
non-race specific tolerance or a non-race specific improved
tolerance. In some aspects, the tolerance, improved tolerance or
susceptibility of a soybean plant to low iron growth conditions can
be quantitated using any suitable means, for example, by assaying
soybean pathology in a field where disease is known to occur
naturally.
[0039] Any of a variety of techniques can be used to identify a
marker allele. It is not intended that the method of allele
detection be limited in any way. Methods for allele detection
typically include molecular identification methods such as
amplification and detection of the marker amplicon. For example, an
allelic form of a polymorphic simple sequence repeat (SSR), or of a
single nucleotide polymorphism (SNP) can be detected, e.g., by an
amplification based technology. In these and other amplification
based detection methods, the marker locus or a portion of the
marker locus is amplified (e.g., via PCR, LCR or transcription
using a nucleic acid isolated from a soybean plant of interest as a
template) and the resulting amplified marker amplicon is detected.
In one example of such an approach, an amplification primer or
amplification primer pair is admixed with genomic nucleic acid
isolated from the first soybean plant or germplasm, wherein the
primer or primer pair is complementary or partially complementary
to at least a portion of the marker locus, and is capable of
initiating DNA polymerization by a DNA polymerase using the soybean
genomic nucleic acid as a template. The primer or primer pair
(e.g., a primer pair provided in FIG. 2 or 3) is extended in a DNA
polymerization reaction having a DNA polymerase and a template
genomic nucleic acid to generate at least one amplicon. In any
case, data representing the detected allele(s) can be transmitted
(e.g., electronically or via infrared, wireless or optical
transmission) to a computer or computer readable medium for
analysis or storage. In some embodiments, plant RNA is the template
for the amplification reaction. In other embodiments, plant genomic
DNA is the template for the amplification reaction. In some
embodiments, the QTL marker is a SNP type marker, and the detected
allele is a SNP allele, and the method of detection is allele
specific hybridization (ASH).
[0040] In some embodiments, the allele that is detected is a
favorable allele that positively correlates with tolerance or
improved tolerance. In the case where more than one marker is
selected, an allele is selected for each of the markers; thus, two
or more alleles are selected. In some embodiments, it can be the
case that a marker locus will have more than one advantageous
allele, and in that case, either allele can be selected.
[0041] It will be appreciated that the ability to identify QTL
marker loci alleles that correlate with tolerance, improved
tolerance or susceptibility of a soybean plant to low iron growth
conditions provides a method for selecting plants that have
favorable marker loci as well. That is, any plant that is
identified as comprising a desired marker locus (e.g., a marker
allele that positively correlates with tolerance) can be selected
for, while plants that lack the locus, or that have a locus that
negatively correlates with tolerance, can be selected against Thus,
in one method, subsequent to identification of a marker locus, the
methods include selecting (e.g., isolating) the first soybean plant
or germplasm, or selecting a progeny of the first plant or
germplasm. In some embodiments, the resulting selected first
soybean plant or germplasm can be crossed with a second soybean
plant or germplasm (e.g., an elite or exotic soybean, depending on
characteristics that are desired in the progeny).
[0042] Similarly, in other embodiments, if an allele is correlated
with tolerance or improved tolerance to low iron growth conditions,
the method can include introgressing the allele into a second
soybean plant or germplasm to produce an introgressed soybean plant
or germplasm. In some embodiments, the second soybean plant or
germplasm will typically display reduced tolerance to low iron
growth conditions as compared to the first soybean plant or
germplasm, while the introgressed soybean plant or germplasm will
display an increased tolerance to low iron growth conditions as
compared to the second plant or germplasm. An introgressed soybean
plant or germplasm produced by these methods are also a feature of
the invention.
[0043] In other aspects, various mapping populations are used to
determine the linked markers of the invention. In one embodiment,
the mapping population used is the population derived from the
cross UP1C6-43/90B73. In other embodiments, other mapping
populations can be used. In other aspects, various software is used
in determining linked marker loci. For example, TASSEL, GeneFlow
and MapManager all find use with the invention. In some
embodiments, such as when software is used in the linkage analysis,
the detected allele information (i.e., the data) is electronically
transmitted or electronically stored, for example, in a computer
readable medium.
[0044] In addition to introgressing selected marker alleles into
desired genetic backgrounds, transgenic approaches can also be used
to produce plants or germplasm that are tolerant to low iron growth
conditions. For example, in some aspects, the invention provides
methods of producing a soybean plant having tolerance or improved
tolerance to low iron growth conditions, the methods comprising
introducing an exogenous nucleic acid into a target soybean plant
or progeny thereof, wherein the exogenous nucleic acid is derived
from a nucleotide sequence that is linked to at least one favorable
allele of one or more marker locus that is associated with
tolerance or improved tolerance to low iron growth conditions. In
some embodiments, the marker locus can be selected from: S60210-TB,
SAC1006, SATT391, SAC1724, SATT307, P13073A-1, P10598A-1, SATT334,
SATT510, SATT335, P5219A-1, P7659A-2, SAT.sub.--117, SATT191,
S60143-TB, SATT451, SATT367, SATT495, P10649C-3, SATT613, SATT257,
SATT581 and SATT153, as well as any other marker that is closely
linked (e.g., demonstrating not more than 10% recombination
frequency) to these QTL markers; and furthermore, any marker locus
that is located within the chromosomal QTL intervals flanked by and
including:
[0045] (a) S60210-TB and SATT391 (LG-C1);
[0046] (b) P10598A-1 and SATT334 (LG-F);
[0047] (c) SATT510 and SATT335 (LG-F);
[0048] (d) P5219A-1 and P7659A-2 (LG-G);
[0049] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0050] (f) SATT451 and SATT367 (LG-I);
[0051] (g) SATT495 and P10649C-3 (LG-L); and
[0052] (h) SATT250 and SATT346 (LG-M).
[0053] In some embodiments, preferred QTL markers used in these
transgenic plant methods are selected from SAC1724, SATT307,
P13073A-1, P10598A-1, SATT334, SATT495, P10649C-3, SATT613 and
SATT257.
[0054] In some embodiments, a plurality of maker loci can be used
to construct the transgenic plant. Which QTL markers are used in
combination is not particularly limited. The QTL markers used in
combinations can be any of the makers listed in FIG. 1, any other
marker that is linked to the markers in FIG. 1 (e.g., the linked
markers as determined from FIGS. 5 and 6, or determined from the
SOYBASE resource), or any markers selected from the QTL intervals
described herein.
[0055] Any of a variety of methods can be used to provide the
exogenous nucleic acid to the soybean plant. In one method, the
nucleotide sequence is isolated by positional cloning, and is
identified by linkage to the favorable allele. The precise
composition of the exogenous nucleic acid can vary; in one
embodiment, the exogenous nucleic acid corresponds to an open
reading frame (ORF) that encodes a polypeptide that, when expressed
in a soybean plant, results in the soybean plant having tolerance
or improved tolerance to iron-deficient growth conditions. The
exogenous nucleic acid optionally comprises an expression vector to
provide for expression of the exogenous nucleic acid in the
plant.
[0056] In other aspects, various mapping populations are used to
determine the linked markers that find use in constructing the
transgenic plant. In one embodiment, the mapping population used is
the population derived from the cross UP1C6-43/90B73. In other
embodiments, other populations can be used. In other aspects,
various software is used in determining linked marker loci used to
construct the transgenic plant. For example, TASSEL, GeneFlow or
MapManager-QTX all find use with the invention.
[0057] Systems for identifying a soybean plant predicted to have
tolerance or improved tolerance to iron-deficient growth conditions
are also a feature of the invention. Typically, the system can
include a set of marker primers and/or probes configured to detect
at least one favorable allele of one or more marker locus
associated with tolerance or improved tolerance to iron-deficient
growth conditions, wherein the marker locus or loci are selected
from: S60210-TB, SAC1006, SATT391, SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT510, SATT335, P5219A-1, P7659A-2,
SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367, SATT495,
P10649C-3, SATT613, SATT257, SATT581 and SATT153, as well as any
other marker that is closely linked (e.g., demonstrating not more
than 10% recombination frequency) to these QTL markers; and
furthermore, any marker locus that is located within the
chromosomal QTL intervals flanked by and including:
[0058] (a) S60210-TB and SATT391 (LG-C1);
[0059] (b) P10598A-1 and SATT334 (LG-F);
[0060] (c) SATT510 and SATT335 (LG-F);
[0061] (d) P5219A-1 and P7659A-2 (LG-G);
[0062] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0063] (f) SATT451 and SATT367 (LG-I);
[0064] (g) SATT495 and P10649C-3 (LG-L); and
[0065] (h) SATT250 and SATT346 (LG-M);
In some embodiments, preferred QTL markers used in these transgenic
plant methods are selected from SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT495, P10649C-3, SATT613 and SATT257.
[0066] Where a system that performs marker detection or correlation
is desired, the system can also include 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/or system instructions
that correlate the presence or absence of the favorable allele with
the predicted tolerance. The precise configuration of the detector
will depend on the type of label used to detect the marker allele.
Typical embodiments include light detectors, radioactivity
detectors, and the like. Detection of the light emission or other
probe label is indicative of the presence or absence of a marker
allele. Similarly, the precise form of the instructions can vary
depending on the components of the system, e.g., they can be
present as system software in one or more integrated unit of the
system, or can be present in one or more computers or computer
readable media operably coupled to the detector. In one typical
embodiment, the system instructions include at least one look-up
table that includes a correlation between the presence or absence
of the favorable allele and predicted tolerance, improved tolerance
or susceptibility.
[0067] In some embodiments, the system can be comprised of separate
elements or can be integrated into a single unit for convenient
detection of markers alleles and for performing marker-tolerance
trait correlations. In some embodiments, the system can also
include a sample, for example, genomic DNA, amplified genomic DNA,
cDNA, amplified cDNA, RNA, or amplified RNA from soybean or from a
selected soybean plant tissue.
[0068] Kits are also a feature of the invention. For example, a kit
can include appropriate primers or probes for detecting tolerance
associated marker loci and instructions in using the primers or
probes for detecting the marker loci and correlating the loci with
predicted low iron tolerance. The kits can further include
packaging materials for packaging the probes, primers or
instructions, controls such as control amplification reactions that
include probes, primers or template nucleic acids for
amplifications, molecular size markers, or the like.
[0069] In other aspects, the invention provides nucleic acid
compositions that are the novel EST-derived SSR QTL markers of the
invention. For example, the invention provides compositions
comprising an amplification primer pair capable of initiating DNA
polymerization by a DNA polymerase on a soybean nucleic acid
template to generate a soybean marker amplicon, where the marker
amplicon corresponds to a soybean marker selected from S60210-TB,
S60143-TB and S60392-TB, and further where the composition
comprises a primer pair that is specific for the marker.
DEFINITIONS
[0070] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
embodiments, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting. As used in this specification and the appended 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, reference to "plant," "the
plant" or "a plant" also includes a plurality of plants; also,
depending on the context, use of the term "plant" can also include
genetically similar or identical progeny of that plant; use of the
term "a nucleic acid" optionally includes, as a practical matter,
many copies of that nucleic acid molecule; similarly, the term
"probe" optionally (and typically) encompasses many similar or
identical probe molecules.
[0071] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation. Numeric ranges recited within the
specification are inclusive of the numbers defining the range and
include each integer or any 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 to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0072] A "plant" can be a whole plant, any part thereof, or a cell
or tissue culture derived from a plant. Thus, the term "plant" can
refer to any of: whole plants, plant components or organs (e.g.,
leaves, stems, roots, etc.), plant tissues, seeds, plant cells,
and/or progeny of the same. A plant cell is a cell of a plant,
taken from a plant, or derived through culture from a cell taken
from a plant. Thus, the term "soybean plant" includes whole soybean
plants, soybean plant cells, soybean plant protoplast, soybean
plant cell or soybean tissue culture from which soybean plants can
be regenerated, soybean plant calli, soybean plant clumps and
soybean plant cells that are intact in soybean plants or parts of
soybean plants, such as soybean seeds, soybean pods, soybean
flowers, soybean cotyledons, soybean leaves, soybean stems, soybean
buds, soybean roots, soybean root tips and the like.
[0073] "Germplasm" refers to genetic material of or from an
individual (e.g., a plant), a group of individuals (e.g., a plant
line, variety or family), or a clone derived from a line, variety,
species, or culture. The germplasm can be part of an organism or
cell, or can be separate from the organism or cell. In general,
germplasm provides genetic material with a specific molecular
makeup that provides a physical foundation for some or all of the
hereditary qualities of an organism or cell culture. As used
herein, germplasm includes cells, seed or tissues from which new
plants may be grown, or plant parts, such as leafs, stems, pollen,
or cells, that can be cultured into a whole plant.
[0074] The term "allele" refers to one of two or more different
nucleotide sequences that occur at a specific locus. For example, a
first allele can occur on one chromosome, while a second allele
occurs 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. A
"favorable allele" is the allele at a particular locus that
confers, or contributes to, an agronomically desirable phenotype,
e.g., tolerance to Phytophthora infection, or alternatively, is an
allele that allows the identification of susceptible plants that
can be removed from a breeding program or planting. A favorable
allele of a marker is a marker allele that segregates with the
favorable phenotype, or alternatively, segregates with susceptible
plant phenotype, therefore providing the benefit of identifying
disease-prone plants. A favorable allelic form of a chromosome
segment is a chromosome segment that includes a nucleotide sequence
that contributes to superior agronomic performance at one or more
genetic loci physically located on the chromosome segment. "Allele
frequency" refers to the frequency (proportion or percentage) at
which an allele is present at a locus within an individual, within
a line, or within a population of lines. For example, for an allele
"A," diploid individuals of genotype "AA," "Aa," or "aa" have
allele frequencies of 1.0, 0.5, or 0.0, respectively. One can
estimate the allele frequency within a line by averaging the allele
frequencies of a sample of individuals from that line. Similarly,
one can calculate the allele frequency within a population of lines
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 can be expressed as a count of
individuals or lines (or any other specified grouping) containing
the allele.
[0075] An allele "positively" correlates with a trait when it is
linked to it and when presence of the allele is an indictor that
the desired trait or trait form will occur in a plant comprising
the allele. An allele negatively correlates with a trait when it is
linked to it and when presence of the allele is an indicator that a
desired trait or trait form will not occur in a plant comprising
the allele.
[0076] An individual is "homozygous" if the individual has only one
type of allele at a given locus (e.g., a diploid individual has a
copy of the same allele at a locus for each of two homologous
chromosomes). An individual is "heterozygous" if more than one
allele type is present at a given locus (e.g., a diploid individual
with one copy each of two different alleles). The term
"homogeneity" indicates that members of a group have the same
genotype at one or more specific loci. In contrast, the term
"heterogeneity" is used to indicate that individuals within the
group differ in genotype at one or more specific loci.
[0077] A "locus" is a chromosomal region where a polymorphic
nucleic acid, trait determinant, gene or marker is located. Thus,
for example, a "gene locus" is a specific chromosome location in
the genome of a species where a specific gene can be found.
[0078] The term "quantitative trait locus" or "QTL" refers to a
polymorphic genetic locus 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.
[0079] The terms "marker," "molecular marker," "marker nucleic
acid," and "marker locus" refer to a nucleotide sequence or encoded
product thereof (e.g., a protein) used as a point of reference when
identifying a linked locus. A marker can be derived from genomic
nucleotide sequence or from expressed nucleotide sequences (e.g.,
from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide.
The term also refers to nucleic acid sequences complementary to or
flanking the marker sequences, such as nucleic acids used as probes
or primer pairs capable of amplifying the marker sequence. A
"marker probe" is a nucleic acid sequence or molecule that can be
used to identify the presence of a marker locus, e.g., a nucleic
acid probe that is complementary to a marker locus sequence.
Alternatively, in some aspects, a marker probe refers to a probe of
any type that is able to distinguish (i.e., genotype) the
particular allele that is present at a marker locus. Nucleic acids
are "complementary" when they specifically hybridize in solution,
e.g., according to Watson-Crick base pairing rules. A "marker
locus" is a locus that can be used to track the presence of a
second linked locus, e.g., a linked locus that encodes or
contributes to expression of a phenotypic trait. For example, a
marker locus can be used to monitor segregation of alleles at a
locus, such as a QTL, that are genetically or physically linked to
the marker locus. Thus, a "marker allele," alternatively an "allele
of a marker locus" is one of a plurality of polymorphic nucleotide
sequences found at a marker locus in a population that is
polymorphic for the marker locus. In some aspects, the present
invention provides marker loci correlating with tolerance to
Phytophthora infection in soybean. Each of the identified markers
is expected to be in close physical and genetic proximity
(resulting in physical and/or genetic linkage) to a genetic
element, e.g., a QTL, that contributes to tolerance.
[0080] "Genetic markers" are nucleic acids that are polymorphic in
a population and where the alleles of which can be detected and
distinguished by one or more analytic methods, e.g., RFLP, AFLP,
isozyme, SNP, SSR, and the like. The terms "genetic marker" and
"molecular marker" refer to a genetic locus (a "marker locus") that
can be used as a point of reference when identifying a genetically
linked locus such as a QTL. Such a marker is also referred to as a
QTL marker. The term also refers to nucleic acid sequences
complementary to the genomic sequences, such as nucleic acids used
as probes.
[0081] Markers corresponding to genetic polymorphisms between
members of a population can be detected by methods well-established
in the art. These include, e.g., PCR-based sequence specific
amplification methods, detection of restriction fragment length
polymorphisms (RFLP), detection of isozyme markers, detection of
polynucleotide polymorphisms by allele specific hybridization
(ASH), detection of amplified variable sequences of the plant
genome, detection of self-sustained sequence replication, detection
of simple sequence repeats (SSRs), detection of single nucleotide
polymorphisms (SNPs), or detection of amplified fragment length
polymorphisms (AFLPs). Well established methods are also know for
the detection of expressed sequence tags (ESTs) and SSR markers
derived from EST sequences and randomly amplified polymorphic DNA
(RAPD).
[0082] A "genetic map" is a description of genetic linkage
relationships among loci on one or more chromosomes (or linkage
groups) within a given species, generally depicted in a
diagrammatic or tabular form. "Genetic mapping" is the process of
defining the linkage relationships of loci through the use of
genetic markers, populations segregating for the markers, and
standard genetic principles of recombination frequency. A "genetic
map location" is a location on a genetic map relative to
surrounding genetic markers on the same linkage group where a
specified 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, e.g., contigs). A physical map of the
genome does not take into account the genetic behavior (e.g.,
recombination frequencies) between different points on the physical
map.
[0083] A "genetic recombination frequency" is the frequency of a
crossing over event (recombination) between two genetic loci.
Recombination frequency can be observed by following the
segregation of markers and/or traits following meiosis. A genetic
recombination frequency can be expressed in centimorgans (cM),
where one cM is the distance between two genetic markers that show
a 1% recombination frequency (i.e., a crossing-over event occurs
between those two markers once in every 100 cell divisions).
[0084] As used herein, the term "linkage" is used to describe the
degree with which one marker locus is "associated with" another
marker locus or some other locus (for example, a tolerance
locus).
[0085] As used herein, linkage equilibrium describes a situation
where two markers independently segregate, i.e., sort among progeny
randomly. Markers that show linkage equilibrium are considered
unlinked (whether or not they lie on the same chromosome).
[0086] As used herein, linkage disequilibrium describes a situation
where two markers segregate in a non-random manner, i.e., have a
recombination frequency of less than 50% (and by definition, are
separated by less than 50 cM on the same linkage group). Markers
that show linkage disequilibrium are considered linked. Linkage
occurs when the marker locus and a linked locus are found together
in progeny plants more frequently than not together in the progeny
plants. As used herein, linkage can be between two markers, or
alternatively between a marker and a phenotype. A marker locus can
be associated with (linked to) a trait, e.g., a marker locus can be
associated with tolerance or improved tolerance to a plant pathogen
when the marker locus is in linkage disequilibrium with the
tolerance trait. The degree of linkage of a molecular marker to a
phenotypic trait (e.g., a QTL) is measured, e.g., as a statistical
probability of co-segregation of that molecular marker with the
phenotype.
[0087] As used herein, the linkage relationship between a molecular
marker and a phenotype is given as a "probability" or "adjusted
probability." The probability value is the statistical likelihood
that the particular combination of a phenotype and the presence or
absence of a particular marker allele is random. Thus, the lower
the probability score, the greater the likelihood that a phenotype
and a particular marker will co-segregate. In some aspects, the
probability score is considered "significant" or "nonsignificant."
In some embodiments, a probability score of 0.05 (p=0.05, or a 5%
probability) of random assortment is considered a significant
indication of co-segregation. However, the present invention is not
limited to this particular standard, and an acceptable probability
can be any probability of less than 50% (p=0.5). For example, a
significant probability can be less than 0.25, less than 0.20, less
than 0.15, or less than 0.1.
[0088] The term "linkage disequilibrium" refers to a non-random
segregation of genetic loci or traits (or both). In either case,
linkage disequilibrium implies that the relevant loci are within
sufficient physical proximity along a length of a chromosome so
that they segregate together with greater than random (i.e.,
non-random) frequency (in the case of co-segregating traits, the
loci that underlie the traits are in sufficient proximity to each
other). Linked loci co-segregate more than 50% of the time, e.g.,
from about 51% to about 100% of the time. The term "physically
linked" is sometimes used to indicate that two loci, e.g., two
marker loci, are physically present on the same chromosome.
[0089] Advantageously, the two linked loci are located in close
proximity such that recombination between homologous chromosome
pairs does not occur between the two loci during meiosis with high
frequency, e.g., such that linked loci co-segregate at least about
90% of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%, 99.75%, or more of the time.
[0090] The phrase "closely linked," in the present application,
means that recombination between two linked loci occurs with a
frequency of equal to or less than about 10% (i.e., are separated
on a genetic map by not more than 10 cM). Put another way, the
closely linked loci co-segregate at least 90% of the time. Marker
loci are especially useful in the present invention when they
demonstrate a significant probability of co-segregation (linkage)
with a desired trait (e.g., pathogenic tolerance). For example, in
some aspects, these markers can be termed linked QTL markers. In
other aspects, especially useful molecular markers are those
markers that are linked or closely linked to QTL markers.
[0091] In some aspects, linkage can be expressed as any desired
limit or range. For example, in some embodiments, two linked loci
are two loci that are separated by less than 50 cM map units. In
other embodiments, linked loci are two loci that are separated by
less than 40 cM. In other embodiments, two linked loci are two loci
that are separated by less than 30 cM. In other embodiments, two
linked loci are two loci that are separated by less than 25 cM. In
other embodiments, two linked loci are two loci that are separated
by less than 20 cM. In other embodiments, two linked loci are two
loci that are separated by less than 15 cM. In some aspects, it is
advantageous to define a bracketed range of linkage, for example,
between 10 and 20 cM, or between 10 and 30 cM, or between 10 and 40
cM.
[0092] The more closely a marker is linked to a second locus, the
better an indicator for the second locus that marker becomes. Thus,
in one embodiment, closely linked loci such as a marker locus and a
second locus (e.g., a QTL marker) display an inter-locus
recombination frequency of 10% or less, preferably about 9% or
less, still more preferably about 8% or less, yet more preferably
about 7% or less, still more preferably about 6% or less, yet more
preferably about 5% or less, still more preferably about 4% or
less, yet more preferably about 3% or less, and still more
preferably about 2% or less. In highly preferred embodiments, the
relevant loci (e.g., a marker locus and a QTL marker) display a
recombination a frequency of about 1% or less, e.g., about 0.75% or
less, more preferably about 0.5% or less, or yet more preferably
about 0.25% or less. Two loci that are localized to the same
chromosome, and at such a distance that recombination between the
two loci occurs at a frequency of less than 10% (e.g., about 9%,
8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are
also said to be "proximal to" each other. In some cases, two
different markers can have the same genetic map coordinates. In
that case, the two markers are in such close proximity to each
other that recombination occurs between them with such low
frequency that it is undetectable.
[0093] When referring to the relationship between two genetic
elements, such as a genetic element contributing to tolerance and a
proximal marker, "coupling" phase linkage indicates the state where
the "favorable" allele at the tolerance locus is physically
associated on the same chromosome strand as the "favorable" allele
of the respective linked marker locus. In coupling phase, both
favorable alleles are inherited together by progeny that inherit
that chromosome strand. In "repulsion" phase linkage, the
"favorable" allele at the locus of interest (e.g., a QTL for
tolerance) is physically linked with an "unfavorable" allele at the
proximal marker locus, and the two "favorable" alleles are not
inherited together (i.e., the two loci are "out of phase" with each
other).
[0094] As used herein, the terms "chromosome interval" or
"chromosome segment" designate a contiguous linear span of genomic
DNA that resides in planta on a single chromosome. The genetic
elements or genes located on a single chromosome interval are
physically linked The size of a chromosome interval is not
particularly limited.
[0095] In some aspects, for example in the context of the present
invention, generally the genetic elements located within a single
chromosome interval are also genetically linked, typically within a
genetic recombination distance of; for example, less than or equal
to 20 centimorgan (cM), or alternatively, less than or equal to 10
cM. That is, two genetic elements within a single chromosome
interval undergo recombination at a frequency of less than or equal
to 20% or 10%
[0096] In one aspect, any marker of the invention is linked
(genetically and physically) to any other marker that is at or less
than 50 cM distant. In another aspect, any marker of the invention
is closely linked (genetically and physically) to any other marker
that is in close proximity, e.g., at or less than 10 cM distant.
Two closely linked markers on the same chromosome can be positioned
9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each
other.
[0097] The phrases "low iron," "low-available iron," "low soluble
iron," "low iron conditions," "low iron growth conditions," "iron
shortage" or "iron deficiency" or the like refer to conditions
where iron availability is less than optimal for soybean growth,
and can cause plant pathology, e.g., IDC, due to the lack of
metabolically-available iron. It is recognized that under "iron
deficient" conditions, the absolute concentration of atomic iron
may be sufficient, but the form of the iron (e.g., its
incorporation into various molecular structures) and other
environmental factors may make the iron unavailable for plant use.
For example, high carbonate levels. High pH, high salt content,
herbicide applications, cool temperatures saturated soils or other
environmental factors can decrease iron solubility, and reduce the
solubilized forms of iron that the plant requires for uptake. One
of skill in the art is familiar with assays to measure iron content
of soil, as well as those concentrations of iron that are optimal
or sub-optimal for plant growth.
[0098] "Tolerance" or "improved tolerance" in a soybean plant to
low-available iron growth conditions is an indication that the
soybean plant is less affected by low-available iron conditions
with respect to yield, survivability and/or other relevant
agronomic measures, compared to a less tolerant, more "susceptible"
plant. Tolerance is a relative term, indicating that a "tolerant"
plant survives and/or produces better yield of soybean in
low-available iron growth conditions compared to a different (less
tolerant) plant (e.g., a different soybean strain) grown in similar
low-available iron conditions. That is, the low-available iron
growth conditions cause a reduced decrease in soybean survival
and/or yield in a tolerant soybean plant, as compared to a
susceptible soybean plant. As used in the art, iron-deficiency
"tolerance" is sometimes used interchangeably with iron-deficiency
"resistance."
[0099] One of skill will appreciate that soybean plant tolerance to
low-available iron conditions varies widely, and can represent a
spectrum of more-tolerant or less-tolerant phenotypes. However, by
simple observation, one of skill can generally determine the
relative tolerance or susceptibility of different plants, plant
lines or plant families under low-available iron conditions, and
furthermore, will also recognize the phenotypic gradations of
"tolerant."
[0100] In one example, a plant's tolerance can be approximately
quantitated using a chlorosis scoring system. In such a system, a
plant that is grown in a known iron-deficient area, or in
low-available iron experimental conditions, and is assigned a
tolerance rating of between 1 (highly susceptible; most or all
plants dead; those that live are stunted and have little living
tissue) to 9 (highly tolerant; yield and survivability not
significantly affected; all plants normal green color). See also,
Dahiya and Singh (1979) "Effect of salinity, alkalinity and iron
sources on availability of iron," Plant and Soil 51:13-18.
[0101] The term "crossed" or "cross" in the context of this
invention means the fusion of gametes via pollination to produce
progeny (e.g., cells, seeds or plants). The term encompasses both
sexual crosses (the pollination of one plant by another) and
selfing (self-pollination, e.g., when the pollen and ovule are from
the same plant).
[0102] The term "introgression" refers to the transmission of a
desired allele of a genetic locus from one genetic background to
another. For example, introgression of a desired allele at a
specified locus can be transmitted 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 desired allele in its genome.
Alternatively, for example, transmission of an allele can occur by
recombination between two donor genomes, e.g., in a fused
protoplast, where at least one of the donor protoplasts has the
desired allele in its genome. The desired allele can be, e.g., a
selected allele of a marker, a QTL, a transgene, or the like. In
any case, offspring comprising the desired allele can be repeatedly
backcrossed to a line having a desired genetic background and
selected for the desired allele, to result in the allele becoming
fixed in a selected genetic background.
[0103] A "line" or "strain" is a group of individuals of identical
parentage that are generally inbred to some degree and that are
generally homozygous and homogeneous at most loci (isogenic or near
isogenic). A "subline" refers to an inbred subset of descendents
that are genetically distinct from other similarly inbred subsets
descended from the same progenitor. Traditionally, a "subline" has
been derived by inbreeding the seed from an individual soybean
plant selected at the F3 to F5 generation until the residual
segregating loci are "fixed" or homozygous across most or all loci.
Commercial soybean varieties (or lines) are typically produced by
aggregating ("bulking") the self-pollinated progeny of a single F3
to F5 plant from a controlled cross between 2 genetically different
parents. While the variety typically appears uniform, the
self-pollinating variety derived from the selected plant eventually
(e.g., F8) becomes a mixture of homozygous plants that can vary in
genotype at any locus that was heterozygous in the originally
selected F3 to F5 plant. In the context of the invention,
marker-based sublines, that differ from each other based on
qualitative polymorphism at the DNA level at one or more specific
marker loci, are derived by genotyping a sample of seed derived
from individual self-pollinated progeny derived from a selected
F3-F5 plant. The seed sample can be genotyped directly as seed, or
as plant tissue grown from such a seed sample. Optionally, seed
sharing a common genotype at the specified locus (or loci) are
bulked providing a subline that is genetically homogenous at
identified loci important for a trait of interest (yield,
tolerance, etc.).
[0104] An "ancestral line" is a parent line used as a source of
genes e.g., for the development of elite lines. An "ancestral
population" is a group of ancestors that have contributed the bulk
of the genetic variation that was used to develop elite lines.
"Descendants" are the progeny of ancestors, and may be separated
from their ancestors by many generations of breeding. For example,
elite lines are the descendants of their ancestors. A "pedigree
structure" defines the relationship between a descendant and each
ancestor that gave rise to that descendant. A pedigree structure
can span one or more generations, describing relationships between
the descendant and it's parents, grand parents, great-grand
parents, etc.
[0105] An "elite line" or "elite strain" is an agronomically
superior line that has resulted from many cycles of breeding and
selection for superior agronomic performance. Numerous elite lines
are available and known to those of skill in the art of soybean
breeding. An "elite population" is an assortment of elite
individuals or lines that can be used to represent the state of the
art in terms of agronomically superior genotypes of a given crop
species, such as soybean. Similarly, an "elite germplasm" or elite
strain of germplasm is an agronomically superior germplasm,
typically derived from and/or capable of giving rise to a plant
with superior agronomic performance, such as an existing or newly
developed elite line of soybean.
[0106] In contrast, an "exotic soybean strain" or an "exotic
soybean germplasm" is a strain or germplasm derived from a soybean
not belonging to an available elite soybean line or strain of
germplasm. In the context of a cross between two soybean plants or
strains of germplasm, an exotic germplasm is not closely related by
descent to the elite germplasm with which it is crossed. Most
commonly, the exotic germplasm is not derived from any known elite
line of soybean, but rather is selected to introduce novel genetic
elements (typically novel alleles) into a breeding program.
[0107] The term "amplifying" in the context of nucleic acid
amplification is any process whereby additional copies of a
selected nucleic acid (or a transcribed form thereof) are produced.
Typical amplification methods include various polymerase based
replication methods, including the polymerase chain reaction (PCR),
ligase mediated methods such as the ligase chain reaction (LCR) and
RNA polymerase based amplification (e.g., by transcription)
methods. An "amplicon" is an amplified nucleic acid, e.g., a
nucleic acid that is produced by amplifying a template nucleic acid
by any available amplification method (e.g., PCR, LCR,
transcription, or the like).
[0108] A "genomic nucleic acid" is a nucleic acid that corresponds
in sequence to a heritable nucleic acid in a cell. Common examples
include nuclear genomic DNA and amplicons thereof. A genomic
nucleic acid is, in some cases, different from a spliced RNA, or a
corresponding cDNA, in that the spliced RNA or cDNA is processed,
e.g., by the splicing machinery, to remove introns. Genomic nucleic
acids optionally comprise non-transcribed (e.g., chromosome
structural sequences, promoter regions, enhancer regions, etc.)
and/or non-translated sequences (e.g., introns), whereas spliced
RNA/cDNA typically do not have non-transcribed sequences or
introns. A "template nucleic acid" is a nucleic acid that serves as
a template in an amplification reaction (e.g., a polymerase based
amplification reaction such as PCR, a ligase mediated amplification
reaction such as LCR, a transcription reaction, or the like). A
template nucleic acid can be genomic in origin, or alternatively,
can be derived from expressed sequences, e.g., a cDNA or an
EST.
[0109] An "exogenous nucleic acid" is a nucleic acid that is not
native to a specified system (e.g., a germplasm, plant, variety,
etc.), with respect to sequence, genomic position, or both. As used
herein, the terms "exogenous" or "heterologous" as applied to
polynucleotides or polypeptides typically refers to molecules that
have been artificially supplied to a biological system (e.g., a
plant cell, a plant gene, a particular plant species or variety or
a plant chromosome under study) and are not native to that
particular biological system. The terms can indicate that the
relevant material originated from a source other than a naturally
occurring source, or can refer to molecules having a non-natural
configuration, genetic location or arrangement of parts.
[0110] In contrast, for example, a "native" or "endogenous" gene is
a gene that does not contain nucleic acid elements encoded by
sources other than the chromosome or other genetic element on which
it is normally found in nature. An endogenous gene, transcript or
polypeptide is encoded by its natural chromosomal locus, and not
artificially supplied to the cell.
[0111] The term "recombinant" in reference to a nucleic acid or
polypeptide indicates that the material (e.g., a recombinant
nucleic acid, gene, polynucleotide, polypeptide, etc.) has been
altered by human intervention. Generally, the arrangement of parts
of a recombinant molecule is not a native configuration, or the
primary sequence of the recombinant polynucleotide or polypeptide
has in some way been manipulated. The alteration to yield the
recombinant material can be performed on the material within or
removed from its natural environment or state. For example, a
naturally occurring nucleic acid becomes a recombinant nucleic acid
if it is altered, or if it is transcribed from DNA which has been
altered, by means of human intervention performed within the cell
from which it originates. A gene sequence open reading frame is
recombinant if that nucleotide sequence has been removed from it
natural context and cloned into any type of artificial nucleic acid
vector. Protocols and reagents to produce recombinant molecules,
especially recombinant nucleic acids, are common and routine in the
art. The term recombinant can also refer to an organism that
harbors recombinant material, e.g., a plant that comprises a
recombinant nucleic acid is considered a recombinant plant. In some
embodiments, a recombinant organism is a transgenic organism.
[0112] The term "introduced" when referring to translocating a
heterologous or exogenous nucleic acid into a cell refers to the
incorporation of the nucleic acid into the cell using any
methodology. The term encompasses such nucleic acid introduction
methods as "transfection," "transformation" and "transduction."
[0113] As used herein, the term "vector" is used in reference to
polynucleotide or other molecules that transfer nucleic acid
segment(s) into a cell. The term "vehicle" is sometimes used
interchangeably with "vector." A vector optionally comprises parts
which mediate vector maintenance and enable its intended use (e.g.,
sequences necessary for replication, genes imparting drug or
antibiotic resistance, a multiple cloning site, operably linked
promoter/enhancer elements which enable the expression of a cloned
gene, etc.). Vectors are often derived from plasmids,
bacteriophages, or plant or animal viruses. A "cloning vector" or
"shuttle vector" or "subcloning vector" contains operably linked
parts that facilitate subcloning steps (e.g., a multiple cloning
site containing multiple restriction endonuclease sites).
[0114] The term "expression vector" as used herein refers to a
vector comprising operably linked polynucleotide sequences that
facilitate expression of a coding sequence in a particular host
organism (e.g., a bacterial expression vector or a plant expression
vector). Polynucleotide sequences that facilitate expression in
prokaryotes typically include, e.g., a promoter, an operator
(optional), and a ribosome binding site, often along with other
sequences. Eukaryotic cells can use promoters, enhancers,
termination and polyadenylation signals and other sequences that
are generally different from those used by prokaryotes.
[0115] The term "transgenic plant" refers to a plant that comprises
within its cells a heterologous polynucleotide. Generally, the
heterologous polynucleotide is stably integrated within the genome
such that the polynucleotide is passed on to successive
generations. The heterologous polynucleotide may be integrated into
the genome alone or as part of a recombinant expression cassette.
"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 heterologous nucleic acid including
those transgenic organisms or cells initially so altered, as well
as those created by crosses or asexual propagation from the initial
transgenic organism or cell. The term "transgenic" as used herein
does not encompass the alteration of the genome (chromosomal or
extra-chromosomal) by conventional plant breeding methods (e.g.,
crosses) or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0116] "Positional cloning" is a cloning procedure in which a
target nucleic acid is identified and isolated by its genomic
proximity to marker nucleic acid. For example, a genomic nucleic
acid clone can include part or all 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 or sequencing can be used to
identify and or isolate subsequences of the clone that are located
near the marker.
[0117] A specified nucleic acid is "derived from" a given nucleic
acid when it is constructed using the given nucleic acid's
sequence, or when the specified nucleic acid is constructed using
the given nucleic acid. For example, a cDNA or EST is derived from
an expressed mRNA.
[0118] The term "genetic element" or "gene" refers to a heritable
sequence of DNA, i.e., a genomic sequence, with functional
significance. The term "gene" can also be used to refer to, e.g., a
cDNA and/or a mRNA encoded by a genomic sequence, as well as to
that genomic sequence.
[0119] The term "genotype" is the genetic constitution of an
individual (or group of individuals) at one or more genetic loci,
as contrasted with the observable trait (the phenotype). Genotype
is defined by the allele(s) of one or more known loci that the
individual has inherited from its parents. The term genotype can be
used to refer to an individual's genetic constitution at a single
locus, at multiple loci, or, more generally, the term genotype can
be used to refer to an individual's genetic make-up for all the
genes in its genome. A "haplotype" is the genotype of an individual
at a plurality of genetic loci. Typically, the genetic loci
described by a haplotype are physically and genetically linked,
i.e., on the same chromosome segment.
[0120] The terms "phenotype," or "phenotypic trait" or "trait"
refers to one or more trait of an organism. The phenotype can be
observable to the naked eye, or by any other means of evaluation
known in the art, e.g., microscopy, biochemical analysis, genomic
analysis, an assay for a particular disease resistance, etc. In
some cases, a phenotype is directly controlled by a single gene or
genetic locus, i.e., a "single gene trait." In other cases, a
phenotype is the result of several genes. A "quantitative trait
loci" (QTL) is a genetic domain that is polymorphic and effects a
phenotype that can be described in quantitative terms, e.g.,
height, weight, oil content, days to germination, disease
resistance, etc, and, therefore, can be assigned a "phenotypic
value" which corresponds to a quantitative value for the phenotypic
trait. A QTL can act through a single gene mechanism or by a
polygenic mechanism.
[0121] A "molecular phenotype" is a phenotype detectable at the
level of a population of (one or more) molecules. Such molecules
can be nucleic acids such as genomic DNA or RNA, proteins, or
metabolites. For example, a molecular phenotype can be an
expression profile for one or more gene products, e.g., at a
specific stage of plant development, in response to an
environmental condition or stress, etc. Expression profiles are
typically evaluated at the level of RNA or protein, e.g., on a
nucleic acid array or "chip" or using antibodies or other binding
proteins.
[0122] The term "yield" refers to the productivity per unit area of
a particular plant product of commercial value. For example, yield
of soybean is commonly measured in bushels of seed per acre or
metric tons of seed per hectare per season. Yield is affected by
both genetic and environmental factors. "Agronomics," "agronomic
traits," and "agronomic performance" refer to the traits (and
underlying genetic elements) of a given plant variety that
contribute to yield over the course of growing season. Individual
agronomic traits include emergence vigor, vegetative vigor, stress
tolerance, disease resistance or tolerance, herbicide resistance,
branching, flowering, seed set, seed size, seed density,
standability, threshability and the like. Yield is, therefore, the
final culmination of all agronomic traits.
[0123] A "set" of markers or probes refers to a collection or group
of markers or probes, or the data derived therefrom, used for a
common purpose, e.g., identifying soybean plants with a desired
trait (e.g., tolerance to Phytophthora infection). Frequently, data
corresponding to the markers or probes, or data derived from their
use, is stored in an electronic medium. While each of the members
of a set possess utility with respect to the specified purpose,
individual markers selected from the set as well as subsets
including some, but not all of the markers, are also effective in
achieving the specified purpose.
[0124] A "look up table" is a table that correlates one form of
data to another, or one or more forms of data with a predicted
outcome that the data is relevant to. For example, a look up table
can include a correlation between allele data and a predicted trait
that a plant comprising a given allele is likely to display. These
tables can be, and typically are, multidimensional, e.g., taking
multiple alleles into account simultaneously, and, optionally,
taking other factors into account as well, such as genetic
background, e.g., in making a trait prediction.
[0125] A "computer readable medium" is an information storage media
that can be accessed by a computer using an available or custom
interface. Examples include memory (e.g., ROM or RAM, flash memory,
etc.), optical storage media (e.g., CD-ROM), magnetic storage media
(computer hard drives, floppy disks, etc.), punch cards, and many
others that are commercially available. Information can be
transmitted between a system of interest and the computer, or to or
from the computer to or from the computer readable medium for
storage or access of stored information. This transmission can be
an electrical transmission, or can be made by other available
methods, such as an IR link, a wireless connection, or the
like.
[0126] "System instructions" are instruction sets that can be
partially or fully executed by the system. Typically, the
instruction sets are present as system software.
BRIEF DESCRIPTION OF THE FIGURES
[0127] FIG. 1 provides a table listing soybean markers
demonstrating linkage disequilibrium with low available iron
tolerance phenotype as determined by intergroup allele frequency
distribution analysis, association mapping analysis and QTL
interval mapping (including marker regression analysis) methods.
The table indicates the marker type SSR (simple sequence repeat;
genomic or EST) or SNP (single nucleotide polymorphism), the
chromosome on which the marker is located and its approximate
genetic map position relative to other known markers, given in cM,
with position zero being the first (most distal) marker on the
chromosome, as provided in the integrated genetic map in FIG. 6.
Also shown are the soybean populations used in the analysis and the
statistical probability of random segregation of the marker and the
tolerance phenotype given as an adjusted probability taking into
account the variability and false positives of multiple tests.
Results from QTL interval mapping are provided, with the
significance values given as a likelihood ratio statistic
(LRS).
[0128] FIG. 2 provides a table listing the genomic and EST SSR
markers that demonstrated linkage disequilibrium with the low iron
tolerance phenotype and the sequences of the left and right PCR
primers used in the SSR marker locus genotyping analysis. Also
shown is the pigtail sequence used on the 5' end of the right
primer, and the number of nucleotides in the tandem repeating
element in the SSR.
[0129] FIG. 3 provides a table listing the SNP markers that
demonstrated linkage disequilibrium with the low iron tolerance
phenotype. The table provides the sequences of the PCR primers used
to generate a SNP-containing amplicon, and the allele-specific
probes that were used to identify the SNP allele in an
allele-specific hybridization assay (ASH assay).
[0130] FIG. 4 provides an allele dictionary of the characterized
alleles of the SSR markers that demonstrated linkage disequilibrium
with the low iron tolerance phenotype. Each allele is defined by
the size of a PCR amplicon generated from soybean genomic DNA or
mRNA using the primers listed in FIG. 2. Sizes of the PCR amplicons
are indicated in base pairs (bp).
[0131] FIG. 5 provides a table listing genetic markers that are
closely linked to the low iron tolerance markers identified by the
present invention.
[0132] FIG. 6 provides an integrated genetic map for approximately
750 soybean markers, including both SSR-type and SNP-type markers.
These markers are distributed over each soybean chromosome. The
chromosome number, as well as the equivalent historical chromosome
name are indicated. The genetic map positions of the markers are
indicated in centiMorgans (cM), typically with position zero being
the first (most distal) marker on the chromosome.
DETAILED DESCRIPTION
[0133] Iron deficiency chlorosis (IDC or FEC) is a soybean disease
causing severe losses in viability and reductions in yield. The
disease is caused by poor iron availability in soil, and is
strongly influenced by environmental factors that control iron
availability (e.g., environmental factors that reduce iron
solubility result in reduced iron availability in the soil). Yield
losses can be minimized by the field application of iron-rich
fertilizers such as livestock manure or making foliar applications
of iron-containing materials. However, one of the most effective
and most environmentally friendly approaches to overcoming this
disease is through the selection of soybean varieties that are
tolerant to the iron-deficient growth conditions.
[0134] The identification and selection of soybean plants that show
tolerance to iron-deficient growth conditions using MAS can provide
an effective and environmentally friendly approach to overcoming
losses caused by this disease. The present invention provides
soybean marker loci that demonstrate statistically significant
co-segregation with tolerance to iron-deficient growth conditions.
Detection of these loci or additional linked loci can be used in
marker assisted soybean breeding programs to produce tolerant
plants, or plants with improved tolerance. The linked SSR and SNP
markers identified herein are provided in FIG. 1. These markers
include S60210-1B, SAC1006, SATT391, SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT510, SATT335, P5219A-1, P7659A-2,
SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367, SATT495,
P10649C-3, SATT613, SATT257, SATT581 and SATT153.
[0135] Each of the SSR-type markers display a plurality of alleles
that can be visualized as different sized PCR amplicons, as
summarized in the SSR allele dictionary in FIG. 4. The PCR primers
that are used to generate the SSR-marker amplicons are provided in
FIG. 2. The alleles of SNP-type markers are determined using an
allele-specific hybridization protocol, as known in the art. The
PCR primers used to amplify the SNP domain, and the allele-specific
probes used to genotype the locus are provided in FIG. 3.
[0136] As recognized in the art, any other marker that is linked to
a QTL marker (e.g., a disease tolerance marker) also finds use for
that same purpose. Examples of additional markers that are linked
to the disease tolerance markers recited herein are provided. For
example, a linked marker can be determined from the soybean
consensus genetic map provided in FIG. 6. Additional closely linked
markers are further provided in FIG. 5. It is not intended,
however, that linked markers finding use with the invention be
limited to those recited in FIG. 5 or 6.
[0137] The invention also provides chromosomal QTL intervals that
correlate with tolerance to low-iron conditions. Any marker located
within these intervals finds use as a marker for iron-deficiency
tolerance. These intervals include:
[0138] (a) S60210-TB and SATT391 (LG-C1);
[0139] (b) P10598A-1 and SATT334 (LG-F);
[0140] (c) SATT510 and SATT335 (LG-F);
[0141] (d) P5219A-1 and P7659A-2 (LG-G);
[0142] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0143] (f) SATT451 and SATT367 (LG-I);
[0144] (g) SATT495 and P10649C-3 (LG-L); and
[0145] (h) SATT250 and SATT346 (LG-M).
[0146] Methods for identifying soybean plants or germplasm that
carry preferred alleles of tolerance marker loci are a feature of
the invention. In these methods, any of a variety of marker
detection protocols are used to identify marker loci, depending on
the type of marker loci. Typical methods for marker detection
include amplification and detection of the resulting amplified
markers, e.g., by PCR, LCR, transcription based amplification
methods, or the like. These include ASH, SSR detection, RFLP
analysis and many others.
[0147] Although particular marker alleles can show co-segregation
with a disease tolerance or susceptibility phenotype, it is
important to note that the marker locus is not necessarily part of
the QTL locus responsible for the tolerance or susceptibility. For
example, it is not a requirement that the marker polynucleotide
sequence be part of a gene that imparts disease resistance (for
example, be part of the gene open reading frame). The association
between a specific marker allele with the tolerance or
susceptibility phenotype is due to the original "coupling" linkage
phase between the marker allele and the QTL tolerance or
susceptibility allele in the ancestral soybean line from which the
tolerance or susceptibility allele originated. Eventually, with
repeated recombination, crossing over events between the marker and
QTL locus can change this orientation. For this reason, the
favorable marker allele may change depending on the linkage phase
that exists within the tolerant parent used to create segregating
populations. This does not change the fact the genetic marker can
be used to monitor segregation of the phenotype. It only changes
which marker allele is considered favorable in a given segregating
population.
[0148] Identification of soybean plants or germplasm that include a
marker locus or marker loci linked to a tolerance trait or traits
provides a basis for performing marker assisted selection of
soybean. Soybean plants that comprise favorable markers or
favorable alleles are selected for, while soybean plants that
comprise markers or alleles that are negatively correlated with
tolerance can be selected against. Desired markers and/or alleles
can be introgressed into soybean having a desired (e.g., elite or
exotic) genetic background to produce an introgressed tolerant
soybean plant or germplasm. In some aspects, it is contemplated
that a plurality of tolerance markers are sequentially or
simultaneous selected and/or introgressed. The combinations of
tolerance markers that are selected for in a single plant is not
limited, and can include any combination of markers recited in FIG.
1, any markers linked to the markers recited in FIG. 1, or any
markers located within the QTL intervals defined herein.
[0149] As an alternative to standard breeding methods of
introducing traits of interest into soybean (e.g., introgression),
transgenic approaches can also be used. In these methods, exogenous
nucleic acids that encode traits linked to markers are introduced
into target plants or germplasm. For example, a nucleic acid that
codes for a tolerance trait is cloned, e.g., via positional cloning
and introduced into a target plant or germplasm.
[0150] Verification of iron-deficiency tolerance can be performed
by available tolerance assay protocols, as known in the art and
discussed in more detail below. For example, see Dahiya and Singh
(1979) "Effect of salinity, alkalinity and iron sources on
availability of iron," Plant and Soil 51:13-18. Tolerance assays
are useful to verify that the tolerance trait still segregates with
the marker in any particular plant or population, and, of course,
to measure the degree of tolerance improvement achieved by
introgressing or recombinantly introducing the trait into a desired
background.
[0151] Systems, including automated systems for selecting plants
that comprise a marker of interest and/or for correlating presence
of the marker with tolerance are also a feature of the invention.
These systems can include probes relevant to marker locus
detection, detectors for detecting labels on the probes,
appropriate fluid handling elements and temperature controllers
that mix probes and templates and/or amplify templates, and systems
instructions that correlate label detection to the presence of a
particular marker locus or allele.
[0152] Kits are also a feature of the invention. For example, a kit
can include appropriate primers or probes for detecting tolerance
associated marker loci and instructions in using the primers or
probes for detecting the marker loci and correlating the loci with
predicted low iron tolerance. The kits can further include
packaging materials for packaging the probes, primers or
instructions, controls such as control amplification reactions that
include probes, primers or template nucleic acids for
amplifications, molecular size markers, or the like.
Tolerance Markers and Favorable Alleles
[0153] In traditional linkage analysis, no direct knowledge of the
physical relationship of genes on a chromosome is required.
Mendel's first law is that factors of pairs of characters are
segregated, meaning that alleles of a diploid trait separate into
two gametes and then into different offspring. Classical linkage
analysis can be thought of as a statistical description of the
relative frequencies of cosegregation of different traits. Linkage
analysis is the well characterized descriptive framework of how
traits are grouped together based upon the frequency with which
they segregate together. That is, if two non-allelic traits are
inherited together with a greater than random frequency, they are
said to be "linked." The frequency with which the traits are
inherited together is the primary measure of how tightly the traits
are linked, i.e., traits which are inherited together with a higher
frequency are more closely linked than traits which are inherited
together with lower (but still above random) frequency. Traits are
linked because the genes which underlie the traits reside on the
same chromosome. The further apart on a chromosome the genes
reside, the less likely they are to segregate together, because
homologous chromosomes recombine during meiosis. Thus, the further
apart on a chromosome the genes reside, the more likely it is that
there will be a crossing over event during meiosis that will result
in two genes segregating separately into progeny.
[0154] A common measure of linkage is the frequency with which
traits cosegregate. This can be expressed as a percentage of
cosegregation (recombination frequency) or, also commonly, in
centiMorgans (cM). The cM is named after the pioneering geneticist
Thomas Hunt Morgan and is a unit of measure of genetic
recombination frequency. One cM is equal to a 1% chance that a
trait at one genetic locus will be separated from a trait at
another locus due to crossing over in a single generation (meaning
the traits segregate together 99% of the time). Because chromosomal
distance is approximately proportional to the frequency of crossing
over events between traits, there is an approximate physical
distance that correlates with recombination frequency. For example,
in soybean, 1 cM correlates, on average, to about 400,000 base
pairs (400 Kb).
[0155] Marker loci are themselves traits and can be assessed
according to standard linkage analysis by tracking the marker loci
during segregation. Thus, in the context of the present invention,
one cM is equal to a 1% chance that a marker locus will be
separated from another locus (which can be any other trait, e.g.,
another marker locus, or another trait locus that encodes a QTL),
due to crossing over in a single generation. The markers herein, as
described in FIG. 1, e.g., S60210-TB, SAC1006, SATT391, SAC1724,
SATT307, P13073A-1, P10598A-1, SATT334, SATT510, SATT335, P5219A-1,
P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367,
SATT495, P10649C-3, SATT613, SATT257, SATT581 and SATT153, as well
as any of the chromosome intervals
[0156] (a) S60210-TB and SATT391 (LG-C1);
[0157] (b) P10598A-1 and SATT334 (LG-F);
[0158] (c) SATT510 and SATT335 (LG-F);
[0159] (d) P5219A-1 and P7659A-2 (LG-G);
[0160] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0161] (f) SATT451 and SATT367 (LG-I);
[0162] (g) SATT495 and P10649C-3 (LG-L); and
[0163] (h) SATT250 and SATT346 (LG-M),
have been found to correlate with tolerance, improved tolerance or
susceptibility to low iron growth conditions in soybean. This means
that the markers are sufficiently proximal to a tolerance QTL that
they can be used as a predictor for the tolerance trait. This is
extremely useful in the context of marker assisted selection (MAS),
discussed in more detail herein. In brief, soybean plants or
germplasm can be selected for markers or marker alleles that
positively correlate with tolerance, without actually raising
soybean and measuring for tolerance or improved tolerance (or,
contrawise, soybean plants can be selected against if they possess
markers that negatively correlate with tolerance or improved
tolerance). MAS is a powerful shortcut to selecting for desired
phenotypes and for introgressing desired traits into cultivars of
soybean (e.g., introgressing desired traits into elite lines). MAS
is easily adapted to high throughput molecular analysis methods
that can quickly screen large numbers of plant or germplasm genetic
material for the markers of interest and is much more cost
effective than raising and observing plants for visible traits.
[0164] In some embodiments, the most preferred QTL markers are a
subset of the markers provided in FIG. 1. For example, the most
preferred markers can be selected from SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT495, P10649C-3, SATT613 and SATT257.
[0165] When referring to the relationship between two genetic
elements, such as a genetic element contributing to tolerance and a
proximal marker, "coupling" phase linkage indicates the state where
the "favorable" allele at the tolerance locus is physically
associated on the same chromosome strand as the "favorable" allele
of the respective linked marker locus. In coupling phase, both
favorable alleles are inherited together by progeny that inherit
that chromosome strand. In "repulsion" phase linkage, the
"favorable" allele at the locus of interest (e.g., a QTL for
tolerance) is physically linked with an "unfavorable" allele at the
proximal marker locus, and the two "favorable" alleles are not
inherited together (i.e., the two loci are "out of phase" with each
other).
[0166] A favorable allele of a marker is that allele of the marker
that co-segregates with a desired phenotype (e.g., disease
tolerance). As used herein, a QTL marker has a minimum of one
favorable allele, although it is possible that the marker might
have two or more favorable alleles found in the population. Any
favorable allele of that marker can be used advantageously for the
identification and construction of tolerant soybean lines.
Optionally, one, two, three or more favorable allele(s) of
different markers are identified in, or introgressed into a plant,
and can be selected for or against during MAS. Desirably, plants or
germplasm are identified that have at least one such favorable
allele that positively correlates with tolerance or improved
tolerance.
[0167] Alternatively, a marker allele that co-segregates with
disease susceptibility also finds use with the invention, since
that allele can be used to identify and counter select
disease-susceptible plants. Such an allele can be used for
exclusionary purposes during breeding to identify alleles that
negatively correlate with tolerance, to eliminate susceptible
plants or germplasm from subsequent rounds of breeding.
[0168] In some embodiments of the invention, a plurality of marker
alleles are simultaneously selected for in a single plant or a
population of plants. In these methods, plants are selected that
contain favorable alleles from more than one tolerance marker, or
alternatively, favorable alleles from more than one tolerance
marker are introgressed into a desired soybean germplasm. One of
skill in the art recognizes that the simultaneous selection of
favorable alleles from more than one disease tolerance marker in
the same plant is likely to result in an additive (or even
synergistic) protective effect for the plant.
[0169] One of skill recognizes that the identification of favorable
marker alleles is germplasm-specific. The determination of which
marker alleles correlate with tolerance (or susceptibility) is
determined for the particular germplasm under study. One of skill
recognizes that methods for identifying the favorable alleles are
routine and well known in the art, and furthermore, that the
identification and use of such favorable alleles is well within the
scope of the invention. Furthermore still, identification of
favorable marker alleles in soybean populations other than the
populations used or described herein is well within the scope of
the invention.
[0170] Amplification primers for amplifying SSR-type marker loci
are a feature of the invention. Another feature of the invention
are primers specific for the amplification of SNP domains (SNP
markers), and the probes that are used to genotype the SNP
sequences. FIGS. 2 and 3 provide specific primers for locus
amplification and probes for detecting amplified marker loci are
provided. However, one of skill will immediately recognize that
other sequences to either side of the given primers can be used in
place of the given primers, so long as the primers can amplify a
region that includes the allele to be detected. Further, it will be
appreciated that the precise probe to be used for detection can
vary, e.g., any probe that can identify the region of a marker
amplicon to be detected can be substituted for those examples
provided herein. Further, the configuration of the amplification
primers and detection probes can, of course, vary. Thus, the
invention is not limited to the primers and probes specifically
recited herein.
[0171] In some aspects, methods of the invention utilize an
amplification step to detect/genotype a marker locus. However, it
will be appreciated that amplification is not a requirement for
marker detection--for example, one can directly detect unamplified
genomic DNA simply by performing a Southern blot on a sample of
genomic DNA. Procedures for performing Southern blotting,
amplification (PCR, LCR, or the like) and many other nucleic acid
detection methods are well established and are taught, e.g., in
Sambrook et al., Molecular Cloning--A Laboratory Manual (3rd Ed.),
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
2000 ("Sambrook"); Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(supplemented through 2002) ("Ausubel")) and PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis) Additional details regarding
detection of nucleic acids in plants can also be found, e.g., in
Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific
Publishers, Inc.
[0172] Separate detection probes can also be omitted in
amplification/detection methods, e.g., by performing a real time
amplification reaction that detects product formation by
modification of the relevant amplification primer upon
incorporation into a product, incorporation of labeled nucleotides
into an amplicon, or by monitoring changes in molecular rotation
properties of amplicons as compared to unamplified precursors
(e.g., by fluorescence polarization).
[0173] Typically, molecular markers are detected by any established
method available in the art, including, without limitation, allele
specific hybridization (ASH) or other methods for detecting single
nucleotide polymorphisms (SNP), amplified fragment length
polymorphism (AFLP) detection, amplified variable sequence
detection, randomly amplified polymorphic DNA (RAPD) detection,
restriction fragment length polymorphism (RFLP) detection,
self-sustained sequence replication detection, simple sequence
repeat (SSR) detection, single-strand conformation polymorphisms
(SSCP) detection, isozyme markers detection, or the like. While the
exemplary markers provided in the figures and tables herein are
either SSR or SNP (ASH) markers, any of the aforementioned marker
types can be employed in the context of the invention to identify
chromosome segments encompassing genetic element that contribute to
superior agronomic performance (e.g., tolerance or improved
tolerance).
QTL Chromosome Intervals
[0174] In some aspects, the invention provides QTL chromosome
intervals, where a QTL (or multiple QTLs) that segregate with low
iron tolerance are contained in those intervals. A variety of
methods well known in the art are available for identifying
chromosome intervals (also as described in detail in EXAMPLE 3).
The boundaries of such chromosome intervals are drawn to encompass
markers that will be linked to one or more QTL. In other words, the
chromosome interval is drawn such that any marker that lies within
that interval (including the terminal markers that define the
boundaries of the interval) can be used as markers for disease
tolerance. Each interval comprises at least one QTL, and
furthermore, may indeed comprise more than one QTL. Close proximity
of multiple QTL in the same interval may obfuscate the correlation
of a particular marker with a particular QTL, as one marker may
demonstrate linkage to more than one QTL. Conversely, e.g., if two
markers in close proximity show co-segregation with the desired
phenotypic trait, it is sometimes unclear if each of those markers
identifying the same QTL or two different QTL. Regardless,
knowledge of how many QTL are in a particular interval is not
necessary to make or practice the invention.
[0175] The present invention provides soybean chromosome intervals,
where the markers within those intervals demonstrate co-segregation
with tolerance to low iron environmental conditions. Thus, each of
these intervals comprises at least one low iron tolerance QTL.
These intervals are:
TABLE-US-00001 Linkage Method(s) of Group Flanking Markers
Identification C1 S60210-TB and SATT391 Marker Clustering F
P10598A-1 and SATT334 QTL Interval Mapping and Marker Clustering F
SATT510 and SATT335 QTL Interval Mapping and Marker Clustering G
P5219A-1 and P7659A-2 Marker Clustering G SAT_117 and S60143-TB
Marker Clustering I SATT451 and SATT367 Marker Clustering L SATT495
and P10649C-3 Marker Clustering M SATT250 and SATT346 QTL Interval
Mapping
[0176] Each of the intervals described above shows a clustering of
markers that co-segregate with iron-deficiency tolerance. This
clustering of markers occurs in relatively small domains on the
linkage groups, indicating the presence of one or more QTL in those
chromosome regions. QTL intervals were drawn to encompass the
markers that co-segregate with environmental low iron tolerance.
The intervals are defined by the markers on their termini, where
the interval encompasses all the markers that map within the
interval as well as the markers that define the termini
[0177] In two cases, intervals that were drawn on LG-F by a marker
clustering effect were further independently confirmed by a QTL
mapping analysis, as described in detail in EXAMPLE 3. In those
experiments, markers on that that domain of LG-F show a significant
likelihood ratio statistic (LRS) for the presence of one or more
QTL responsible for the low-iron tolerance trait. Optionally,
because the two intervals on LG-F are relatively close together,
these two intervals can be viewed as a single interval that contain
one (or more) tolerance QTL.
Genetic Maps
[0178] As one of skill in the art will recognize, recombination
frequencies (and as a result, genetic map positions) in any
particular population are not static. The genetic distances
separating two markers (or a marker and a QTL) can vary depending
on how the map positions are determined. For example, variables
such as the parental mapping populations used, the software used in
the marker mapping or QTL mapping, and the parameters input by the
user of the mapping software can contribute to the QTL/marker
genetic map relationships. However, it is not intended that the
invention be limited to any particular mapping populations, use of
any particular software, or any particular set of software
parameters to determine linkage of a particular marker or
chromosome interval with the low iron tolerance phenotype. It is
well within the ability of one of ordinary skill in the art to
extrapolate the novel features described herein to any soybean gene
pool or population of interest, and using any particular software
and software parameters. Indeed, observations regarding tolerance
markers and chromosome intervals in populations in addition to
those described herein are readily made using the teaching of the
present disclosure.
[0179] Mapping Populations
[0180] Any suitable soybean strains can be used to generate mapping
data or for marker association studies. A large number of commonly
used soybean lines (e.g., commercial varieties) and mapping
populations are known in the art. Additional strains finding use
with the invention are also described in the present disclosure.
Useful soybean mapping populations and lines include but are not
limited to:
TABLE-US-00002 Mapping Population Description/Reference UP1C6-43
.times. 90B73 UP1C6-43 is a public line from the University of
Nebraska. 90B73 is notably susceptible to FEC, and is described in
Plant Variety Protection Act, Certificate No. 200000152 for Soybean
`90B73,` issued May 8, 2001; see also, U.S. Pat. No. 6,316,700,
issued Nov. 13, 2001, to Hedges. P1082 .times. 90B73 P1082 is
described in Plant Variety Protection Act, Certificate No. 8200115,
issued May 26, 1982. 90B73 is described in Plant Variety Protection
Act, Certificate No. 200000152 for Soybean `90B73` issued May 8,
2001; see also, U.S. Pat. No. 6,316,700, issued Nov. 13, 2001, to
Hedges. Minsoy .times. Noir 1 Recombinant Inbred Line (RIL)
population derived by single seed descent, consisting of 240
F7-derived RILs. Described in Lark et al., (1993) "A genetic map of
soybean (Glycine max L.) and using an intraspecific cross of two
cultivars: Minosy and Noir 1," Theor. Appl. Genet., 86: 901-906;
Mansur and Orf (1995) "Evaluation of soybean recombinant inbreds
for agronomic performance in northern USA and Chile," Crop Sci.,
35: 422-425; Mansur et al., (1996) "Genetic mapping of agronomic
traits using recombinant inbred lines of soybean," Crop Sci., 36:
1327-1336. Developed at the University of Utah. See also Intl.
Patent Appl. No. WO 98/49887, filed May 1, 1998. Minsoy .times.
Archer RIL population derived by single seed descent, consisting of
233 F7- derived RILs. Described in Mansur and Orf (1995)
"Evaluation of soybean recombinant inbreds for agronomic
performance in northern USA and Chile," Crop Sci., 35: 422-425;
Mansur et al., (1996) "Genetic mapping of agronomic traits using
recombinant inbred lines of soybean," Crop Sci., 36: 1327-1336.
Developed at the University of Utah. See also Intl. Patent Appl.
No. WO 98/49887, filed May 1, 1998. Noir 1 .times. Archer RIL
Population derived by single seed descent, consisting of 240 F7-
derived RILs. Described in Mansur and Orf (1995) "Evaluation of
soybean recombinant inbreds for agronomic performance in northern
USA and Chile," Crop Sci., 35: 422-425; Mansur et al., (1996)
"Genetic mapping of agronomic traits using recombinant inbred lines
of soybean," Crop Sci., 36: 1327-1336. Developed at the University
of Utah. See also Intl. Patent Appl. No. WO 98/49887, filed May 1,
1998. Clark .times. Harosoy Population derived from the cross of
near isogenic lines (NILs) of the cultivars Clark and Harosoy. The
population consists of derivatives of 57 F2 plants (see, Shoemaker
and Specht (1995) "Integration of the soybean molecular and
classical genetic linkage groups," Crop Sci., 35: 436-446).
Developed at the University of Nebraska. A81-356022 .times.
PI468916 This is an F2-derived mapping population from the
interspecific cross of the A81-356022 (Glycine max) and PI468.916
(G. soja). The population consists of 59 F2 plant derivatives and
has been described in detail (Keim et al., (1990) "RFLP mapping in
soybean: association between marker loci and variation in
quantitative traits," Genetics 126: 735-742; Shoemaker and Specht
(1995) "Integration of the soybean molecular and classical genetic
linkage groups," Crop Sci., 35: 436-446; Shoemaker and Olson (1993)
Molecular linkage map of soybean (Glycine max L. Merr.).
p.6.131-6.138, in Genetic maps: Locus maps of complex genomes
[O'Brien (ed.)] Cold Spring Harbor Laboratory Press, New York).
Commonly referred to as the USDA/Iowa State University Population
(MS). OX715 .times. P9242 OX715 is a public variety. P9242 is
described in Plant Variety Protection Act, Certificate No. 9300238
for Soybean `9242` issued May 30, 1997. Sloan, Williams, Harosoy
and See, Burnham et al., Crop Sci., "Quantitative Trait Loci for
Partial Conrad Resistance to Phytophthora sojae in Soybean," 43:
1610-1617 (various RILs derived from (2003); Weiss and Stevenson,
Agron. J., 47: 541-543 (1955); crosses of the above cultivars)
Bernard and Lindahl, Crop Sci., 43: 101-105 (1972); Bahrenfus and
Fehr, Crop Sci., 20: 673 (1980); Fehr et al., Crop Sci., 29: 830
(1989). Bert, Marcus, Corsoy, A92- See, Glover and Scott,
"Heritability and Phenotypic Variation of 627030, Simpson, OT92-1,
Tolerance to Phytophthora Root Rot of Soybean," Crop Sci.,
Hendricks, Freeborn, Surge, 38: 1495-1500 (1998); and additional
references made therein. Kenwood 94 (various RILs derived from
crosses of the above cultivars) Essex .times. Forrest See, Yuan et
al., "Quantitative trait loci in two soybean recombinant Flyer
.times. Hartwig inbred line populations segregating for yield and
disease resistance," Crop Sci., 42: 271-277 (2002). Williams
.times. PI399073 US Patent Appl. No. 2004/0034890, published Feb.
19, 2004; US S 19-90 .times. PI399073 Patent Appl. No.
2004/0261144, published Dec. 23, 2004. 9163 .times. 92B05 P9163 is
a commercially available Pioneer variety described in Plant Variety
Protection Act, Certificate No. 9600053. 92B05 is a commercially
available Pioneer variety described in Plant Variety Protection
Act, Certificate No. 9900092 for Soybean `92B05` issued Sep. 21,
2000; see also, U.S. Pat. No. 5,942,668, issued Aug. 24, 1999 to
Grace et al. 9362 .times. 93B41 P9362 is a commercially available
Pioneer variety described in Plant Variety Protection Act,
Certificate No. 9400098. 93B41 is a commercially available Pioneer
variety described in Plant Variety Protection Act, Certificate No.
9800068; see also, U.S. Pat. No. 5,750,853, issued May 12, 1998 to
Fuller et al. 93B35 Described in Plant Variety Protection Act,
Certificate No. 200000035, issued Apr. 24, 2001. See also, U.S.
Pat. No. 6,153,818, issued Nov. 28, 2000. 93B53 Described in Plant
Variety Protection Act, Certificate No. 9900101, issued Oct. 27,
2000. See also, U.S. Pat. No. 6,075,182, issued Jun. 13, 2000.
93M11 Described in Plant Variety Protection Act, Certificate No.
200400080, issued Aug. 16, 2004. See also, U.S. Pat. No. 6,855,875,
issued Feb. 15, 2005. 93B68 Described in Plant Variety Protection
Act, Certificate No. 200200084, issued Jun. 10, 2002. See also, US
Patent Appl. Serial No. 10/271,115. 93B72 Described in Plant
Variety Protection Act, Certificate No. 200100071, issued May 8,
2001. See also, U.S. Pat. No. 6,566,589, issued May 20, 2003. 94B53
Described in Plant Variety Protection Act, Certificate No.
200000031, issued May 8, 2001. See also, U.S. Pat. No. 6,235,976,
issued May 22, 2001. 94M80 Described in pending Plant Variety
Protection Act, Certificate No. 200500084, filed Jan. 18, 2005. See
also, pending US Patent Appl. Serial No. 10/768,275, filed Jan. 30,
2005. 9492 Described in Plant Variety Protection Act, Certificate
No. 9800077, issued Sep. 12, 2001. See also, U.S. Pat. No.
5,792,907, issued Aug. 11, 1998.
[0181] Mapping Software
[0182] A variety of commercial software is available for genetic
mapping and marker association studies (e.g., QTL mapping). This
software includes but is not limited to:
TABLE-US-00003 Software Description/References JoinMap .RTM.
VanOoijen, and Voorrips (2001) "JoinMap 3.0 software for the
calculation of genetic linkage maps," Plant Research International,
Wageningen, the Netherlands; and, Stam "Construction of integrated
genetic linkage maps by means of a new computer package: JoinMap,"
The Plant Journal 3(5): 739-744 (1993) MapQTL .RTM. J. W.
vanOoijen, "Software for the mapping of quantitative trait loci in
experimental populations," Kyazma B. V., Wageningen, Netherlands
MapManager QT Manly and Olson, "Overview of QTL mapping software
and introduction to Map Manager QT," Mamm. Genome 10: 327-334
(1999) MapManager QTX Manly, Cudmore and Meer, "MapManager QTX,
cross-platform software for genetic mapping," Mamm. Genome 12:
930-932 (2001) GeneFlow .RTM. and GENEFLOW, Inc. (Alexandria, VA)
QTLocate .TM. TASSEL (Trait Analysis by aSSociation, Evolution, and
Linkage) by Edward Buckler, and information about the program can
be found on the Buckler Lab web page at the Institute for Genomic
Diversity at Cornell University.
[0183] Unified Genetic Maps
[0184] "Unified," "consensus" or "integrated" genetic maps have
been created that incorporate mapping data from two or more
sources, including sources that used different mapping populations
and different modes of statistical analysis. The merging of genetic
map information increases the marker density on the map, as well as
improving map resolution. These improved maps can be advantageously
used in marker assisted selection, map-based cloning, provide an
improved framework for positioning newly identified molecular
markers and aid in the identification of QTL chromosome intervals
and clusters of advantageously-linked markers.
[0185] In some aspects, a consensus map is derived by simply
overlaying one map on top of another. In other aspects, various
algorithms, e.g., JoinMap.RTM. analysis, allows the combination of
genetic mapping data from multiple sources, and reconciles
discrepancies between mapping data from the original sources. See,
Van Ooijen, and Voorrips (2001) "JoinMap 3.0 software for the
calculation of genetic linkage maps," Plant Research International,
Wageningen, the Netherlands; and, Stam (1993) "Construction of
integrated genetic linkage maps by means of a new computer package:
JoinMap," The Plant Journal 3(5):739-744.
[0186] FIG. 6 provides a composite genetic map that incorporates
mapping information from various sources. This map was derived
using the USDA/Iowa State University mapping population data (as
described in Cregan et al., "An Integrated Genetic Linkage Map of
the Soybean Genome" Crop Science 39:1464-1490 [1999]; and see
references therein) as a framework. Additional markers, as they
became known, have been continuously added to that map, including
public SSR markers, EST-derived markers, and SNP markers. This map
contains approximately 750 soybean markers that are distributed
over each of the soybean chromosomes. The markers that are on this
map are known in the art (i.e., have been previously described;
see, e.g., the SOYBASE on-line resource for extensive listings of
these markers and descriptions of the individual markers) or are
described herein.
[0187] Additional integrated maps are known in the art. See, e.g.,
Cregan et al., "An Integrated Genetic Linkage Map of the Soybean
Genome" Crop Science 39:1464-1490 (1999); and also International
Application No. PCT/US2004/024919 by Sebastian, filed Jul. 27,
2004, entitled "Soybean Plants Having Superior Agronomic
Performance and Methods for their Production").
[0188] Song et al. provides another integrated soybean genetic map
that incorporates mapping information from five different mapping
populations (Song et al., "A New Integrated Genetic Linkage Map of
the Soybean," Theor. Appl. Genet., 109:122-128 [2004]). This
integrated map contains approximately 1,800 soybean markers,
including SSR and SNP-type markers, as well as EST markers, RPLP
markers, AFLP, RAPD, isozyme and classical markers (e.g., seed coat
color). The markers that are on this map are known in the art and
have been previously characterized. This information is also
available at the website for the Soybean Genomics and Improvement
Laboratory (SGIL) at the USDA Beltsville Agricultural Research
Center (BARC). See, specifically, the description of projects in
the Cregan Laboratory on that website.
[0189] The soybean integrated linkage map provided in Song et al.
(2004) is based on the principle described by Stam (1993)
"Construction of integrated genetic linkage maps by means of a new
computer package: JoinMap," The Plant Journal 3(5):739-744; and Van
Ooijen and Voorrips (2001) "JoinMap 3.0 software for the
calculation of genetic linkage maps," Plant Research International,
Wageningen, the Netherlands. Mapping information from five soybean
populations was used in the map integration, and also used to place
recently identified SSR markers onto the soybean genome. These
mapping populations were Minsoy.times.Noir 1 (MN),
Minsoy.times.Archer (MA), Noir 1.times.Archer (NA),
Clark.times.Harosoy (CH) and A81-356022.times.PI468916 (MS). The
JoinMap.RTM. analysis resulted in a map with 20 linkage groups
containing a total of 1849 markers, including 1015 SSRs, 709 RFLPs,
73 RAPDs, 24 classical traits, six AFLPs, ten isozymes and 12
others. Among the mapped SSR markers were 417 previously
uncharacterized SSRs.
[0190] Initially, LOD scores and pairwise recombination frequencies
between markers were calculated. A LOD of 5.0 was used to create
groups in the MS, MA, NA populations and LOD 4.0 in the MN and CH
populations. The map of each linkage group was then integrated.
Recombination values were converted to genetic distances using the
Kosambi mapping function.
Linked Markers
[0191] From the present disclosure and widely recognized in the
art, it is clear that any genetic marker that has a significant
probability of co-segregation with a phenotypic trait of interest
(e.g., in the present case, a pathogen tolerance or improved
tolerance trait) can be used as a marker for that trait. As list of
useful QTL markers provided by the present invention is provided in
FIG. 1.
[0192] In addition to the QTL markers noted in FIG. 1, additional
markers linked to (showing linkage disequilibrium with) the QTL
markers can also be used to predict the tolerance or improved
tolerance trait in a soybean plant. In other words, any other
marker showing less than 50% recombination frequency (separated by
a genetic distance less than 50 cM) with a QTL marker of the
invention (e.g., the markers provided in FIG. 1) is also a feature
of the invention. Any marker that is linked to a QTL marker can
also be used advantageously in marker-assisted selection for the
particular trait.
[0193] Genetic markers that are linked to QTL markers (e.g., QTL
markers provided in FIG. 1) are particularly useful when they are
sufficiently proximal (e.g., closely linked) to a given QTL marker
so that the genetic marker and the QTL marker display a low
recombination frequency. In the present invention, such closely
linked markers are a feature of the invention. As defined herein,
closely linked markers display a recombination frequency of about
10% or less (e.g., the given marker is within 10 cM of the QTL).
Put another way, these closely linked loci 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.
[0194] Thus, in other embodiments, closely linked loci such as a
QTL marker locus and a second locus display an inter-locus
cross-over frequency of about 10% or less, preferably about 9% or
less, still more preferably about 8% or less, yet more preferably
about 7% or less, still more preferably about 6% or less, yet more
preferably about 5% or less, still more preferably about 4% or
less, yet more preferably about 3% or less, and still more
preferably about 2% or less. In highly preferred embodiments, 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, e.g.,
about 0.75% or less, more preferably about 0.5% or less, or yet
more preferably about 0.25% or less. Thus, the loci are about 10
cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2cM, 1cM, 0.75 cM,
0.5 cM or 0.25 cM or less apart. Put another way, two loci that are
localized to the same chromosome, and at such a distance that
recombination between the two loci occurs at a frequency of less
than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%,
0.5%, 0.25%, or less) are said to be "proximal to" each other.
[0195] In some aspects, linked markers (including closely linked
markers) of the invention are determined by review of a genetic
map, for example, the integrated genetic map shown in FIG. 6. For
example, it is shown herein that the linkage group L marker SATT613
correlates with at least one low-iron tolerance QTL. Markers that
are linked to SATT613 (e.g., within 50 cM) can be determined from
the map provided in FIG. 6. For example, markers on linkage group L
that are linked to SATT613 include:
TABLE-US-00004 Chrom. Position Marker Name (cM) SATT495 L 4.5
SATT232 L 7.4 SATT446 L 9.2 P10649C-3 L 12.5 SATT182 L 13.4 SAT_301
L 15.6 SAT_071 L 19.7 SATT238 L 19.7 SATT388 L 22.2 SATT143 L 31.8
SAT_134 L 32.4 SATT523 L 32.4 SATT278 L 33.2 SATT418 L 33.9 SATT711
L 34.0 SATT398 L 34.6 SATT497 L 42.3 SATT313 L 43.9 SATT613 L 45.1
SATT284 L 47.7 SATT462 L 49.3 SAT_340 L 65.2 SATT156 L 65.8 SATT481
L 65.8 SCT_010 L 68.5 S60392-TB L 70.0 SATT076 L 72.3 SATT265 L
75.0 SATT527 L 75.7 SATT561 L 75.7 SATT166 L 77.1 SATT448 L 78.0
SATT678 L 80.9 SAT_099 L 89.4 SAG1055 L 95.0
[0196] In other aspects, closely linked markers of the invention
can be determined by review of this same genetic map. For example,
markers that are closely linked (e.g., separated by not more than
10 cM) to SATT613 on linkage group L include:
TABLE-US-00005 Chrom. Position Marker Name (cM) SATT497 L 42.3
SATT313 L 43.9 SATT613 L 45.1 SATT284 L 47.7 SATT462 L 49.3
[0197] Similarly, linked markers (including closely linked markers)
of the invention can be determined by review of any suitable
soybean genetic map. For example, the integrated genetic map
described in Song et al. (2004) also provides a means to identify
linked (including closely linked) markers. See, Song et al., "A New
Integrated Genetic Linkage Map of the Soybean," Theor. Appl.
Genet., 109:122-128 [2004]; see also the website for the Soybean
Genomics and Improvement Laboratory (SGIL) at the USDA Beltsville
Agricultural Research Center (BARC), and see specifically the
description of projects in the Cregan Laboratory on that website.
That genetic map incorporates a variety of genetic markers that are
known in the art or alternatively are described in that reference.
Detailed descriptions of numerous markers, including many of those
described in Song et al. (2004) can be found at the SOYBASE website
resource.
[0198] For example, according to the Song et al. (2004) integrated
genetic map, markers on linkage group L that are closely linked to
SATT613 include: A264.sub.--1, RGA.sub.--7, Satt523, Sat.sub.--134,
i8.sub.--2, A450.sub.--2, A106.sub.--1, Sat.sub.--405, Satt143,
B124.sub.--2, A459.sub.--1, Satt398, Satt694, Sat.sub.--195,
Sat.sub.--388, Satt652, Satt711, Sat.sub.--187, Satt418, Satt278,
Sat.sub.--397, Sat.sub.--191, Sat.sub.--320, A204.sub.--2, Satt497,
G214.sub.--17, Satt313, B164.sub.--1, G214.sub.--16, Satt613,
A023.sub.--1, Satt284, AW508247, Satt462, L050.sub.--7,
E014.sub.--1 and A071.sub.--5.
[0199] It is not intended that the determination of linked or
closely linked markers be limited to the use of any particular
soybean genetic map. Indeed, a large number of soybean genetic maps
is available and are well known to one of skill in the art. Another
map that finds use with the invention in this respect is the
integrated soybean genetic maps found on the SOYBASE website
resource. Alternatively still, the determination of linked and
closely linked markers can be made by the generation of an
experimental dataset and linkage analysis.
[0200] It is not intended that the identification of markers that
are linked (e.g., within about 50 cM or within about 10 cM) to the
low iron tolerance QTL markers identified herein be limited to any
particular map or methodology. The integrated genetic map provided
in FIG. 6 serves only as example for identifying linked markers.
Indeed, linked markers as defined herein can be determined from any
genetic map known in the art (an experimental map or an integrated
map), or alternatively, can be determined from any new mapping
dataset.
[0201] It is noted that lists of linked and closely linked markers
may vary between maps and methodologies due to various factors.
First, the markers that are placed on any two maps may not be
identical, and furthermore, some maps may have a greater marker
density than another map. Also, the mapping populations,
methodologies and algorithms used to construct genetic maps can
differ. One of skill in the art recognizes that one genetic map is
not necessarily more or less accurate than another, and
furthermore, recognizes that any soybean genetic map can be used to
determine markers that are linked and closely linked to the QTL
markers of the present invention.
Techniques for Marker Detection
[0202] The invention provides molecular markers that have a
significant probability of co-segregation with QTL that impart a
low iron tolerance phenotype. These QTL markers find use in marker
assisted selection for desired traits (tolerance or improved
tolerance), and also have other uses. It is not intended that the
invention be limited to any particular method for the detection of
these markers.
[0203] Markers corresponding to genetic polymorphisms between
members of a population can be detected by numerous methods
well-established in the art (e.g., PCR-based sequence specific
amplification, restriction fragment length polymorphisms (RFLPs),
isozyme markers, allele specific hybridization (ASH), amplified
variable sequences of the plant genome, self-sustained sequence
replication, simple sequence repeat (SSR), single nucleotide
polymorphism (SNP), random amplified polymorphic DNA ("RAPD") or
amplified fragment length polymorphisms (AFLP). In one additional
embodiment, the presence or absence of a molecular marker is
determined simply through nucleotide sequencing of the polymorphic
marker region. This method is readily adapted to high throughput
analysis as are the other methods noted above, e.g., using
available high throughput sequencing methods such as sequencing by
hybridization.
[0204] In general, the majority of genetic markers rely on one or
more property of nucleic acids for their detection. For example,
some techniques for detecting genetic markers utilize hybridization
of a probe nucleic acid to nucleic acids corresponding to the
genetic marker (e.g., amplified nucleic acids produced using
genomic soybean DNA as a template). Hybridization formats,
including but not limited to solution phase, solid phase, mixed
phase, or in situ hybridization assays are useful for allele
detection. An extensive guide to the hybridization of nucleic acids
is found in Tijssen (1993) Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Acid Probes
Elsevier, New York, as well as in Sambrook, Berger and Ausubel
(herein).
[0205] For example, markers that comprise restriction fragment
length polymorphisms (RFLP) are detected, e.g., by hybridizing a
probe which is typically a sub-fragment (or a synthetic
oligonucleotide corresponding to a sub-fragment) of the nucleic
acid to be detected to restriction digested genomic DNA. The
restriction enzyme is selected to provide restriction fragments of
at least two alternative (or polymorphic) lengths in different
individuals or populations. Determining one or more restriction
enzyme that produces informative fragments for each cross is a
simple procedure, well known in the art. After separation by length
in an appropriate matrix (e.g., agarose or polyacrylamide) and
transfer to a membrane (e.g., nitrocellulose, nylon, etc.), the
labeled probe is hybridized under conditions which result in
equilibrium binding of the probe to the target followed by removal
of excess probe by washing.
[0206] Nucleic acid probes to the marker loci can be cloned and/or
synthesized. Any suitable label can be used with a probe of the
invention. Detectable labels suitable for use with nucleic acid
probes include, for example, any composition detectable by
spectroscopic, radioisotopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. Useful
labels include biotin for staining with labeled streptavidin
conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes,
and colorimetric labels. Other labels include ligands which bind to
antibodies labeled with fluorophores, chemiluminescent agents, and
enzymes. A probe can also constitute radiolabelled PCR primers that
are used to generate a radiolabelled amplicon. Labeling strategies
for labeling nucleic acids and corresponding detection strategies
can be found, e.g., in Haugland (1996) Handbook of Fluorescent
Probes and Research Chemicals Sixth Edition by Molecular Probes,
Inc. (Eugene Oreg.); or Haugland (2001) Handbook of Fluorescent
Probes and Research Chemicals Eigth Edition by Molecular Probes,
Inc. (Eugene Oreg.) (Available on CD ROM).
[0207] Amplification-Based Detection Methods
[0208] PCR, RT-PCR and LCR are in particularly broad use as
amplification and amplification-detection methods for amplifying
nucleic acids of interest (e.g., those comprising marker loci),
facilitating detection of the markers. Details regarding the use of
these and other amplification methods can be found in any of a
variety of standard texts, including, e.g., Sambrook, Ausubel,
Berger and Croy, herein. 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 suitable for restriction
digestion, PCR expansion and sequencing using reverse transcriptase
and a polymerase ("Reverse Transcription-PCR, or "RT-PCR"). See
also, Ausubel, Sambrook and Berger, above.
[0209] Real Time Amplification/Detection Methods
[0210] In one aspect, real time PCR or LCR is performed on the
amplification mixtures described herein, e.g., using molecular
beacons or TaqMan.TM. probes. A molecular beacon (MB) is an
oligonucleotide or PNA which, under appropriate hybridization
conditions, self-hybridizes to form a stem and loop structure. The
MB has a label and a quencher at the termini of the oligonucleotide
or PNA; thus, under conditions that permit intra-molecular
hybridization, the label is typically quenched (or at least altered
in its fluorescence) by the quencher. Under conditions where the MB
does not display intra-molecular hybridization (e.g., when bound to
a target nucleic acid, e.g., to a region of an amplicon during
amplification), the MB label is unquenched. Details regarding
standard methods of making and using MBs are well established in
the literature and MBs are available from a number of commercial
reagent sources. See also, e.g., Leone et al. (1995) "Molecular
beacon probes combined with amplification by NASBA enable
homogenous real-time detection of RNA." Nucleic Acids Res.
26:2150-2155; Tyagi and Kramer (1996) "Molecular beacons: probes
that fluoresce upon hybridization" Nature Biotechnology 14:303-308;
Blok and Kramer (1997) "Amplifiable hybridization probes containing
a molecular switch" Mol Cell Probes 11:187-194; Hsuih et al. (1997)
"Novel, ligation-dependent PCR assay for detection of hepatitis C
in serum" J Clin Microbiol 34:501-507; Kostrikis et al. (1998)
"Molecular beacons: spectral genotyping of human alleles" Science
279:1228-1229; Sokol et al. (1998) "Real time detection of DNA:RNA
hybridization in living cells" Proc. Natl. Acad. Sci. U.S.A.
95:11538-11543; Tyagi et al. (1998) "Multicolor molecular beacons
for allele discrimination" Nature Biotechnology 16:49-53; Bonnet et
al. (1999) "Thermodynamic basis of the chemical specificity of
structured DNA probes" Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176;
Fang et al. (1999) "Designing a novel molecular beacon for
surface-immobilized DNA hybridization studies" J. Am. Chem. Soc.
121:2921-2922; Maras et al. (1999) "Multiplex detection of
single-nucleotide variation using molecular beacons" Genet. Anal.
Biomol. Eng. 14:151-156; and Vet et al. (1999) "Multiplex detection
of four pathogenic retroviruses using molecular beacons" Proc.
Natl. Acad. Sci. U.S.A. 96:6394-6399. Additional details regarding
MB construction and use is found in the patent literature, e.g.,
U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al. entitled
"Detectably labeled dual conformation oligonucleotide probes,
assays and kits;" U.S. Pat. No. 6,150,097 to Tyagi et al (Nov. 21,
2000) entitled "Nucleic acid detection probes having non-FRET
fluorescence quenching and kits and assays including such probes"
and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000),
entitled "Wavelength-shifting probes and primers and their use in
assays and kits."
[0211] PCR detection and quantification using dual-labeled
fluorogenic oligonucleotide probes, commonly referred to as
"TaqMan.TM." probes, can also be performed according to the present
invention. These probes are composed of short (e.g., 20-25 base)
oligodeoxynucleotides that are labeled with two different
fluorescent dyes. On the 5' terminus of each probe is a reporter
dye, and on the 3' terminus of each probe a quenching dye is found.
The oligonucleotide probe sequence is complementary to an internal
target sequence present in a PCR amplicon. When the probe is
intact, energy transfer occurs between the two fluorophores and
emission from the reporter is quenched by the quencher by FRET.
During the extension phase of PCR, the probe is cleaved by 5'
nuclease activity of the polymerase used in the reaction, thereby
releasing the reporter from the oligonucleotide-quencher and
producing an increase in reporter emission intensity. Accordingly,
TaqMan.TM. probes are oligonucleotides that have a label and a
quencher, where the label is released during amplification by the
exonuclease action of the polymerase used in amplification. This
provides a real time measure of amplification during synthesis. A
variety of TaqMan.TM. reagents are commercially available, e.g.,
from Applied Biosystems (Division Headquarters in Foster City,
Calif.) as well as from a variety of specialty vendors such as
Biosearch Technologies (e.g., black hole quencher probes).
[0212] Additional Details Regarding Amplified Variable Sequences,
SSR, AFLP ASH, SNPs and Isozyme Markers
[0213] Amplified variable sequences refer to amplified sequences of
the plant genome which 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. Preferably, DNA from the plant serves
as a template for amplification with primers that flank a variable
sequence of DNA. The variable sequence is amplified and then
sequenced.
[0214] Alternatively, self-sustained sequence replication can be
used to identify genetic markers. Self-sustained sequence
replication refers to a method of nucleic acid amplification using
target nucleic acid sequences which are replicated exponentially in
vitro under substantially isothermal conditions by using three
enzymatic activities involved in retroviral replication: (1)
reverse transcriptase, (2) Rnase H, and (3) 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.
[0215] Amplified fragment length polymophisms (AFLP) can also be
used as genetic markers (Vos et al. (1995) Nucl Acids Res 23:4407).
The phrase "amplified fragment length polymorphism" refers to
selected restriction fragments which are amplified before or after
cleavage by a restriction endonuclease. The amplification step
allows easier detection of specific restriction fragments. AFLP
allows the detection large numbers of polymorphic markers and has
been used for genetic mapping of plants (Becker et al. (1995) Mol
Gen Genet 249:65; and Meksem et al. (1995) Mol Gen Genet
249:74).
[0216] Allele-specific hybridization (ASH) can be used to identify
the genetic markers of the invention. 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 is via an isotopic or non-isotopic label attached
to the probe.
[0217] For each polymorphism, two or more different ASH probes are
designed to have identical DNA sequences except at the polymorphic
nucleotides. Each probe will have exact homology with one allele
sequence so that the range of probes can distinguish all the known
alternative allele sequences. Each probe is hybridized to the
target DNA. With appropriate probe design and hybridization
conditions, a single-base mismatch between the probe and target DNA
will prevent hybridization. In this manner, only one of the
alternative probes will hybridize to a target sample that is
homozygous or homogenous for an allele. Samples that are
heterozygous or heterogeneous for two alleles will hybridize to
both of two alternative probes.
[0218] ASH markers are 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 the lack of hybridization. ASH probe and target
molecules are optionally RNA or DNA; the target molecules are any
length of nucleotides beyond the sequence that is complementary to
the probe; the probe is designed to hybridize with either strand of
a DNA target; the probe ranges in size to conform to variously
stringent hybridization conditions, etc.
[0219] PCR allows the target sequence for ASH to be amplified from
low concentrations of nucleic acid in relatively small volumes.
Otherwise, the target sequence from genomic DNA is digested with a
restriction endonuclease and size separated by gel electrophoresis.
Hybridizations typically occur with the target sequence bound to
the surface of a membrane or, as described in U.S. Pat. No.
5,468,613, the ASH probe sequence may be bound to a membrane.
[0220] In one embodiment, ASH data are typically obtained by
amplifying nucleic acid fragments (amplicons) from genomic DNA
using PCR, transferring the amplicon target DNA to a membrane in a
dot-blot format, hybridizing a labeled oligonucleotide probe to the
amplicon target, and observing the hybridization dots by
autoradiography.
[0221] Single nucleotide polymorphisms (SNP) are markers that
consist of a shared sequence differentiated on the basis of a
single nucleotide. Typically, this distinction is detected by
differential migration patterns of an amplicon comprising the SNP
on e.g., an acrylamide gel. However, alternative modes of
detection, such as hybridization, e.g., ASH, or RFLP analysis are
also appropriate.
[0222] Isozyme markers can be employed as genetic markers, e.g., to
track markers other than the tolerance markers herein, or to track
isozyme markers linked to the markers herein. Isozymes are multiple
forms of enzymes that differ from one another in their amino acid,
and therefore their nucleic acid sequences. Some isozymes are
multimeric enzymes containing slightly different subunits. Other
isozymes are either multimeric or monomeric but have been cleaved
from the proenzyme at different sites in the amino acid sequence.
Isozymes can be characterized and analyzed at the protein level, or
alternatively, isozymes which differ at the nucleic acid level can
be determined. In such cases any of the nucleic acid based methods
described herein can be used to analyze isozyme markers.
[0223] Additional Details Regarding Nucleic Acid Amplification
[0224] As noted, nucleic acid amplification techniques such as PCR
and LCR are well known in the art and can be applied to the present
invention to amplify and/or detect nucleic acids of interest, such
as nucleic acids comprising marker loci. Examples of techniques
sufficient to direct persons of skill through such in vitro
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), are found in the
references noted above, e.g., Innis, Sambrook, Ausubel, Berger and
Croy. Additional details are found in Mullis et al. (1987) U.S.
Pat. No. 4,683,202; Arnheim & Levinson (Oct. 1, 1990) C&EN
36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990)
Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin.
Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van
Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene
4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and
Malek (1995) Biotechnology 13: 563-564. Improved methods of
amplifying large nucleic acids by PCR, which is useful in the
context of positional cloning, are further summarized in Cheng et
al. (1994) Nature 369: 684, and the references therein, in which
PCR amplicons of up to 40 kb are generated.
[0225] Detection of Markers For Positional Cloning
[0226] In some embodiments, a nucleic acid probe is used to detect
a nucleic acid that comprises a marker sequence. Such probes can be
used, for example, in positional cloning to isolate nucleotide
sequences linked to the marker nucleotide sequence. It is not
intended that the nucleic acid probes of the invention be limited
to any particular size. In some embodiments, nucleic acid probe is
at least 20 nucleotides in length, or alternatively, at least 50
nucleotides in length, or alternatively, at least 100 nucleotides
in length, or alternatively, at least 200 nucleotides in
length.
[0227] A hybridized probe is detected using, autoradiography,
fluorography or other similar detection techniques depending on the
label to be detected. Examples of specific hybridization protocols
are widely available in the art, see, e.g., Berger, Sambrook, and
Ausubel, all herein.
[0228] Probe/Primer Synthesis Methods
[0229] In general, synthetic methods for making oligonucleotides,
including probes, primers, molecular beacons, PNAs, LNAs (locked
nucleic acids), etc., are well known. For example, oligonucleotides
can be synthesized chemically according to the solid phase
phosphoramidite triester method described by Beaucage and Caruthers
(1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using a
commercially available automated synthesizer, e.g., as described in
Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.
Oligonucleotides, including modified oligonucleotides can also be
ordered from a variety of commercial sources known to persons of
skill. There are many commercial providers of oligo synthesis
services, and thus this is a broadly accessible technology. Any
nucleic acid can be custom ordered from any of a variety of
commercial sources, such as The Midland Certified Reagent Company,
The Great American Gene Company, ExpressGen Inc., Operon
Technologies Inc. (Alameda, Calif.) and many others. Similarly,
PNAs can be custom ordered from any of a variety of sources, such
as PeptidoGenic, HTI Bio-products, Inc., BMA Biomedicals Ltd
(U.K.), Bio.cndot.Synthesis, Inc., and many others.
[0230] In Silico Marker Detection
[0231] In alternative embodiments, in silico methods can be used to
detect the marker loci of interest. For example, the sequence of a
nucleic acid comprising the marker locus of interest can be stored
in a computer. The desired marker locus sequence or its homolog can
be identified using an appropriate nucleic acid search algorithm as
provided by, for example, in such readily available programs as
BLAST, or even simple word processors.
Amplification Primers for Marker Detection
[0232] In some preferred embodiments, the molecular markers of the
invention are detected using a suitable PCR-based detection method,
where the size or sequence of the PCR amplicon is indicative of the
absence or presence of the marker (e.g., a particular marker
allele). In these types of methods, PCR primers are hybridized to
the conserved regions flanking the polymorphic marker region. As
used in the art, PCR primers used to amplify a molecular marker are
sometimes termed "PCR markers" or simply "markers."
[0233] It will be appreciated that, although many specific examples
of primers are provided herein (see, FIGS. 2 and 3), suitable
primers to be used with the invention can be designed using any
suitable method. It is not intended that the invention be limited
to any particular primer or primer pair. For example, primers can
be designed using any suitable software program, such as
LASERGENE.RTM..
[0234] In some embodiments, the primers of the invention are
radiolabelled, or labeled by any suitable means (e.g., using a
non-radioactive fluorescent tag), to allow for rapid visualization
of the different size amplicons following an amplification reaction
without any additional labeling step or visualization step. In some
embodiments, the primers are not labeled, and the amplicons are
visualized following their size resolution, e.g., following agarose
gel electrophoresis. In some embodiments, ethidium bromide staining
of the PCR amplicons following size resolution allows visualization
of the different size amplicons.
[0235] It is not intended that the primers of the invention be
limited to generating an amplicon of any particular size. For
example, the primers used to amplify the marker loci and alleles
herein are not limited to amplifying the entire region of the
relevant locus. The primers can generate an amplicon of any
suitable length that is longer or shorter than those given in the
allele definitions in FIG. 4. In some embodiments, marker
amplification produces an amplicon at least 20 nucleotides in
length, or alternatively, at least 50 nucleotides in length, or
alternatively, at least 100 nucleotides in length, or
alternatively, at least 200 nucleotides in length. Marker alleles
in addition to those recited in FIG. 4 also find use with the
present invention.
Marker Assisted Selection and Breeding of Plants
[0236] 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
are used to identify plants that contain a desired genotype at one
or more loci, and that are expected to transfer the desired
genotype, along with a desired phenotype to their progeny. Genetic
markers can be used to identify plants that contain a desired
genotype at one locus, or at several unlinked or linked loci (e.g.,
a haplotype), and that would be expected to transfer the desired
genotype, along with a desired phenotype to their progeny. The
present invention provides the means to identify plants,
particularly soybean plants, that are tolerant, exhibit improved
tolerance or are susceptible to low iron growth conditions by
identifying plants having a specified allele at one of those loci,
e.g., S60210-TB, SAC1006, SATT391, SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT510, SATT335, P5219A-1, P7659A-2,
SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367, SATT495,
P10649C-3, SATT613, SATT257, SATT581 and/or SATT153. Similarly, by
identifying plants lacking the desired marker locus, susceptible or
less tolerant plants can be identified, and, e.g., eliminated from
subsequent crosses. Similarly, these marker loci can be
introgressed into any desired genomic background, germplasm, plant,
line, variety, etc., as part of an overall MAS breeding program
designed to enhance soybean yield.
[0237] The invention also provides chromosome QTL intervals that
find equal use in MAS to select plants that demonstrate low iron
tolerance or improved tolerance. Similarly, the QTL intervals can
also be used to counter-select plants that are susceptible or have
reduced tolerance to low iron growth conditions. Any marker that
maps within the QTL interval (including the termini of the
intervals) finds use with the invention. These intervals are
defined by the following pairs of markers:
[0238] (a) 560210-TB and SATT391 (LG-C1);
[0239] (b) P10598A-1 and SATT334 (LG-F);
[0240] (c) SATT510 and SATT335 (LG-F);
[0241] (d) P5219A-1 and P7659A-2 (LG-G);
[0242] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0243] (f) SATT451 and SATT367 (LG-I);
[0244] (g) SATT495 and P10649C-3 (LG-L); and
[0245] (h) SATT250 and SATT346 (LG-M).
[0246] In general, MAS uses polymorphic markers that have been
identified as having a significant likelihood of co-segregation
with a tolerance trait. Such markers are presumed to map near a
gene or genes that give the plant its tolerance phenotype, and are
considered indicators for the desired trait, and are termed QTL
markers. Plants are tested for the presence of a desired allele in
the QTL marker. The most preferred markers (or marker alleles) are
those that have the strongest association with the tolerance
trait.
[0247] Linkage analysis is used to determine which polymorphic
marker allele demonstrates a statistical likelihood of
co-segregation with the tolerance phenotype (thus, a "tolerance
marker allele"). Following identification of a marker allele for
co-segregation with the tolerance phenotype, it is possible to use
this marker for rapid, accurate screening of plant lines for the
tolerance allele without the need to grow the plants through their
life cycle and await phenotypic evaluations, and furthermore,
permits genetic selection for the particular tolerance allele even
when the molecular identity of the actual tolerance QTL is unknown.
Tissue samples can be taken, for example, from the first leaf of
the plant and screened with the appropriate molecular marker, and
it is rapidly determined which progeny will advance. Linked markers
also remove the impact of environmental factors that can often
influence phenotypic expression.
[0248] A polymorphic QTL marker locus can be used to select plants
that contain the marker allele (or alleles) that correlate with the
desired tolerance phenotype, typically called marker-assisted
selection (MAS). In brief, a nucleic acid corresponding to the
marker nucleic acid allele is detected in a biological sample from
a plant to be selected. This detection can 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,
hybridization of primers followed by PCR amplification of a region
of the marker, or the like. A variety of procedures for detecting
markers are described herein, e.g., in the section entitled
"TECHNIQUES FOR MARKER DETECTION." After the presence (or absence)
of a particular marker allele in the biological sample is verified,
the plant is selected, e.g., used to make progeny plants by
selective breeding.
[0249] Soybean plant breeders desire combinations of tolerance loci
with genes for high yield and other desirable traits to develop
improved soybean varieties. Screening large numbers of samples by
non-molecular methods (e.g., trait evaluation in soybean plants)
can be expensive, time consuming, and unreliable. Use of the
polymorphic markers described herein, when genetically-linked to
tolerance loci, provide an effective method for selecting resistant
varieties in breeding programs. For example, one advantage of
marker-assisted selection over field evaluations for tolerance
resistance is that MAS can be done at any time of year, regardless
of the growing season. Moreover, environmental effects are largely
irrelevant to marker-assisted selection.
[0250] When a population is segregating for multiple loci affecting
one or multiple traits, e.g., multiple loci involved in tolerance,
or multiple loci each involved in tolerance or resistance to
different diseases, the efficiency of MAS compared to phenotypic
screening becomes even greater, because all of the loci can be
evaluated in the lab together from a single sample of DNA. In the
present instance, the S60210-TB, SAC1006, SATT391, SAC1724,
SATT307, P13073A-1, P10598A-1, SATT334, SATT510, SATT335, P5219A-1,
P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367,
SATT495, P10649C-3, SATT613, SATT257, SATT581 and SATT153 markers,
as well as any of the chromosome intervals:
[0251] (a) S60210-TB and SATT391 (LG-C1);
[0252] (b) P10598A-1 and SATT334 (LG-F);
[0253] (c) SATT510 and SATT335 (LG-F);
[0254] (d) P5219A-1 and P7659A-2 (LG-G);
[0255] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0256] (f) SATT451 and SATT367 (LG-I);
[0257] (g) SATT495 and P10649C-3 (LG-L); and
[0258] (h) SATT250 and SATT346 (LG-M),
can be assayed simultaneously or sequentially in a single sample or
population of samples.
[0259] Another use of MAS in plant breeding is to assist the
recovery of the recurrent parent genotype by backcross breeding.
Backcross breeding is the process of crossing a progeny back to one
of its parents or parent lines. Backcrossing is usually done for
the purpose of introgressing one or a few loci from a donor parent
(e.g., a parent comprising desirable tolerance marker loci) into an
otherwise desirable genetic background from the recurrent parent
(e.g., an otherwise high yielding soybean line). The more cycles of
backcrossing that are done, the greater the genetic contribution of
the recurrent parent to the resulting introgressed variety. This is
often necessary, because tolerant plants may be otherwise
undesirable, e.g., due to low yield, low fecundity, or the like. In
contrast, strains which are the result of intensive breeding
programs may have excellent yield, fecundity or the like, merely
being deficient in one desired trait such as tolerance to low iron
growth conditions.
[0260] The presence and/or absence of a particular genetic marker
or allele, e.g., S60210-TB, SAC1006, SATT391, SAC1724, SATT307,
P13073A-1, P10598A-1, SATT334, SATT510, SATT335, P5219A-1,
P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367,
SATT495, P10649C-3, SATT613, SATT257, SATT581 and/or SATT153
markers, as well as any of the chromosome intervals
[0261] (a) S60210-TB and SATT391 (LG-C1);
[0262] (b) P10598A-1 and SATT334 (LG-F);
[0263] (c) SATT510 and SATT335 (LG-F);
[0264] (d) P5219A-1 and P7659A-2 (LG-G);
[0265] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0266] (f) SATT451 and SATT367 (LG-I);
[0267] (g) SATT495 and P10649C-3 (LG-L); and
[0268] (h) SATT250 and SATT346 (LG-M),
in the genome of a plant is made by any method noted herein. If the
nucleic acids from the plant are positive for a desired genetic
marker allele, the plant can be self fertilized to create a true
breeding line with the same genotype, or it can be crossed with a
plant with the same marker or with other desired characteristics to
create a sexually crossed hybrid generation.
[0269] Introgression of Favorable Alleles--Efficient Backcrossing
of Tolerance Markers into Elite Lines
[0270] One application of MAS, in the context of the present
invention is to use the tolerance or improved tolerance markers to
increase the efficiency of an introgression or backcrossing effort
aimed at introducing a tolerance QTL into a desired (typically high
yielding) background. In marker assisted backcrossing of specific
markers (and associated QTL) from a donor source, e.g., to an elite
or exotic genetic background, one selects among backcross progeny
for the donor trait and then uses repeated backcrossing to the
elite or exotic line to reconstitute as much of the elite/exotic
background's genome as possible.
[0271] Thus, the markers and methods of the present invention can
be utilized to guide marker assisted selection or breeding of
soybean varieties with the desired complement (set) of allelic
forms of chromosome segments associated with superior agronomic
performance (tolerance, along with any other available markers for
yield, disease resistance, etc.). Any of the disclosed marker
alleles can be introduced into a soybean line via introgression, by
traditional breeding (or introduced via transformation, or both) to
yield a soybean plant with superior agronomic performance. The
number of alleles associated with tolerance that can be introduced
or be present in a soybean plant of the present invention ranges
from 1 to the number of alleles disclosed herein, each integer of
which is incorporated herein as if explicitly recited.
[0272] The present invention also extends to a method of making a
progeny soybean plant and these progeny soybean plants, per se. The
method comprises crossing a first parent soybean plant with a
second soybean plant and growing the female soybean plant under
plant growth conditions to yield soybean plant progeny. Methods of
crossing and growing soybean plants are well within the ability of
those of ordinary skill in the art. Such soybean plant progeny can
be assayed for alleles associated with tolerance and, thereby, the
desired progeny selected. Such progeny plants or seed can be sold
commercially for soybean production, used for food, processed to
obtain a desired constituent of the soybean, or further utilized in
subsequent rounds of breeding. At least one of the first or second
soybean plants is a soybean plant of the present invention in that
it comprises at least one of the allelic forms of the markers of
the present invention, such that the progeny are capable of
inheriting the allele.
[0273] Often, a method of the present invention is applied to at
least one related soybean plant such as from progenitor or
descendant lines in the subject soybean plants pedigree such that
inheritance of the desired tolerance allele can be traced. The
number of generations separating the soybean 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
soybean plant will be subject to the method (i.e., one generation
of separation).
[0274] Introgression of Favorable Alleles--Incorporation of
"Exotic" Germplasm while Maintaining Breeding Progress
[0275] Genetic diversity is important for long term genetic gain in
any breeding program. With limited diversity, genetic gain will
eventually plateau when all of the favorable alleles have been
fixed within the elite population. One objective 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.
[0276] For example, the markers of the present invention can be
used for MAS in crosses involving elite x exotic soybean lines by
subjecting the segregating progeny to MAS to maintain major yield
alleles, along with the tolerance marker alleles herein.
Positional Cloning
[0277] The molecular marker loci and alleles of the present
invention, e.g., S60210-TB, SAC1006, SATT391, SAC1724, SATT307,
P13073A-1, P10598A-1, SATT334, SATT510, SATT335, P5219A-1,
P7659A-2, SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367,
SATT495, P10649C-3, SATT613, SATT257, SATT581 and SATT153 markers,
as well as any of the chromosome intervals
[0278] (a) S60210-TB and SATT391 (LG-C1);
[0279] (b) P10598A-1 and SATT334 (LG-F);
[0280] (c) SATT510 and SATT335 (LG-F);
[0281] (d) P5219A-1 and P7659A-2 (LG-G);
[0282] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0283] (f) SATT451 and SATT367 (LG-I);
[0284] (g) SATT495 and P10649C-3 (LG-L); and
[0285] (h) SATT250 and SATT346 (LG-M),
can be used, as indicated previously, to identify a tolerance QTL,
which can be cloned by well established procedures, e.g., as
described in detail in Ausubel, Berger and Sambrook, herein.
[0286] These tolerance clones are first identified by their genetic
linkage to markers of the present invention. Isolation of a nucleic
acid of interest is achieved by any number of methods as discussed
in detail in such references as Ausubel, Berger and Sambrook,
herein, and Clark, Ed. (1997) Plant Molecular Biology: A Laboratory
Manual Springer-Verlag, Berlin.
[0287] For example, "positional gene cloning" uses the proximity of
a tolerance marker to physically define an isolated chromosomal
fragment containing a tolerance QTL gene. The isolated chromosomal
fragment can be produced by such well known methods as digesting
chromosomal DNA with one or more restriction enzymes, or by
amplifying a chromosomal region in a polymerase chain reaction
(PCR), or any suitable alternative amplification reaction. The
digested or amplified fragment is typically ligated into a vector
suitable for replication, and, e.g., expression, of the inserted
fragment. Markers that are adjacent to an open reading frame (ORF)
associated with a phenotypic trait can hybridize to a DNA clone
(e.g., a clone from a genomic DNA library), thereby identifying a
clone on which an ORF (or a fragment of an ORF) is located. If the
marker is more distant, a fragment containing the open reading
frame is identified by successive rounds of screening and isolation
of clones which together comprise a contiguous sequence of DNA, a
process termed "chromosome walking", resulting in a "contig" or
"contig map." Protocols sufficient to guide one of skill through
the isolation of clones associated with linked markers are found
in, e.g. Berger, Sambrook and Ausubel, all herein.
Generation of Transgenic Cells and Plants
[0288] The present invention also relates to host cells and
organisms which are transformed with nucleic acids corresponding to
tolerance QTL identified according to the invention. For example,
such nucleic acids include chromosome intervals (e.g., genomic
fragments), ORFs and/or cDNAs that encode a tolerance or improved
tolerance trait. Additionally, the invention provides for the
production of polypeptides that provide tolerance or improved
tolerance by recombinant techniques.
[0289] General texts which describe molecular biological techniques
for the cloning and manipulation of nucleic acids and production of
encoded polypeptides include Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular
Cloning--A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2004 or later) ("Ausubel")). These texts describe
mutagenesis, the use of vectors, promoters and many other relevant
topics related to, e.g., the generation of clones that comprise
nucleic acids of interest, e.g., marker loci, marker probes, QTL
that segregate with marker loci, etc.
[0290] Host cells are genetically engineered (e.g., transduced,
transfected, transformed, etc.) with the vectors of this invention
(e.g., vectors, such as expression vectors which comprise an ORF
derived from or related to a tolerance QTL) which can be, for
example, a cloning vector, a shuttle vector or an expression
vector. Such vectors are, for example, in the form of a plasmid, a
phagemid, an agrobacterium, a virus, a naked polynucleotide (linear
or circular), or a conjugated polynucleotide. Vectors can be
introduced into bacteria, especially for the purpose of propagation
and expansion. The vectors are also introduced into plant tissues,
cultured plant cells or plant protoplasts by a variety of standard
methods known in the art, including but not limited to
electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82;
5824), infection by viral vectors such as cauliflower mosaic virus
(CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors
(Academic Press, New York, pp. 549-560; Howell U.S. Pat. No.
4,407,956), high velocity ballistic penetration by small particles
with the nucleic acid either within the matrix of small beads or
particles, or on the surface (Klein et al. (1987) Nature 327; 70),
use of pollen as vector (WO 85/01856), or use of Agrobacterium
tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA
fragments are cloned. The T-DNA plasmid is transmitted to plant
cells upon infection by Agrobacterium tumefaciens, and a portion is
stably integrated into the plant genome (Horsch et al. (1984)
Science 233; 496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA
80; 4803). Additional details regarding nucleic acid introduction
methods are found in Sambrook, Berger and Ausubel, infra. The
method of introducing a nucleic acid of the present invention into
a host cell is not critical to the instant invention, and it is not
intended that the invention be limited to any particular method for
introducing exogenous genetic material into a host cell. Thus, any
suitable method, e.g., including but not limited to the methods
provided herein, which provides for effective introduction of a
nucleic acid into a cell or protoplast can be employed and finds
use with the invention.
[0291] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for such activities as, for
example, activating promoters or selecting transformants. These
cells can optionally be cultured into transgenic plants. In
addition to Sambrook, Berger and Ausubel, all infra, Plant
regeneration from cultured protoplasts is described in Evans et al.
(1983) "Protoplast Isolation and Culture," Handbook of Plant Cell
Cultures 1, 124-176 (MacMillan Publishing Co., New York; Davey
(1983) "Recent Developments in the Culture and Regeneration of
Plant Protoplasts," Protoplasts, pp. 12-29, (Birkhauser, Basel);
Dale (1983) "Protoplast Culture and Plant Regeneration of Cereals
and Other Recalcitrant Crops," Protoplasts pp. 31-41, (Birkhauser,
Basel); Binding (1985) "Regeneration of Plants," Plant Protoplasts,
pp. 21-73, (CRC Press, Boca Raton, Fla.). Additional details
regarding plant cell culture and regeneration include Payne et al.
(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley
& Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995)
Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer
Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Plant
Molecular Biolgy (1993) R. R. D. Croy, Ed. Bios Scientific
Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in
general are also set forth in Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla. Additional
information for cell culture is found in available commercial
literature such as the Life Science Research Cell Culture Catalogue
(1998) from Sigma-Aldrich, Inc (St Louis, Mo.) ("Sigma-LSRCCC")
and, e.g., the Plant Culture Catalogue and supplement (e.g., 1997
or later) also from Sigma-Aldrich, Inc (St Louis, Mo.)
("Sigma-PCCS").
[0292] The present invention also relates to the production of
transgenic organisms, which may be bacteria, yeast, fungi, animals
or plants, transduced with the nucleic acids of the invention
(e.g., nucleic acids comprising the marker loci and/or QTL noted
herein). A thorough discussion of techniques relevant to bacteria,
unicellular eukaryotes and cell culture is found in references
enumerated herein and are briefly outlined as follows. Several
well-known methods of introducing target nucleic acids into
bacterial cells are available, any of which may be used in the
present invention. These include: fusion of the recipient cells
with bacterial protoplasts containing the DNA, treatment of the
cells with liposomes containing the DNA, electroporation,
projectile bombardment (biolistics), carbon fiber delivery, and
infection with viral vectors (discussed further, below), etc.
Bacterial cells can be used to amplify the number of plasmids
containing DNA constructs of this invention. The bacteria are grown
to log phase and the plasmids within the bacteria can be isolated
by a variety of methods known in the art (see, for instance,
Sambrook). In addition, a plethora of kits are commercially
available for the purification of plasmids from bacteria. For their
proper use, follow the manufacturer's instructions (see, for
example, EasyPrep.TM., FlexiPrep.TM., both from Pharmacia Biotech;
StrataClean.TM., from Stratagene; and, QIAprep.TM. from Qiagen).
The isolated and purified plasmids are then further manipulated to
produce other plasmids, used to transfect plant cells or
incorporated into Agrobacterium tumefaciens related vectors to
infect plants. Typical vectors contain transcription and
translation terminators, transcription and translation initiation
sequences, and promoters useful for regulation of the expression of
the particular target nucleic acid. The vectors optionally comprise
generic expression cassettes containing at least one independent
terminator sequence, sequences permitting replication of the
cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle
vectors) and selection markers for both prokaryotic and eukaryotic
systems. Vectors are suitable for replication and integration in
prokaryotes, eukaryotes, or preferably both. See, Giliman &
Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731;
Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel,
Sambrook, Berger (all infra). A catalogue of Bacteria and
Bacteriophages useful for cloning is provided, e.g., by the ATCC,
e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992)
Gherna et al. (eds) published by the ATCC. Additional basic
procedures for sequencing, cloning and other aspects of molecular
biology and underlying theoretical considerations are also found in
Watson et al. (1992) Recombinant DNA, Second Edition, Scientific
American Books, NY. In addition, essentially any nucleic acid (and
virtually any labeled nucleic acid, whether standard or
non-standard) can be custom or standard ordered from any of a
variety of commercial sources, such as the Midland Certified
Reagent Company (Midland, Tex.), The Great American Gene Company
(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon
Technologies Inc. (Alameda, Calif.) and many others.
[0293] Introducing Nucleic Acids into Plants.
[0294] Embodiments of the present invention pertain to the
production of transgenic plants comprising the cloned nucleic
acids, e.g., isolated ORFs and cDNAs encoding tolerance genes.
Techniques for transforming plant cells with nucleic acids are
widely available and can be readily adapted to the invention. In
addition to Berger, Ausubel and Sambrook, all infra, useful general
references for plant cell cloning, culture and regeneration include
Jones (ed) (1995) Plant Gene Transfer and Expression
Protocols--Methods in Molecular Biology, Volume 49 Humana Press
Towata N.J.; Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y. (Payne);
and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ
Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag
(Berlin Heidelberg New York) (Gamborg). A variety of cell culture
media are described in Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas).
Additional information for plant cell culture is found in available
commercial literature such as the Life Science Research Cell
Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)
(Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and
supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.)
(Sigma-PCCS). Additional details regarding plant cell culture are
found in Croy, (ed.) (1993) Plant Molecular Biology, Bios
Scientific Publishers, Oxford, U.K.
[0295] The nucleic acid constructs of the invention, e.g.,
plasmids, cosmids, artificial chromosomes, DNA and RNA
polynucleotides, are introduced into plant cells, either in culture
or in the organs of a plant by a variety of conventional
techniques. Where the sequence is expressed, the sequence is
optionally combined with transcriptional and translational
initiation regulatory sequences which direct the transcription or
translation of the sequence from the exogenous DNA in the intended
tissues of the transformed plant.
[0296] Isolated nucleic acid acids of the present invention can be
introduced into plants according to any of a variety of techniques
known in the art. Techniques for transforming a wide variety of
higher plant species are also well known and described in widely
available technical, scientific, and patent literature. See, for
example, Weising et al. (1988) Ann. Rev. Genet. 22:421-477.
[0297] The DNA constructs of the invention, for example plasmids,
phagemids, cosmids, phage, naked or variously conjugated-DNA
polynucleotides, (e.g., polylysine-conjugated DNA,
peptide-conjugated DNA, liposome-conjugated DNA, etc.), or
artificial chromosomes, can be introduced directly into the genomic
DNA of the plant cell using techniques such as electroporation and
microinjection of plant cell protoplasts, or the DNA constructs can
be introduced directly to plant cells using ballistic methods, such
as DNA particle bombardment.
[0298] Microinjection techniques for injecting plant, e.g., cells,
embryos, callus and protoplasts, are known in the art and well
described in the scientific and patent literature. For example, a
number of methods are described in Jones (ed) (1995) Plant Gene
Transfer and Expression Protocols--Methods in Molecular Biology,
Volume 49 Humana Press, Towata, N.J., as well as in the other
references noted herein and available in the literature.
[0299] For example, the introduction of DNA constructs using
polyethylene glycol precipitation is described in Paszkowski, et
al., EMBO J. 3:2717 (1984). Electroporation techniques are
described in Fromm, et al., Proc. Natl. Acad. Sci. USA 82:5824
(1985). Ballistic transformation techniques are described in Klein,
et al., Nature 327:70-73 (1987). Additional details are found in
Jones (1995) and Gamborg and Phillips (1995), supra, and in U.S.
Pat. No. 5,990,387.
[0300] Alternatively, and in some cases preferably, Agrobacterium
mediated transformation is employed to generate transgenic plants.
Agrobacterium-mediated transformation techniques, including
disarming and use of binary vectors, are also well described in the
scientific literature. See, for example, Horsch, et al. (1984)
Science 233:496; and Fraley et al. (1984) Proc. Nat'l. Acad. Sci.
USA 80:4803 and recently reviewed in Hansen and Chilton (1998)
Current Topics in Microbiology 240:22 and Das (1998) Subcellular
Biochemistry 29: Plant Microbe Interactions, pp 343-363.
[0301] DNA constructs are optionally combined with suitable T-DNA
flanking regions and introduced into a conventional Agrobacterium
tumefaciens host vector. The virulence functions of the
Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. See, U.S. Pat. No. 5,591,616. Although
Agrobacterium is useful primarily in dicots, certain monocots can
be transformed by Agrobacterium. For instance, Agrobacterium
transformation of maize is described in U.S. Pat. No.
5,550,318.
[0302] Other methods of transfection or transformation include (1)
Agrobacterium rhizogenes-mediated transformation (see, e.g.,
Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, PWJ
Rigby, Ed., London, Academic Press; and Lichtenstein; C. P., and
Draper (1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford,
IRI Press; WO 88/02405, published Apr. 7, 1988, describes the use
of A. rhizogenes strain A4 and its Ri plasmid along with A.
tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA
uptake (see, e.g., Freeman et al. (1984) Plant Cell Physiol.
25:1353), (3) the vortexing method (see, e.g., Kindle (1990) Proc.
Natl. Acad. Sci., (USA) 87:1228.
[0303] DNA can also be introduced into plants by direct DNA
transfer into pollen as described by Zhou et al. (1983) Methods in
Enzymology, 101:433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo
et al. (1988) Plant Mol. Biol. Reporter 6:165. Expression of
polypeptide coding genes can be obtained by injection of the DNA
into reproductive organs of a plant as described by Pena et al.
(1987) Nature 325:274. DNA can also be injected directly into the
cells of immature embryos and the desiccated embryos rehydrated as
described by Neuhaus et al. (1987) Theor. Appl. Genet. 75:30; and
Benbrook et al. (1986) in Proceedings Bio Expo Butterworth,
Stoneham, Mass., pp. 27-54. A variety of plant viruses that can be
employed as vectors are known in the art and include cauliflower
mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco
mosaic virus.
[0304] Generation/Regeneration of Transgenic Plants
[0305] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant that possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker
which has been introduced together with the desired nucleotide
sequences. Plant regeneration from cultured protoplasts is
described in Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg
and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg New York); Evans et al. (1983) Protoplasts Isolation and
Culture, Handbook of Plant Cell Culture pp. 124-176, Macmillian
Publishing Company, New York; and Binding (1985) Regeneration of
Plants, Plant Protoplasts pp. 21-73, CRC Press, Boca Raton.
Regeneration can also be obtained from plant callus, explants,
somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth.
12:145; McGranahan, et al. (1990) Plant Cell Rep. 8:512) organs, or
parts thereof. Such regeneration techniques are described generally
in Klee et al. (1987)., Ann. Rev. of Plant Phys. 38:467-486.
Additional details are found in Payne (1992) and Jones (1995), both
supra, and Weissbach and Weissbach, eds. (1988) Methods for Plant
Molecular Biology Academic Press, Inc., San Diego, Calif. This
regeneration and growth process includes the steps of selection of
transformant cells and shoots, rooting the transformant shoots and
growth of the plantlets in soil. These methods are adapted to the
invention to produce transgenic plants bearing QTLs and other genes
isolated according to the methods of the invention.
[0306] In addition, the regeneration of plants containing the
polynucleotide of the present invention and introduced by
Agrobacterium into cells of leaf explants can be achieved as
described by Horsch et al. (1985) Science 227:1229-1231. In this
procedure, transformants are grown in the presence of a selection
agent and in a medium that induces the regeneration of shoots in
the plant species being transformed as described by Fraley et al.
(1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803. This procedure
typically produces shoots within two to four weeks and these
transformant shoots are then transferred to an appropriate
root-inducing medium containing the selective agent and an
antibiotic to prevent bacterial growth. Transgenic plants of the
present invention may be fertile or sterile.
[0307] It is not intended that plant transformation and expression
of polypeptides that provide disease resistance, as provided by the
present invention, be limited to soybean species. Indeed, it is
contemplated that the polypeptides that provide disease tolerance
in soybean can also provide disease resistance when transformed and
expressed in other agronomically and horticulturally important
species. Such species include primarily dicots, e.g., 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).
[0308] Additionally, preferred targets for modification with the
nucleic acids of the invention, as well as those specified above,
plants from 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.
[0309] Common crop plants which are targets of the present
invention include 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.
[0310] In construction of recombinant expression cassettes of the
invention, which include, for example, helper plasmids comprising
virulence functions, and plasmids or viruses comprising exogenous
DNA sequences such as structural genes, a plant promoter fragment
is optionally employed which directs expression of a nucleic acid
in any or all tissues of a regenerated plant. Examples of
constitutive promoters include the cauliflower mosaic virus (CaMV)
35S transcription initiation region, the 1'- or 2'-promoter derived
from T-DNA of Agrobacterium tumefaciens, and other transcription
initiation regions from various plant genes known to those of
skill. Alternatively, the plant promoter may direct expression of
the polynucleotide of the invention in a specific tissue
(tissue-specific promoters) or may be otherwise under more precise
environmental control (inducible promoters). Examples of
tissue-specific promoters under developmental control include
promoters that initiate transcription only in certain tissues, such
as fruit, seeds or flowers.
[0311] Any of a number of promoters which direct transcription in
plant cells can be suitable. The promoter can be either
constitutive or inducible. In addition to the promoters noted
above, promoters of bacterial origin that operate in plants include
the octopine synthase promoter, the nopaline synthase promoter and
other promoters derived from native Ti plasmids. See,
Herrara-Estrella et al. (1983), Nature, 303:209. Viral promoters
include the 35S and 19S RNA promoters of cauliflower mosaic virus.
See, Odell et al. (1985) Nature, 313:810. Other plant promoters
include Kunitz trypsin inhibitor promoter (KTI), SCP1, SUP, UCD3,
the ribulose-1,3-bisphosphate carboxylase small subunit promoter
and the phaseolin promoter. The promoter sequence from the E8 gene
and other genes may also be used. The isolation and sequence of the
E8 promoter is described in detail in Deikman and Fischer (1988)
EMBO J. 7:3315. Many other promoters are in current use and can be
coupled to an exogenous DNA sequence to direct expression of the
nucleic acid.
[0312] If expression of a polypeptide from a cDNA is desired, a
polyadenylation region at the 3'-end of the coding region is
typically included. The polyadenylation region can be derived from
the natural gene, from a variety of other plant genes, or from,
e.g., T-DNA.
[0313] The vector comprising the sequences (e.g., promoters or
coding regions) from genes encoding expression products and
transgenes of the invention will typically include a nucleic acid
subsequence, a marker gene which confers a selectable, or
alternatively, a screenable, phenotype on plant cells. For example,
the marker can encode biocide tolerance, particularly antibiotic
tolerance, such as tolerance to kanamycin, G418, bleomycin,
hygromycin, or herbicide tolerance, such as tolerance to
chlorosluforon, or phosphinothricin (the active ingredient in the
herbicides bialaphos or Basta). See, e.g., Padgette et al. (1996)
In: Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis
Publishers, Boca Raton ("Padgette, 1996"). For example, crop
selectivity to specific herbicides can be conferred by engineering
genes into crops that encode appropriate herbicide metabolizing
enzymes from other organisms, such as microbes. See, Vasil (1996)
In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91, CRC Lewis
Publishers, Boca Raton) ("Vasil", 1996).
[0314] One of skill will recognize that after the recombinant
expression cassette is stably incorporated in transgenic plants and
confirmed to be operable, it can be introduced into other plants by
sexual crossing. Any of a number of standard breeding techniques
can be used, depending upon the species to be crossed. In
vegetatively propagated crops, mature transgenic plants can be
propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants. Selection of
desirable transgenics is made and new varieties are obtained and
propagated vegetatively for commercial use. In seed propagated
crops, mature transgenic plants can be self crossed to produce a
homozygous inbred plant. The inbred plant produces seed containing
the newly introduced heterologous nucleic acid. These seeds can be
grown to produce plants that would produce the selected phenotype.
Parts obtained from the regenerated plant, such as flowers, seeds,
leaves, branches, fruit, and the like are included in the
invention, provided that these parts comprise cells comprising the
isolated nucleic acid of the present invention. Progeny and
variants, and mutants of the regenerated plants are also included
within the scope of the invention, provided that these parts
comprise the introduced nucleic acid sequences.
[0315] Transgenic or introgressed plants expressing a
polynucleotide of the present invention can be screened for
transmission of the nucleic acid of the present invention by, for
example, standard nucleic acid detection methods or by immunoblot
protocols. Expression at the RNA level can be determined to
identify and quantitate expression-positive plants. Standard
techniques for RNA analysis can be employed and include RT-PCR
amplification assays using oligonucleotide primers designed to
amplify only heterologous or introgressed RNA templates and
solution hybridization assays using marker or linked QTL specific
probes. Plants can also be analyzed for protein expression, e.g.,
by Western immunoblot analysis using antibodies that recognize the
encoded polypeptides. In addition, in situ hybridization and
immunocytochemistry according to standard protocols can be done
using heterologous nucleic acid specific polynucleotide probes and
antibodies, respectively, to localize sites of expression within
transgenic tissue. Generally, a number of transgenic lines are
usually screened for the incorporated nucleic acid to identify and
select plants with the most appropriate expression profiles.
[0316] A preferred embodiment of the invention is a transgenic
plant that is homozygous for the added heterologous nucleic acid;
e.g., a transgenic plant that contains two added nucleic acid
sequence copies, e.g., a gene at the same locus on each chromosome
of a homologous chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (self-fertilizing) a heterozygous
transgenic plant that contains a single added heterologous nucleic
acid, germinating some of the seed produced and analyzing the
resulting plants produced for altered expression of a
polynucleotide of the present invention relative to a control plant
(e.g., a native, non-transgenic plant). Back-crossing to a parental
plant and out-crossing with a non-transgenic plant can be used to
introgress the heterologous nucleic acid into a selected background
(e.g., an elite or exotic soybean line).
Methods for Identifying Soybean Plants Tolerant to Iron Deficient
Growth Conditions
[0317] Experienced plant breeders can recognize tolerant soybean
plants in the field, and can select the tolerant individuals or
populations for breeding purposes or for propagation. In this
context, the plant breeder recognizes "tolerant" and
"non-tolerant," or "susceptible" soybean plants in fortuitous
naturally-occurring field observations.
[0318] However, plant breeding practitioners will appreciate that
plant tolerance is a phenotypic spectrum consisting of extremes in
tolerance, susceptibility and a continuum of intermediate
phenotypes. Tolerance also varies due to environmental effects.
Evaluation of phenotypes using reproducible assays and tolerance
scoring methods are of value to scientists who seek to identify
genetic loci that impart tolerance, conduct marker assisted
selection to create tolerant soybean populations, and for
introgression techniques to breed a tolerance trait into an elite
soybean line, for example.
[0319] In contrast to fortuitous field observations that classify
plants as either "tolerant" or ":susceptible," various methods are
known in the art for determining (and quantitating) the tolerance
of a soybean plant to iron-deficient growth conditions. These
techniques can be applied to different fields at different times,
or to experimental greenhouse or laboratory settings, and provide
approximate tolerance scores that can be used to characterize a
given strain regardless of growth conditions or location. See, for
example, Diers et al. (1992) "Possible identification of
quantitative trait loci affecting iron efficiency in soybean," J.
Plant Nutr. 15:2127-2136; Dahiya and M. Singh (1979) "Effect of
salinity, alkalinity and iron sources on availability of iron,"
Plant and Soil 51:13-18; and Gonzalez-Vallejo et al. (2000) "Iron
Deficiency Decreases the Fe(III)-Chelate Reducing Activity of Leaf
Protoplasts" Plant Physiol. 122 (2): 337-344.
[0320] The degree of IDC in a particular plant or stand of plants
can be quantitated by using a system to score the severity of the
disease in each plant. A plant strain or a number of plant strains
are planted and grown in a single stand in soil that is known to
produce chlorotic plants as a result of iron deficiency ("field
screens", i.e., in fields that have previously demonstrated IDC),
or alternatively, in controlled nursery conditions. When the assay
is conducted in controlled nursery conditions, defined soil can be
used, where the concentration of iron (e.g., available iron) has
been previously measured. The plants can be scored at maturity, or
at any time before maturity. The scoring system rates each plant on
a scale of one (most susceptible; most severe disease) to nine
(most tolerant; no disease), as follows:
TABLE-US-00006 TABLE 1 Plant or Plant Stand Score Symptoms 1 Most
plants are completely dead. The plants that are still alive are
approximately 10% of normal height, and have very little living
tissue. 2 Most leaves are almost dead, most stems are still green.
Plants are severely stunted (10-20% of normal height). 3 Most
plants are yellow and necrosis is seen on most leaves. Most plants
are approximately 20-40% of normal height. 4 Most plants are
yellow, and necrosis is seen on the edges of less than half the
leaves. Most plants are approximately 50% of normal height. 5 Most
plants are light green to yellow, and no necrosis is seen on the
leaves. Most plants are stunted (50-75% of normal height). 6 More
than half the plants show moderate chlorosis, but no necrosis is
seen on the leaves. 7 Less than half of the plants showing moderate
chlorosis (light green leaves). 8 A few plants are showing very
light chlorosis on one or two leaves. 9 All plants are normal green
color.
[0321] It will be appreciated that any such scale is relative, and
furthermore, there may be variability between practitioners as to
how the individual plants and the entire stand as a whole are
scored. Optionally, the degree of chlorosis can be measured using a
chlorophyll meter, e.g., a Minolta SPAD-502 Chlorophyll Meter,
where readings off a single plant or a stand of plants can be made.
Optionally, multiple readings can be obtained and averaged.
[0322] The IDC scoring of soybean stands can occur at any time. For
example, plots can be scored in the early season, typically mid
July (depending on geographic latitude), so that the results can be
used in making crossing decisions. Alternatively, soybean plots can
be scored in the late season, which generally yields more precise
data.
[0323] In general, while there is a certain amount of subjectivity
to assigning severity measurements for disease symptoms, assignment
to a given scale as noted above is well within the skill of a
practitioner in the field. Measurements can also be averaged across
multiple scorers to reduce variation in field measurements.
[0324] Although protocols using field nurseries known to produce
chlorotic plants can be used in assessing tolerance, it is typical
for tolerance ratings to be based on actual field observations of
fortuitous natural disease incidence, with the information
corresponding to disease incidence for a cultivar being averaged
over many locations and, typically, several seasons of crop
plantings. Optionally, field stands or nursery/greenhouse plantings
can be co-cultivated with IDC susceptibility "reference checks." A
reference check is a planting of soybean strains with known
susceptibilities to IDC, for example, highly tolerant strains and
highly susceptible strains. This parallel planting can aid the
breeder in scoring disease severity by allowing the breeder to
compare the plant pathology in the experimental stands with the
plant pathology in the reference stands.
[0325] When plants are studied in a fortuitous natural field
setting, if there is no chlorosis present, the rating system above
can not be used, because the existence of iron-deficient soil can
not be ascertained. However, if some number of plants demonstrate
IDC symptoms, the growth conditions in that field can be assumed to
be iron-deficient, and the entire stand can be scored as described
above. These scores can accumulate over multiple locations and
years to show disease tolerance for given cultivars. Thus, older
lines can have more years of observation than newer ones etc.
However, relative tolerance measurements between different strains
in the same field at the same time can easily be made using the
scoring system noted above. Furthermore, the tolerance ratings can
be updated and refined each year based on the previous year's
observations in the field. The experiments described herein (see,
Example 1) scored soybean tolerance to iron deficiency using the
scale described above at nursery locations at several locations and
over several years.
[0326] In assessing linkage of markers to tolerance, either
quantitative or qualitative approaches can be used. For example, an
average rating for each line that is a single number (for each
line) from 1 to 9 can be assessed for linkage. This approach is
quantitative and uses the scores from lines that have both marker
data and IDC scores. In an alternative approach, an "intergroup"
comparison of tolerant versus susceptible lines is used. In this
approach, those soybean lines that are considered to be
representative of either the tolerant of susceptible classes are
used for assessing linkage. A list of tolerant lines is
constructed, e.g., having average rating of 6 to 9 on the above
scale (when averaged over years and locations). The susceptible
lines are those with an average rating of 1 to 4 over years and
locations. Only lines that can be reliably placed in the 2 groups
are used. Once a line is included in the group, it is treated as an
equal in that group--i.e. the actual quantitative ratings are not
used.
Automated Detection/Correlation Systems of the Invention
[0327] In some embodiments, the present invention includes an
automated system for detecting markers of the invention and/or
correlating the markers with a desired phenotype (e.g., tolerance).
Thus, a typical system can include a set of marker probes or
primers configured to detect at least one favorable allele of one
or more marker locus associated with tolerance or improved
tolerance to Phytophthora infection. These probes or primers are
configured to detect the marker alleles noted in the tables and
examples herein, e.g., using any available allele detection format,
e.g., solid or liquid phase array based detection,
microfluidic-based sample detection, etc.
[0328] For example, in one embodiment, the marker locus is
S60210-TB, SAC1006, SATT391, SAC1724, SATT307, P13073A-1,
P10598A-1, SATT334, SATT510, SATT335, P5219A-1, P7659A-2,
SAT.sub.--117, SATT191, S60143-TB, SATT451, SATT367, SATT495,
P10649C-3, SATT613, SATT257, SATT581 and/or SATT153 as well as any
of the chromosome intervals:
[0329] (a) S60210-TB and SATT391 (LG-C1);
[0330] (b) P10598A-1 and SATT334 (LG-F);
[0331] (c) SATT510 and SATT335 (LG-F);
[0332] (d) P5219A-1 and P7659A-2 (LG-G);
[0333] (e) SAT.sub.--117 and S60143-TB (LG-G);
[0334] (f) SATT451 and SATT367 (LG-1);
[0335] (g) SATT495 and P10649C-3 (LG-L); and
[0336] (h) SATT250 and SATT346 (LG-M),
and the probe set is configured to detect the locus.
[0337] The typical system includes 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. A wide variety of signal detection apparatus
are available, including photo multiplier tubes,
spectrophotometers, CCD arrays, arrays and array scanners, scanning
detectors, phototubes and photodiodes, microscope stations,
galvo-scanns, microfluidic nucleic acid amplification detection
appliances and the like. The precise configuration of the detector
will depend, in part, on the type of label used to detect the
marker allele, as well as the instrumentation that is most
conveniently obtained for the user. Detectors that detect
fluorescence, phosphorescence, radioactivity, pH, charge,
absorbance, luminescence, temperature, magnetism or the like can be
used. Typical detector embodiments include light (e.g.,
fluorescence) detectors or radioactivity detectors. For example,
detection of a light emission (e.g., a fluorescence emission) or
other probe label is indicative of the presence or absence of a
marker allele. Fluorescent detection is especially preferred and is
generally used for detection of amplified nucleic acids (however,
upstream and/or downstream operations can also be performed on
amplicons, which can involve other detection methods). In general,
the detector detects one or more label (e.g., light) emission from
a probe label, which is indicative of the presence or absence of a
marker allele.
[0338] The detector(s) optionally monitors one or a plurality of
signals from an amplification reaction. For example, the detector
can monitor optical signals which correspond to "real time"
amplification assay results.
[0339] System instructions that correlate the presence or absence
of the favorable allele with the predicted tolerance are also a
feature of the invention. For example, the instructions can include
at least one look-up table that includes a correlation between the
presence or absence of the favorable alleles and the predicted
tolerance or improved tolerance. The precise form of the
instructions can vary depending on the components of the system,
e.g., they can be present as system software in one or more
integrated unit of the system (e.g., a microprocessor, computer or
computer readable medium), or can be present in one or more units
(e.g., computers or computer readable media) operably coupled to
the detector. As noted, in one typical embodiment, the system
instructions include at least one look-up table that includes a
correlation between the presence or absence of the favorable
alleles and predicted tolerance or improved tolerance. The
instructions also typically include instructions providing 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.
[0340] The system typically includes components for storing or
transmitting computer readable data representing or designating the
alleles detected by the methods of the present invention, e.g., in
an automated system. The computer readable media can include cache,
main, and storage memory and/or other electronic data storage
components (hard drives, floppy drives, storage drives, etc.) for
storage of computer code. Data representing alleles detected by the
method of the present invention can also be electronically,
optically, magnetically o transmitted in a computer data signal
embodied in a transmission medium over a network such as an
intranet or internet or combinations thereof. The system can also
or alternatively transmit data via wireless, IR, or other available
transmission alternatives.
[0341] 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.
[0342] The phrase "allele detection/correlation system" in the
context of this invention refers to a system in which data entering
a computer corresponds to physical objects or processes external to
the computer, e.g., a marker allele, and a process that, within a
computer, causes a physical transformation of the input signals to
different output signals. In other words, the input data, e.g.,
amplification of a particular marker allele is transformed to
output data, e.g., the identification of the allelic form of a
chromosome segment. The process within the computer is a set of
instructions, or "program," by which positive amplification or
hybridization signals are recognized by the integrated system and
attributed to individual samples as a genotype. Additional programs
correlate the identity of individual samples with phenotypic values
or marker alleles, e.g., statistical methods. In addition there are
numerous e.g., C/C++ programs for computing, Delphi and/or Java
programs for GUI interfaces, and productivity tools (e.g.,
Microsoft Excel and/or SigmaPlot) for charting or creating look up
tables of relevant allele-trait correlations. Other useful software
tools in the context of the integrated systems of the invention
include statistical packages such as SAS, Genstat, Matlab,
Mathematica, and S-Plus and genetic modeling packages such as
QU-GENE. Furthermore, additional programming languages such as
visual basic are also suitably employed in the integrated systems
of the invention.
[0343] For example, tolerance marker allele values assigned to a
population of progeny descending from crosses between elite lines
are recorded in a computer readable medium, thereby establishing a
database corresponding tolerance alleles with unique identifiers
for members of the population of progeny. Any file or folder,
whether custom-made or commercially available (e.g., from Oracle or
Sybase) suitable for recording data in a computer readable medium
is acceptable as a database in the context of the present
invention. Data regarding genotype for one or more molecular
markers, e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP, isozyme markers or
other markers as described herein, are similarly recorded in a
computer accessible database. Optionally, marker data is obtained
using an integrated system that automates one or more aspects of
the assay (or assays) used to determine marker(s) genotype. In such
a system, input data corresponding to genotypes for molecular
markers are relayed from a detector, e.g., an array, a scanner, a
CCD, or other detection device directly to files in a computer
readable medium accessible to the central processing unit. A set of
system instructions (typically embodied in one or more programs)
encoding the correlations between tolerance and the alleles of the
invention is then executed by the computational device to identify
correlations between marker alleles and predicted trait
phenotypes.
[0344] Typically, the system also includes a user input device,
such as a keyboard, a mouse, a touchscreen, or the like, for, e.g.,
selecting files, retrieving data, reviewing tables of maker
information, etc., and an output device (e.g., a monitor, a
printer, etc.) for viewing or recovering the product of the
statistical analysis.
[0345] Thus, in one aspect, the invention provides an integrated
system comprising a computer or computer readable medium comprising
set of files and/or a database with at least one data set that
corresponds to the marker alleles herein. The system also includes
a user interface allowing a user to selectively view one or more of
these databases. In addition, standard text manipulation software
such as word processing software (e.g., Microsoft Word.TM. or Corel
WordPerfect.TM.) and database or spreadsheet software (e.g.,
spreadsheet software such as Microsoft Excel.TM., Corel Quattro
Pro.TM., or database programs such as Microsoft Access.TM. or
Paradox.TM.) can be used in conjunction with a user interface
(e.g., a GUI in a standard operating system such as a Windows,
Macintosh, Unix or Linux system) to manipulate strings of
characters corresponding to the alleles or other features of the
database.
[0346] The systems optionally include components for sample
manipulation, e.g., incorporating robotic devices. For example, 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, is optionally operably
linked to the digital computer (or to an additional computer in the
integrated 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 is commonly a feature
of the integrated system. Many such automated robotic fluid
handling systems are commercially available. For example, a variety
of automated systems are available from Caliper Technologies
(Hopkinton, Mass.), which utilize various Zymate systems, which
typically include, e.g., robotics and fluid handling modules.
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, e.g., from Beckman Coulter, Inc.
(Fullerton, Calif.). As an alternative to conventional robotics,
microfluidic systems for performing fluid handling and detection
are now widely available, e.g., from Caliper Technologies Corp.
(Hopkinton, Mass.) and Agilent technologies (Palo Alto,
Calif.).
[0347] Systems for molecular marker analysis of the present
invention can, thus, include a digital computer with one or more of
high-throughput liquid control software, image analysis software
for analyzing data from marker labels, data interpretation
software, a robotic liquid control armature for transferring
solutions from a source to a destination operably linked to the
digital computer, an input device (e.g., a computer keyboard) for
entering data to the digital computer to control high throughput
liquid transfer by the robotic liquid control armature and,
optionally, an image scanner for digitizing label signals from
labeled probes hybridized, e.g., to markers on a solid support
operably linked to the digital computer. The image scanner
interfaces with the image analysis software to provide a
measurement of, e.g., nucleic acid probe label intensity upon
hybridization to an arrayed sample nucleic acid population (e.g.,
comprising one or more markers), where the probe label intensity
measurement is interpreted by the data interpretation software to
show whether, and to what degree, the labeled probe hybridizes to a
marker nucleic acid (e.g., an amplified marker allele). The data so
derived is then correlated with sample identity, to determine the
identity of a plant with a particular genotype(s) for particular
markers or alleles, e.g., to facilitate marker assisted selection
of soybean plants with favorable allelic forms of chromosome
segments involved in agronomic performance (e.g., tolerance or
improved tolerance).
[0348] Optical images, e.g., hybridization patterns viewed (and,
optionally, recorded) by a camera or other recording device (e.g.,
a photodiode and data storage device) are optionally further
processed in any of the embodiments herein, e.g., 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, e.g., using PC (Intel x86 or
pentium chip-compatible DOS.TM., OS2.TM. WINDOWS.TM., WINDOWS
NT.TM. or WINDOWS95.TM. based machines), MACINTOSH.TM., LINUX, or
UNIX based (e.g., SUN.TM. work station) computers.
EXAMPLES
[0349] The following examples are offered to illustrate, but not to
limit, the claimed 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 or parameters that can be altered without departing from
the spirit of the invention or the scope of the appended
claims.
Example 1
Intergroup Allele Frequency Distribution Analysis
[0350] Two independent allele frequency distribution analyses were
undertaken to identify soybean genetic marker loci associated with
tolerance to low-iron infection. By identifying such genetic
markers, marker assisted selection (MAS) can be used to improve the
efficiency of breeding for improved tolerance of soybean to
iron-deficient growth conditions.
Soybean Lines and Tolerance Scoring
[0351] The plant varieties used in the analysis were from diverse
sources, including elite germplasm, commercially released cultivars
and other public lines representing a broad range of germplasm. The
lines used in the study had a broad maturity range varying from
group 0 to group 6.
[0352] Two groups of soybean lines were assembled for each analysis
based on their phenotypic extremes in tolerance to iron-deficient
growth conditions, where the plants were sorted into either highly
susceptible or highly tolerant varieties. The classifications of
tolerant and susceptible were based solely on observations of
fortuitous, naturally occurring fields displaying disease incidence
in greenhouse and field tests over several years. The degree of
plant tolerance to iron-poor growth conditions varied widely, as
measured using a scale from one (highly susceptible) to nine
(highly tolerant). Generally, a score of two (2) indicated the most
susceptible strains, and a score of seven (7) was assigned to the
most tolerant lines. A score of one (1) was generally not used, as
soybean strains with such extremely high susceptibility were not
typically propagated. Tolerance scores of eight (8) and nine (9)
were reserved for tolerance levels that are very rare and generally
not observed in existing germplasm. If no disease was present in a
field, no tolerance scoring was done. However, if a disease did
occur in a specific field location, all of the lines in that
location were scored. Scores for test strains accumulated over
multiple locations and multiple years, and an averaged (e.g.,
consensus) score was ultimately assigned to each line.
[0353] Individual fields showing naturally-occurring FEC were
monitored for disease symptoms. Data collection was made in
successive scorings on multiple days. Scorings continued until
worsening symptoms could no longer be quantified or until the
symptoms are confounded by other factors such as other diseases,
insect pressure, severe weather, or advancing maturity.
[0354] In assessing linkage of markers to tolerance, a qualitative
"intergroup allele frequency distribution" comparison approach was
used. Using this approach, those soybean lines that were considered
to be representative of either the tolerant or susceptible classes
were used for assessing linkage. A list of tolerant lines was
constructed, where strains having a tolerance score of 6 or greater
were considered "tolerant." Similarly, soybean lines with scores of
four or less were collectively considered susceptible. Only lines
that could be reliably placed into the two groups were used. Once a
line is included in the "tolerant" or "susceptible" group, it was
treated as an equal in that group, i.e., the actual quantitative
ratings was not used.
[0355] In one of the analyses, 62 soybean lines were identified
that were considered tolerant in the phenotypic spectrum; these
plants formed the "TOLERANT" group. Also, 64 soybean lines were
identified that were judged to be susceptible to iron-poor growth
conditions; these strains formed the "SUSCEPTIBLE" group. In the
second analysis, there were 32 tolerant lines and 36 susceptible
lines.
Soybean Genotyping
[0356] Each of the tolerant and susceptible lines were genotyped
with SSR and SNP markers that span the soybean genome using
techniques well known in the art. The genotyping protocol consisted
of collecting young leaf tissue from eight individuals from each
tolerant and resistant soybean strain, pooling (i.e., bulking) the
leaf tissue from the eight individuals, and isolating genomic DNA
from the pooled tissue. The soybean genomic DNA was extracted by
the CTAB method, as described in Maroof et al., (1984) Proc. Natl.
Acad. Sci. (USA) 81:8014-8018.
[0357] The isolated genomic DNA was then used in PCR reactions
using amplification primers specific for a large number of markers
that covered all chromosomes in the soybean genome. The length of
the PCR amplicon or amplicons from each PCR reaction were
characterized. The length of the amplicons generated in the PCR
reactions were compared to known allele definitions for the various
markers (see, e.g., FIG. 4), and allele designations were assigned.
SNP-type markers were genotyped using an ASH protocol (see, FIG.
3).
Intergroup Allele Frequency Analysis
[0358] An "Intergroup Allele Frequency Distribution" analysis was
conducted using GeneFlow.TM. version 7.0 software. An intergroup
allele frequency distribution analysis provides a method for
finding non-random distributions of alleles between two phenotypic
groups.
[0359] During processing, a contingency table of allele frequencies
is constructed and from this a G-statistic and probability are
calculated (the G statistic is adjusted by using the William's
correction factor). The probability value is adjusted to take into
account the fact that multiple tests are being done (thus, there is
some expected rate of false positives). The adjusted probability is
proportional to the probability that the observed allele
distribution differences between the two classes would occur by
chance alone. The lower that probability value, the greater the
likelihood that the low iron tolerance phenotype and the marker
will co-segregate. A more complete discussion of the derivation of
the probability values can be found in the GeneFlow.TM. version 7.0
software documentation. See, also, Sokal and Rolf (1981), Biometry:
The Principles and Practices of Statistics in Biological Research,
2nd ed., San Francisco, W. H. Freeman and Co.
[0360] The underlying logic is that markers with significantly
different allele distributions between the tolerant and susceptible
groups (i.e., non random distributions) might be associated with
the trait and can be used to separate them for purposes of marker
assisted selection of soybean lines with previously uncharacterized
tolerance or susceptibility to low iron growth conditions. The
present analysis examined one marker locus at a time and determined
if the allele distribution within the tolerant group is
significantly different from the allele distribution within the
susceptible group. A statistically different allele distribution is
an indication that the marker is linked to a locus that is
associated with reaction to iron-poor conditions. In this analysis,
adjusted probabilities less than approximately 0.10 are considered
highly significant. Allele classes represented by less than 5
observations across both groups were not included in the
statistical analysis. In this analysis, 424 marker loci had enough
observations for analysis.
[0361] This analysis compares the plants' phenotypic score with the
genotypes at the various loci. This type of intergroup analysis
neither generates nor requires any map data. Subsequently, map data
(for example, a composite soybean genetic map) is relevant in that
multiple significant markers that are also genetically linked can
be considered as collaborating evidence that a given chromosomal
region is associated with the trait of interest.
Results
[0362] FIG. 1 provides a table listing the soybean markers that
demonstrated linkage disequilibrium with the low iron
tolerance/susceptibility phenotype. Also indicated in that figure
are the chromosomes on which the markers are located and their
approximate map position relative to other known markers, given in
cM, with position zero being the first (most distal) marker known
at the beginning of the chromosome. These map positions are not
absolute, and represent an estimate of map position. The
statistical probabilities that the marker allele and tolerance
phenotype are segregating independently are reflected in the
adjusted probability values.
[0363] FIG. 2 provides the PCR primer sequences that were used to
genotype the SSR marker loci. FIG. 2 also provides the pigtail
sequence used on the 5' end of the right SSR-marker primers and the
number of nucleotides in the repeating element in the SSR. The
observed alleles that are known to occur for these marker loci are
provided in the allele dictionary in FIG. 4. SNP-type markers were
genotyped using an ASH protocol with appropriate primers and
allele-specific probes (see, FIG. 3).
Discussion
[0364] There are a number of ways to use the information provided
in this analysis for the development of improved soybean varieties.
One application is to use the associated markers (or more based on
a higher probability cutoff value) as candidates for mapping QTL in
specific populations that are segregating for plants having
tolerance to iron poor growth conditions. In this application, one
proceeds with conventional QTL mapping in a segregating population,
but focusing on the markers that are associated with low iron
tolerance, instead of using markers that span the entire genome.
This makes mapping efforts more cost-effective by dramatically
reducing lab resources committed to the project. For example,
instead of screening segregating populations with a large set of
markers that spans the entire genome, one would screen with only
those few markers that met some statistical cutoff in the
intergroup allele association study. This will not only reduce the
cost of mapping but will also eliminate false leads that will
undoubtedly occur with a large set of markers. In any given cross,
it is likely that only a small subset of the associated markers
will actually be correlated with tolerance to low iron conditions.
Once the few relevant markers are identified in any tolerant
parent, future marker assisted selection (MAS) efforts can focus on
only those markers that are important for that source of tolerance.
By pre-selecting lines that have the allele associated with
tolerance via MAS, one can eliminate the undesirable susceptible
lines and concentrate the expensive field testing resources on
lines that have a higher probability of being tolerant to iron-poor
growth conditions.
Example 2
Association Mapping Analysis
[0365] An association mapping strategy was undertaken to identify
soybean genetic markers associated with tolerance to low iron
growth conditions. The study was completed twice, generating two
independent data sets. By identifying such genetic markers, marker
assisted selection (MAS) can be used to improve the efficiency of
breeding for improved tolerance of soybean to low iron growth
conditions. Association mapping is known in the art, and is
described in various sources, e.g., Jorde (2000), Genome Res.,
10:1435-1444; Remington et al. (2001), "Structure of linkage
disequilibrium and phenotype associations in the maize genome,"
Proc Natl Acad Sci USA 98:11479-11484; and Weiss and Clark (2002),
Trends in Genetics 18:19-24.
Association Mapping
[0366] Understanding the extent and patterns of linkage
disequilibrium (LD) in the genome is a prerequisite for developing
efficient association approaches to identify and map quantitative
trait loci (QTL). Linkage disequilibrium (LD) refers to the
non-random association of alleles in a collection of individuals.
When LD is observed among alleles at linked loci, it is measured as
LD decay across a specific region of a chromosome. The extent of
the LD is a reflection of the recombinational history of that
region. The average rate of LD decay in a genome can help predict
the number and density of markers that are required to undertake a
genome-wide association study and provides an estimate of the
resolution that can be expected.
[0367] Association or LD mapping aims to identify significant
genotype-phenotype associations. It has been exploited as a
powerful tool for fine mapping in outcrossing species such as
humans (Corder et al. (1994) "Protective effect of apolipoprotein-E
type-2 allele for late-onset Alzheimer-disease," Nat Genet 7:
180-184; Hastbacka et al., (1992) "Linkage disequilibrium mapping
in isolated founder populations: diastrophic dysplasia in Finland,"
Nat Genet 2:204-211; Kerem et al., (1989) "Identification of the
cystic fibrosis gene: genetic analysis," Science 245:1073-1080) and
maize (Remington et al., (2001) "Structure of linkage
disequilibrium and phenotype associations in the maize genome,"
Proc Natl Acad Sci USA 98:11479-11484; Thornsberry et al. (2001)
"Dwarf8 polymorphisms associate with variation in flowering time,"
Nat Genet 28:286-289; reviewed by Flint-Garcia et al. (2003)
"Structure of linkage disequilibrium in plants," Annu Rev Plant
Biol., 54:357-374), where recombination among heterozygotes is
frequent and results in a rapid decay of LD. In inbreeding species
where recombination among homozygous genotypes is not genetically
detectable, the extent of LD is greater (i.e., larger blocks of
linked markers are inherited together) and this dramatically lowers
the resolution of association mapping (Wall and Pritchard (2003)
"Haplotype blocks and linkage disequilibrium in the human genome,"
Nat Rev Genet 4:587-597).
[0368] The recombinational and mutational history of a population
is a function of the mating habit, as well as the effective size
and age of a population. Large population sizes offer enhanced
possibilities for detecting recombination, while older populations
are generally associated with higher levels of polymorphism, both
of which contribute to observably accelerated rates of LD decay. On
the other hand, smaller effective population sizes, i.e., those
that have experienced a recent genetic bottleneck, tend to show a
slower rate of LD decay, resulting in more extensive haplotype
conservation (Flint-Garcia et al. (2003) "Structure of linkage
disequilibrium in plants," Annu Rev Plant Biol., 54:357-374).
[0369] Elite breeding lines provide a valuable starting point for
association analyses. Association analyses use quantitative
phenotypic scores (e.g., disease tolerance rated from one to nine
for each soybean line) in the analysis (as opposed to looking only
at tolerant versus resistant allele frequency distributions in
intergroup allele distribution types of analysis). The availability
of detailed phenotypic performance data collected by breeding
programs over multiple years and environments for a large number of
elite lines provides a valuable dataset for genetic marker
association mapping analyses. This paves the way for a seamless
integration between research and application and takes advantage of
historically accumulated data sets. However, an understanding of
the relationship between polymorphism and recombination is useful
in developing appropriate strategies for efficiently extracting
maximum information from these resources.
[0370] This type of association analysis neither generates nor
requires any map data, but rather, is independent of map position.
This analysis compares the plants' phenotypic score with the
genotypes at the various loci. Subsequently, any suitable soybean
map (for example, a composite map) can optionally be used to help
observe distribution of the identified QTL markers and/or QTL
marker clustering using previously determined map locations of the
markers.
Soybean Lines and Phenotypic Scoring
[0371] Soybean lines were phenotypically scored based on their
degree of tolerance to low iron growth conditions (in contrast to
simple categorization of "tolerant" or "susceptible"). The plant
varieties used in the analysis were from diverse sources, including
elite germplasm, other commercially released cultivars and
proprietary experimental varieties. The RIL collections comprised
205 Pioneer soybean R3+ lines, or alternatively, 177 Pioneer R3+
lines. The lines used in the study had a broad maturity range
varying from group 0 to group 6.
[0372] The tolerance scoring was based solely on observations in
fortuitous, naturally occurring fields displaying disease incidence
in multienvironmental field tests over several years. The degree of
plant tolerance to low iron growth conditions varied widely, as
measured using a scale from one (1; highly susceptible) to nine (9;
highly tolerant). Generally, a score of two (2) indicated the most
susceptible strains, and a score of seven (8) was assigned to the
most tolerant lines. A score of one (1) was generally not used, as
soybean strains with such extremely high susceptibility were not
typically propagated. A tolerance score of nine (9) was reserved
for tolerance levels that are very rare and generally not observed
in existing germplasm.
[0373] Experimental plants were scored for the low iron
tolerance/susceptibility phenotype according to a scoring scale as
described above. If no disease (chlorosis) was present in a field,
no tolerance scoring was done. However, if disease did occur in a
specific field location, all of the lines in that location were
scored. Tolerance scores for the reference strains accumulated over
multiple locations and years, and an averaged (e.g., consensus)
score was ultimately assigned to each line. Tolerance scores for
the 205 variety collection or the 177 variety collection were
collected over a single grow season.
[0374] Individual fields showing iron chlorosis were monitored for
disease symptoms. Data collection was typically done in multiple
scorings on multiple days. Scorings continued until worsening
symptoms could no longer be quantified or until the symptoms are
confounded by other factors such as other diseases, insect
pressure, severe weather, or advancing maturity.
[0375] In assessing the linkage of markers to tolerance, a
quantitative approach was used, where a tolerance score for each
soybean line was assessed and incorporated into the association
mapping statistical analysis.
Soybean Genotyping
[0376] The independent populations of either 205 or 177 soybean
lines that were scored for disease tolerance were then genotyped.
The 205 member population was genotyped using 287 SSR and ASH
markers. The 177 member population was genotyped using 374 SSR and
ASH markers. These SSR and SNP markers collectively spanned each
chromosome in the plant genome. The genotyping protocol consisted
of collecting young leaf tissue from eight individuals from each
soybean strain, pooling (i.e., bulking) the leaf tissue from the
eight individuals, and isolating genomic DNA from the pooled
tissue. The soybean genomic DNA was extracted by the CTAB method,
as described in Maroof et al., (1984) Proc. Natl. Acad. Sci. (USA)
81:8014-8018.
[0377] The isolated genomic DNA was then used in PCR reactions
using amplification primers specific for a large number of markers
that covered all chromosomes in the soybean genome. The length of
the PCR amplicon or amplicons from each PCR reaction were
characterized. SNP-type markers were genotyped using an ASH
protocol. The length of the amplicons generated in the PCR
reactions were compared to known allele definitions for the various
markers (see FIG. 4), and allele designations for each tested
marker were assigned.
Statistical Methods
[0378] Monomorphic loci are considered uninformative and thus are
eliminated from LD analyses. The monomorphic loci are defined as
those whose gene diversity (1-.SIGMA..sub.i=1.sup.npi, where
p.sub.i is i.sup.th allele frequency in the population of study) is
less than 0.10. Since rare alleles (frequency <0.05) tend to
cause large variances for the estimates of r.sup.2, they were
treated as missing data and pooled together. Marker screening and
partitioning are conducted using PowerMarker software (version
2.72), which was developed by Jack Liu and is available at
http://152.14.14.48.
[0379] The rate of LD decay with genetic distance (cM) was
calculated for pairs of markers on the same chromosome and was
evaluated using linear regression in which the genetic distances
were transformed by taking log.sub.10, as described by McRae et al.
(2002). Population structure was evaluated using Pritchard's
model-based method (Pritchard et al. 2000) and the software,
STRUCTURE (version 2.0; see the web at:
pritch.bsd.uchicago.edu/index.html). This version of the program
controls for linked markers and correlated allelic frequencies
(Falush et al. (2003) "Inference of population structure using
multilocus genotype data linked loci and correlated allele
frequencies," Genetics 164: 1567-1587). It detects population
structure in structured or admixed populations. This method is more
appropriate than conventionally used genetic distance-based method,
because Structure provides the likelihood associated with different
numbers of sub-populations and the estimated percentage of shared
ancestry with each sub-population for each entry.
[0380] Associations of individual SSR markers with tolerance to
Fusarium solani infection were evaluated by logistic regression in
TASSEL (Trait Analysis by aSSociation, Evolution, and Linkage)
using the Structured Association analysis mode. TASSEL is provided
by Edward Buckler, and information about the program can be found
on the Buckler Lab web page at the Institute for Genomic Diversity
at Cornell University. The significance level for each association
was tested using an empirical distribution that was established by
running 5,000 permutations. Modifications of established procedures
were made to accommodate the nature and characteristics of soybean
and the soybean data set, especially with regard to those aspects
that differ from rice.
Results
[0381] FIG. 1 provides a table listing the soybean markers that
demonstrated linkage disequilibrium with the low iron tolerance
phenotype using the Association Mapping method. Also indicated in
that figure are the chromosomes on which the markers are located
and their approximate map position relative to other known markers,
given in cM, with position zero being the first (most distal)
marker known at the beginning of the chromosome. These map
positions are not absolute, and represent an estimate of map
position. The SNP-type markers were detected by an allele specific
hybridization (ASH) method, as known in the art (see, e.g., Coryell
et al., (1999) "Allele specific hybridization markers for soybean,"
Theor. Appl. Genet., 98:690-696). FIG. 2 provides the PCR primer
sequences that were used to genotype these seven marker loci. FIG.
2 also provides the pigtail sequence used on the 5' end of the
right SSR-marker primers and the number of nucleotides in the
repeating element in the SSR. The alleles that are known to occur
for the marker loci are provided in the SSR allele dictionary in
FIG. 4. FIG. 3 provides the PCR amplification primer sequences and
the allele-specific probes that were used to genotype the SNP-type
marker loci.
[0382] The statistical probabilities that the marker allele and
disease tolerance phenotype are segregating independently are
reflected in the adjusted probability values in FIG. 1, which is a
probability (P) derived from 5000 rounds of permutation analysis
between genotype and phenotype. The permutations method for
probability analysis is known in the art, and described in various
sources, for example, Churchill and Doerge (1994), Genetics 138:
963-971; Doerge and Churchill (1996), Genetics 142: 285-294; Lynch
and Walsh (1998) in Genetics and analysis of quantitative traits,
published by Sinauer Associates, Inc. Sunderland, Mass. 01375, p.
441-442.
[0383] The lower the probability value, the more significant is the
association between the marker genotype at that locus and the low
iron tolerance phenotype. A more complete discussion of the
derivation of the probability values can be found in the software
documentation. See, also, Sokal and Rolf (1981), Biometry: The
Principles and Practices of Statistics in Biological Research, 2nd
ed., San Francisco, W. H. Freeman and Co.
Example 3
QTL Interval Mapping and Single Marker Regression Analysis
[0384] Two independent QTL interval mapping and marker regression
analyses were undertaken to identify soybean genetic markers and
chromosome intervals associated with tolerance that allow the plant
to escape the pathology associated with growth in iron-poor
conditions. QTL mapping and marker regression are widely used
method to identify genetic loci that co-segregate with a desired
phenotype. By identifying such genetic loci, marker assisted
selection (MAS) can be used to improve the efficiency of breeding
for improved soybean strains.
Study A
[0385] Materials and Methods
[0386] A mapping population for iron-deficiency tolerance was
created using the mapping population UP1C6-43190B73. The population
had 458 progeny that were used in the mapping.
[0387] Phenotypic scoring took place in replicated plots that were
planted in fields known to promote FEC pathology. Planting sites
near Cottonwood, Minn. and near Glyndon, Minn. were used. All plots
were scored once at late vegetative to early reproductive stage of
growth. Scoring was on a scale of one to nine, where one is
susceptible and nine is tolerant. Phenotypic scoring of each of the
progeny lines was based on two years of collection data, with ten
reps of data for each scored line. The overall means of ten reps
were used for QTL interval mapping.
[0388] Soybean Genotyping
[0389] No genotype information was available for the parent
UP106-43. For the purpose of identifying polymorphic loci, the
first plate was run with all available SSR markers (approximately
550). Based on the collected data, 210 markers that were
potentially polymorphic were selected for further study. These
markers were screened against the rest of four plates. Because of
missing data for one of the parents, manual editing was used in
analyzing the SSR data.
[0390] From the 210 SSR markers screened, 41 of those had no
meaningful data and were dropped from the analysis. Out of the
remaining 169 SSR markers, 143 were mappable. These 143 markers
were mapped to 20 linkage groups with between 2 and 14 markers per
linkage group. Linkage groups A2, B2, D1a, I and N only have two to
four markers per linkage group. Compared to the most comprehensive
public map, this map covers about 50% of the genome.
[0391] QTL Interval Mapping
[0392] QTL mapping analyses were performed on the overall mean of
two years of data of ten repetitions for each observation. One
thousand (1000) permutation tests were used to establish the
threshold for statistical significance (likelihood ratio
statistic--LRS). The LRS threshold for means at P=0.05 is 13.4. The
LRS provides a measure of the linkage between variation in the
phenotype and genetic differences at a particular genetic locus.
LRS values can be converted to LOD scores (logarithm of the odds
ratio) by dividing by 4.61. Generally, the LRS values above 15 are
considered significant for simple interval maps. The term
"likelihood" of "odds" is used to describe the relative probability
of two or more explanations of the sources of variation in a trait.
The probability of these two different explanations (models) can be
computed, and most likely model chosen. If model A is 1000 times
more probable than model B, then the ratio of the odds are 1000:1
and the logarithm of the odds ratio is 3. MapManager-QTXb20 (2004)
was used for the QTL interval mapping.
[0393] Results
[0394] QTL Interval Mapping
[0395] The present study identified two chromosome intervals that
correlate with QTL that associate with tolerance/susceptibility to
iron-deficient growth conditions according to a constrained
additive model. Two QTL were identified for FEC tolerance. One QTL
is on LG-F, which has LRS of 25.2, and explains 5% of total
variation. The flanking markers for this QTL are Satt334 and
Satt510. Another QTL is LG-L, this QTL has LRS of 67.8 and explains
14% of the total variation. The flanking markers for this QTL are
Satt613 and Satt513. This QTL has much larger effect on FEC than
the QTL on LG-F.
[0396] Marker Regression
[0397] Using single marker regression, there are a number of
markers showing association with FEC, as provided in FIG. 1.
Study B
[0398] Materials and Methods
[0399] A mapping population for iron-deficiency tolerance was
created using the mapping population P1082/90B73. The population
had 460 progeny that were used in the mapping.
[0400] Phenotypic scoring of each of the progeny lines was based on
two years of collection data, with ten reps of data for each scored
line. Phenotypic scoring took place in replicated plots that were
planted in fields known to promote FEC pathology. Sites near Ada,
Minn. and near Glyndon, Minn. were used. All plots were scored once
at late vegetative to early reproductive stage of growth. Scoring
was on a scale of one to nine, where one is susceptible and nine is
tolerant. The overall means of ten reps were used for QTL interval
mapping.
[0401] Soybean Genotyping
[0402] A total of 245 markers were analyzed. After review and
editing, 200 SSR marker data were loaded to MapManager-QTX. Out of
the 200 SSRs loaded into the mapping program, 164 were placed into
18 linkage groups. The remaining 36 markers were unlinked although
some of them have linkage group information available. The map
covers about 75% genome. There were no markers placed on LG-I or
LG-N (there are markers genotyped for these two linkage groups, but
because of large gaps, these markers can not be placed
together).
[0403] QTL Interval Mapping
[0404] MapManager was used for both genetic and QTL interval
mapping. The 1000 permutation tests were used to establish the
threshold for statistical significance (likelihood ratio
statistic--LRS). The LRS threshold at P=0.05 was 26.1.
[0405] Results
[0406] A total of six QTL intervals were identified. These QTL are
on LG-C2, LG-D1b, LG-D2, LG-G, LG-L and LG-M. Using single marker
regression, there are a number of markers showing association with
the FEC tolerance phenotype. By reviewing the mapping results from
both interval mapping and marker regression analysis for two
mapping populations, several consistent QTL for FEC were
identified. For example, a QTL interval was identified on LG-M,
which is defined by and includes the termini SATT250 and SATT346.
Furthermore, markers within these intervals were also confirmed.
For example, the EST-SSR marker S60126-TB maps between SATT250 and
SATT346, and also correlates with the low-iron tolerance phenotype
(p=0.0000).
DISCUSSION/CONCLUSIONS
[0407] This present mapping study has identified chromosome
intervals and individual markers that correlate with tolerance to
iron-deficient growth conditions. Markers that lie within these
intervals are useful for use in MAS, as well as other purposes.
[0408] While the foregoing invention has been described in some
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. 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
69122DNAArtificialoligonucleotide primer 1tcattgcgtg attgattttc cg
22222DNAArtificialoligonucleotide primer 2gtttcgctct gagtctccca gg
22322DNAArtificialoligonucleotide primer 3caatcaggtt agtggtccta cc
22420DNAArtificialoligonucleotide primer 4caaaaggttt tcagtggtgg
20524DNAArtificialoligonucleotide primer 5tgctcaaagg gtcaatttct
ttcc 24628DNAArtificialoligonucleotide primer 6tgtgtaattt
ctatcacctt attgtgcc 28725DNAArtificialoligonucleotide primer
7cgactaacac ctttcacttg acttg 25822DNAArtificialoligonucleotide
primer 8gcaggaattt gggggagtct gt 22923DNAArtificialoligonucleotide
primer 9gctggccttt agaacgtctg act
231022DNAArtificialoligonucleotide primer 10cgttggattc gactttttgg
ga 221127DNAArtificialoligonucleotide primer 11gcgttaagaa
tgcatttatg tttagtc 271225DNAArtificialoligonucleotide primer
12gcgagttttt ggttggattg agttg 251328DNAArtificialoligonucleotide
primer 13gcgagtttcg ccgttaccac ctcagctt
281428DNAArtificialoligonucleotide primer 14ccctcttatt tcaccctaag
acctacaa 281520DNAArtificialoligonucleotide primer 15caagctcaag
cctcacacat 201622DNAArtificialoligonucleotide primer 16tgaccagagt
ccaaagttca tc 221722DNAArtificialoligonucleotide primer
17tgctcatgtg gtcctaccca ga 221826DNAArtificialoligonucleotide
primer 18cgctatccct ttgtattttc ttttgc
261924DNAArtificialoligonucleotide primer 19aaagcatttt tggcagtttc
ttgt 242022DNAArtificialoligonucleotide primer 20ggaatgtccc
aagtgtcagc aa 222122DNAArtificialoligonucleotide primer
21gcgatcatgt ctctgccatc ag 222222DNAArtificialoligonucleotide
primer 22cctcttgaaa ccgtgaaacc gt
222322DNAArtificialoligonucleotide primer 23ccccaacaac aacgatcatc
aa 222422DNAArtificialoligonucleotide primer 24tttgtaggta
accaccgcag gc 222526DNAArtificialoligonucleotide primer
25gcgcaattaa aaggataact tatatc 262626DNAArtificialoligonucleotide
primer 26cccctctttg gccctcacac cttctc
262725DNAArtificialoligonucleotide primer 27gcggatatgc cacttctctc
gtgac 252825DNAArtificialoligonucleotide primer 28gcggaatagt
tgccaaacaa taatc 252925DNAArtificialoligonucleotide primer
29tggagattta atatagatgc cgcga 253024DNAArtificialoligonucleotide
primer 30gcaccatgtt ctttttccat caaa
243126DNAArtificialoligonucleotide primer 31gcggaatatg atcattggta
atgtac 263222DNAArtificialoligonucleotide primer 32cggcttcaaa
cggcaaataa tc 223322DNAArtificialoligonucleotide primer
33gaaccacaga ggctgcaact cc 223422DNAArtificialoligonucleotide
primer 34acctggttga agaggtggtg ga
223520DNAArtificialoligonucleotide primer 35cgccagctag ctagtctcat
203624DNAArtificialoligonucleotide primer 36aatttgctcc agtgttttaa
gttt 243722DNAArtificialoligonucleotide primer 37accttcacca
ccaccaccat ct 223822DNAArtificialoligonucleotide primer
38tagtttccgt tgctgggagg ag 223922DNAArtificialoligonucleotide
primer 39gttcggaggg aggaaagtgt tg
224026DNAArtificialoligonucleotide primer 40ccataaaaca tagcaactgt
cgtctc 264126DNAArtificialoligonucleotide primer 41gagcaggaca
ttttttttat ccttga 264225DNAArtificialoligonucleotide primer
42tgcttccatt agtctctcat cctcc 254325DNAArtificialoligonucleotide
primer 43gcgactttct tttcaatttc actcc
254421DNAArtificialoligonucleotide primer 44gcgcaattgt caccaacaca t
214523DNAArtificialoligonucleotide primer 45ccaaagctga gcagctgata
act 234625DNAArtificialoligonucleotide primer 46ccctcactcc
tagattattt gttgt 254726DNAArtificialoligonucleotide primer
47gggttatatc agtttttctt tttgtt 264820DNAArtificialoligonucleotide
primer 48ccatcctcgt tagcatctat 204929DNAArtificialoligonucleotide
primer 49gatggctgtc attgctacag aggagtatc
295038DNAArtificialoligonucleotide primer 50gtgactccaa aggaaagaga
aatgtttctt aaatcatc 385131DNAArtificialoligonucleotide primer
51caattcttgt gggttgaagc cttgttctga c
315230DNAArtificialoligonucleotide primer 52ggaatcaact tcttcgtgag
tgggttgttc 305332DNAArtificialoligonucleotide primer 53cacactatca
acacctattg gtgaccattg ta 325433DNAArtificialoligonucleotide primer
54ggagggtgct tatgtaaatg atgtaaagac cat
335533DNAArtificialoligonucleotide primer 55catgaagctc caccatttgc
tagtacatga aac 335633DNAArtificialoligonucleotide primer
56ccagagttac caaaccatct gtgagaaata tcc
335730DNAArtificialoligonucleotide primer 57gagggctatg ttttcttctc
cagatgtgag 305826DNAArtificialoligonucleotide primer 58aaggtcggct
tggtggttaa aggcag 265914DNAArtificialoligonucleotide probe
59aatgataatt tagt 146013DNAArtificialoligonucleotide probe
60aatgatcatt tag 136112DNAArtificialoligonucleotide probe
61gaatgacttt ga 126213DNAArtificialoligonucleotide probe
62gaatgatttt gac 136313DNAArtificialoligonucleotide probe
63ttatagacac ttg 136412DNAArtificialoligonucleotide probe
64tataggcact tg 126512DNAArtificialoligonucleotide probe
65gaggagatgt ag 126612DNAArtificialoligonucleotide probe
66gaggaaatgt ag 126713DNAArtificialoligonucleotide probe
67tcatctgtga taa 136813DNAArtificialoligonucleotide probe
68tcatgtgtga taa 136913DNAArtificialoligonucleotide probe
69tcatctctga taa 13
* * * * *
References