Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
A METHOD TO IDENTIFY AND BREED CORN WITH
INCREASED KERNEL OIL CONCENTRATION
FIELD OF INVENTION
The invention is in the fields of plant breeding and molecular biology.
More specifically, the invention relates to the identification of corn loci
conferring
increased kernel oil concentration using genetic markers and the use of
genetic
markers as an aid to the identification and breeding of corn with increased
kernel
oil concentration.
BACKGROUND OF INVENTION
Corn is a major crop used as a human food source, an animal feed, and as a
source of carbohydrate, oil, protein, and fiber. It is principally used as an
energy
source in animal feeds, or as a raw material for the recovery of starch,
protein feed
fractions, fiber, flaking grits, flour, and oil.
Most commercial corn produced throughout the United States is produced
from hybrid seed. The production of corn hybrids requires the development of
elite corn inbreds that upon intermating produce agronomically superior
hybrids.
During the development of corn inbreds, plant breeders select for a number of
different traits affecting agronomic performance. These traits include but are
not
limited to stalk strength, lodging, disease resistance, grain moisture and
grain
yield. Agronomic traits tend to be quantitatively measured with continuous
rather
than discrete distributions. It is theorized that quantitative traits are
controlled by
several genes with small and generally equivalent effects. Further, the
observed
phenotype is due partially to this genetic component and an environmental
component.
The heritability of a trait is defined in the broad sense as the ratio of the
genetic variance to the total phenotypic variance. Many agronomic traits
display
low heritability; i.e., the performance of parent plants is a poor predictor
of
offspring performance. Thus, traits with low heritability have small genetic
variance components in comparison with observed variation. The impact on the
plant breeder is that in breeding populations, the value of a plant's genetic
composition is difficult to determine from agronomic trait measurements. In an
attempt to maximize their discriminative abilities, breeders collect multiple
measurements both from individuals related by descent and from many
environments. This strategy is resource intensive because it involves the use
of
extensive trialing to make even small gains in plant improvement. This,
coupled
with the fact that improved corn lines are selected for multiple traits
simultaneously, makes the development of superior corn inbreds both a time-
consuming and an expensive labor.
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The addition of novel traits in a corn breeding program imposes an
additional burden upon the plant breeder. Depending upon the genetic
complexity
of the novel trait (i.e., single gene versus many genes), a signif cant
increase in
time and effort is required to produce elite lines containing novel traits.
One such
trait is kernel oil concentration.
Com with increased kernel oil concentration is important because it
possesses improved feeding value for poultry (Han Y. et ai. ( 1987) Poultry
Sci.
66:103-111 ) and livestock (Nordstrom, J. W. et al. ( 1972) J. An. Sci
3.5(2):357-361 ). Grain from conventional corn hybrids typically contains 4%
oil.
In an effort to increase the kernel oil concentration, a long-term recurrent
selection
program was initiated in the open-pollinated cv. Burr's White by C.G. Hopkins
in
1896. This recurrently-selected population known as Illinois High Oil (IHO),
has
been selected for increased oil concentration for over ninety generations
(Dudley,
J.W. and R.J. Lambert. (1992) Maydica 37:1-7) using modified mass selection.
As a result, oil concentration was increased in the population over 20%. The
germplasm was little used because derived materials had yields substantially
lower than conventional varieties (Alexander, D.E. (1988) In: Proc. 43rd Ann.
Com and Sorghum Res. Conf. Am. Seed Trade Assoc., Washington, D.C.
pp 97-105).
Using thirty-eight open-pollinated cultivars and synthetics, Alexander
initiated a second recurrent selection program (Alexho synthetic) to increase
kernel oil (Alexander, D.E. (1988) In: Corn and Corn Improvement. G.F. Sprague
and J.W. Dudley eds. American Society of Agronomy, Madison WI. Pp 869-880).
Equivalent oil levels to IHO were achieved in twenty-eight cycles using
selection
based upon the oil concentration of single ears and in later generations based
upon
the oil concentration of single kernels. Yield performance of Alexho-derived
material in single cross hybrids (high oil inbred x conventional inbred) is
improved over IHO, presumably due to the greater genetic variability initially
available, although performance was not equivalent to conventional hybrids.
The
development of agronomically elite corn germplasm also containing increased
kernel oil concentration is clearly a challenge using conventional plant
breeding
methods.
Kernel oil concentration can be phenotypically measured using a variety of
analytical methods. Oil concentration displays a non-discrete distribution,
common for quantitatively-inherited traits controlled by several loci. Kernel
oil
measurements select those breeding lines with the highest phenotypic
expression.
Unfortunately, the genetic potential for high oil is limited in most of these
lines
because it is impossible to discriminate between lines based upon their true
genetic composition. This situation is further aggrevated when simultaneous
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selection for agronomic performance is practiced. It would therefore be
advantageous to base selection upon the genotype of the plants in the
population.
Genetic markers, especially nucleic acid markers, may be used to advantage as
an
indirect selection method for complex quantitative traits. Genetic markers
identifying alleles conferring increased oil would therefore be an
advantageous
tool for plant breeding programs developing elite high oil corn germplasm.
There is limited published information on the identification of genetic
markers predictive for increased oil yield. Kahler (Kahler, A.L. (1985) In:
Proc.
40th Ann. Corn and Sorghum Res. Conf. Am. Seed Trade Assoc., Washington
D.C. pp. 66-89) measured isozyme allelic frequency changes following twenty-
five cycles of selection in Alexho synthetic and found eight significant loci.
Most
of these allele frequency changes were also significant for tests measuring
random
genetic drift, making it difficult to conclude that selection based upon these
isozyme alleles would be useful. More recently Goldman et al. (Goldman, LL.,
et
al. (1994) Crop Sci. 34:908-915) and Berke and Rocheford (Berke, T.G. and
Rocheford, T.R. (1995) Crop Sci. 35:1542-1549) used RFLP markers to identify
significant marker loci associated with oil concentration in the Illinois long-
term
selection populations. These studies identified twenty-five and thirty-one
markers
respectively, in populations derived from Burr's White, which were
significantly
associated with increased oil. Some of the regions identified by significant
RFLP
marker loci may be in common between the two studies, however of the fifteen
RFLP markers which were used in both studies, six were in disagreement for
their
effect on oil concentration. In these studies the populations used were
derived
from common ancestry (Burr's White); however, the populations were selected
for
different traits (oil and protein) over many generations. It is not surprising
that
many identified oil loci would be unique to each population analyzed. It is
therefore desirable to identify those genetic markers which are uniquely
predictive
of getznplasm being used in the breeding program.
SUMMARY OF INVENTION
A method is disclosed for reliably and predictably breeding for corn with
increased kernel oil concentration. The method comprises a) using one or more
genetic markers to select a corn plant from a corn breeding population by
marker-
assisted selection, wherein the genetic markers are selected from the group
consisting of s 1375, s 13 84, s 13 94, s 1416, s 1422, s 1432, s 1457, s
1480, s 1476,
s 1478, s 1484, s 1500, s 1513, s 1529, s 1544, s 1545, s 1630, s 1633, s
1647, s 1750,
s 1756, s 1757, s 1767, s 1772, s 1774, s 1780, s 1797, s 1813, s 1816, s
1817, s 1836,
s1853, s1860, sI870, s1921, s1922, s1925, s1931, s1933, s1939, s1946, s1949,
s2054, s2055, s2057, s2058, s2097, s2122, s2I25, s2150, s2156 and s2175; and
b) crossing the selected corn plant with a second corn plant wherein the
progeny
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of the cross displays increased kernel oil concentration. A preferred source
of
high oil corn germplasm is a member of an Alexho synthetic population or a
progeny thereof.
Also disclosed is a method for identifying corn plants or corn lines for use
as parents for creation of a breeding population, the method comprising
a) genotyping corn plants or corn lines with one or more genetic markers
wherein
the genetic markers are selected from the group consisting of x1375, x1384,
s1394,
s I 416, s 1422, s 1432, s I 457, s 1480, s 1476, s 1478, s 1484, s 1500, s 15
I 3, s 1529,
s 1544, s 1545, s 1630, s 1633, s 1647, s 1750, s 1756, s 1757, s 1767, s
1772, s 1774,
x1780, s1797, x1813, x1816, x1817, x1836, x1853, x1860, s1870, x1921, x1922,
x1925, s1931, x1933, x1939, s1946, x1949, s2054, s2055, x2057, x2058, x2097,
x2122, x2125, x2150, x2156 and x2175; and b) identifying corn plants or corn
lines
which, based upon their genotype, are predicted to produce transgressive
segregants for kernel oil concentration.
The present invention provides a method for the identification of and
selection for genes controlling increased corn kernel oil concentration. These
oil
alleles were initially identified in materials composed of or derived from the
Alexho synthetic breeding populations. Further, the method facilitates the use
of
this high oil material in breeding programs with the objective of developing
new
high oil corn germplasm.
Specifically, the method uses genetic markers to predict the oil breeding
value of lines in a corn breeding program. By indirect selection of oil loci
using
these markers, those lines with the greatest genetic potential for increased
kernel
oil concentration are chosen.
According to the method, any type of genetic marker may be used to
identify an association with kernel oil concentration. The method is only
limited
by the ability to measure polymorphism at a given marker locus. Those skilled
in
the art will recognize that the various genetic markers which may be used
includes
but is not limited to restriction fragment length polymorphisms (RFLPs),
random
amplified polymorphic DNAs (RAPDs), simple sequence repeats (SSRs), AFLPs,
various single base pair detection methods, allozymes, and phenotypic markers.
SSR markers useful in the practice of the instant method include s1375, s1384,
s 1394, s 1416, s 1422, s 1432, s 1457, s 1476, s 1478, s 1480, s 1484, s
1500, s 1513,
s 1529, s 1544, s 1545, s I 630, s 1633, s 1647, s 1750, s 1756, s I 757, s
1767, s 1772,
x1774, s1780, s1797, x1813, x1816, x1817, x1836, x1853, x1860, s1870, s1921,
x1922, x1925, x1931, x1933, x1939, x1946, s1949, x2054, x2055, x2057, x2058,
x2097, x2122, s2125, x2150, x2156 and x2175.
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A further embodiment of the present invention are the trait loci controlling
the expression of corn kernel oiI concentration. These loci are identified and
defined (i.e., mapped) by the marker loci of the present invention.
An additional embodiment of the present invention are corn plants and
high oil corn germplasm that are produced using the instant breeding method.
DETAILED DESCRIPTION OF THE INVENTION
Table 1 provides a brief description of the genetic markers that form a part
of the instant invention. Each marker is defined by it's constituent nucleic
acid
primers (forward and reverse) that facilitate amplification of the specific
marker
locus of the corn genome. Also indicated is the required identifier for each
sequence. The identifiers listed in Table 1 correspond to those listed in the
Sequence Listing (infra) as required by 37 C.F.R. ~ 1.821 et seq.
Table 1
Genetic markers useful for defining
the location of trait loci
controllin corn kernel oil concentration
Marker Sequence (S'-3') Primer Type SEQ ID NO.
sI375 TTTATGGGTTGGGAGATACTTG forward 1
AGATGTGTGCGTTTZTGAGAG reverse 2
s1384 TTACGGCCTAGACATTTCGAC fo~~d 3
CACTTGCTTTCAGGTACCCA reverse 4
s1394 CTGCCCAGTCCGTAATGAA forward
TAGATTTATTTTCTGAACGATTGG reverse 6
s 1416GATCTCTCTGAGGCTTGTCC forward 7
TGTAGTTGAGGATGCTCCC reverse g
s 1422AGGCAAGGCTTTCTTCATAC forward 9
CGGACGACGACTGTGTTC reverse 10
s1432 ACATGAGAAACAAGATAGAACCAG forward 11
AAAATGTAAGAACTTGTTTGGGA reverse 12
s1457 ____ _ 13
CTGCTTATTGCTTTCGTCATA forward
TGCTGCACTACTTGAACCTAG reverse 14
s 1476ACACAGAGATGACAA.AAGCAA forward 15
GCAGGCGTGCTATGAGAG _ reverse 16
s1478 .-._._ ___- 17
AGCGGTGAAACCCTTATG forward
CTGTGGCTGGTTCCTCTC reverse 1
g
s1480 GCTCTTGATAAAAAGGCAAGT forward 19
_._._ CTTGTTGTAATGGATGAGTGAG_ reverse 20
__ _ ___ ___
s 1484GCTCGTAGTAGGGGTTACG forward _
21
GACAGCCTCACCTCAAGA reverse 22
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s ACAGATCTTGACACGTACATACC forward 23
1500
GGACGTGTATCCTCAAATCAT reverse 24
s CAGCGAATACTGAATAACGC forward 25
1513
TGTTGGATGAGCACTGAAC reverse 26
s TGTTCTCAACAACCACCG forward 27
1529
CGTTTAGCGATATCATTTTCC reverse 28
_____
s GATCCTACCAAAATCTTATAGGC forward 29
1544
ACAGCTAGCCAAGATCTGATT reverse 30
s CGATACTAATGGAAGCCCTAA forward 31
1545
ATGGCCCATTAAGTTTATCAC reverse 32
s1630AAAGCGTAGTCGGAAAGC forward 33
ACCAATGATCTTTACGCAGAT reverse 34
si633TAATCAGAGCGTACATCAGGA forward 35
AGGGCATCAATCAAGAATG reverse 36
s1647GAGACTTI"TGAGGAGAAAGCA forward 37
GATCAAAAGAGCAAAAGGAGA reverse 38
si750AACTGATGAATACCTTCCCAG forward 39
TGATTAACTTCTCCCTTTGGT reverse 40
s1756TCGGCACAACATATGAGTTAC forward 41
CCCCCATAGAGAGAGATAGAG reverse 42
s1757AAGCACGGCCCAATAGAAT forward 43
AGGATGTCCCTAGCTTTATTG reverse 44
s TCATTGCCCAAAGTGTTG forward 45
1767
CTCATCACCCCTCCAGAG reverse 46
s1772GATCCACGCCATTTAAAC forward 47
TGATACTCTGGTGCATGTTC reverse 48
s1774GATCGCTCCGATCTATCC forward 49
AGCGGCATCTATGTTCTATG reverse 50
s1780CCCAGTGCGAAGAGACTC forward 51
ACACCTGCTCTGCACCAC reverse 52
si797CTAACCCACGACGACCCT forward 53
GCATGAGTGCATGTGCAT reverse 54
si813CTGCCACATGCTTTTCTG forward 55
CTGTAAAGAAGCTGGTCTGGA reverse 56
si816TTCTCCTCATGGATGCGT forward 57
CTATTTGGAAGTATGGGCTTCA reverse 58
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s 1817GAGGGCATCTATGTGCAAC . forward
GCTCAGAAGTTGCGTTTATG . reverse 60
s 1836TTCCTTCACGTTTCTCTGTTAA _ 61
forward
CACATAAACCTAATGGGGTACA reverse 62
s1853 CCCAAAGGCGATACCTATT forward 63
_-__-.__,. CCCACTTTCTCACTCTTTTCT - reverse 6
4
-__..._. .. ________
s 1860GAGGTGAGTACTATGCAAATGC forward _
_
65
CAGGCTTACCTAGCCTTCTC reverse 66
s 1870CTATGGATGGCTGCTTGC forward 67
_._____________._."GTCAGGCAGCAGAATGTG __ ___...___reverse6g
s1921 AAACCGTCCAGCGACTAC forward
GGAAGAACCAATCCCATATCT reverse
s1922 AACATCCTGTCGGAAACAG forward 71
TCATCACGTCTCTCTTTCAAC reverse 72
s 1925TTGTGGCAGAATCTCAAATTA forward 73
CGACTGGTGACATGTGAAG reverse 74
s 1931AGTGAGGAAAGAATATGCTGG forward 75
__ TGGACTGAGAAACTGATTTGA reverse 76
s1933 CACAAATGTGAAGGTAAACACT forward 77
AATGGTACGGTTCAGGATG reverse 7g
s1939 AGATGACGCACGGAACAC forward 79
_ AGCATCATGTAGCAGGAGG reverse g0
s 1946TTGCAGCACTGTCGTAGTC fo,-~,~.d g 1
GCGCGAGTGGAGTAGTAAG reverse g2
s 1949AAGATTATGCAGATGAGACACC forward g3
GTTCCATGCTTTCCTTGG reverse g4
s2054 GCCGATACCATGTAAGAGAAT forward g5
CTCTGGGCTCTGTGTTAGAGT reverse g6
s2055 CTGCTTTCTCTGTTCCAGC forward g7
_-. __AATCGCTTACTTGTAACCCAC reverse gg
s2057 AAGAACGTACGTCCCATAAAG __ _
____ g9
' forward
CAAGGTAAAGTGACAAAGCAG reverse 90
s2058 GTTCAGGATGAGGCGGAA forward 91
___. GTGATCATCGCAG_GA_GA_CC_____ reverse 92
____
s2097 _ __ 93
GGAGCCTGGAGTGAGAAC __ __
forward '
CATGCTCACCTAACGTGG reverse 94
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s2122ATCTGAACACTTGAGCAACAA forward 95
ATAGACCGGACCCATCAC reverse 96
s2125CGAACAGCGGGTACACCT forward 97
GAGGTCAGCTTCCTCGATCT reverse __98 __
_
s2150GGAATCGTTCCTCCACAC forward 99
CTTCCTCGGTGTCAGACG reverse _100
_ __ __
s2156ATGGAAACATCAAAGTGGATT forward 101
TGCTACCCTGATGACCTGAT reverse 102
s2175ACCACTAGTCTCATATGAAGGG forward 103
GGTAGGTGGGTAGGGGTT reverse 104
For the purposes of this invention, we define the following terms:
Corn. Any variety, cultivar, or population of Zea mays L.
S Elite. This term characterizes a plant or variety possessing favorable
traits,
such as, but not limited to high yield, good grain quality, and disease
resistance.
This enables its use in commercial production of seed or grain at a profit.
The
term also characterizes parents giving rise to such plants or varieties.
High Oil Com Germplasm. This term characterizes corn plants which,
when either self pollinated or used as either the male or the female parent in
a
variety of outcrossing combinations, produce kernels with increased oil when
compared to kernels produced by non-high oil germplasm. Examples of high oil
corn germplasm include but are not limited to open-pollinated varieties,
hybrids,
synthetics, inbred lines, races, and populations or corn plants derived from
one of
the aforementioned.
Variety or cultivar. These terms refer to a group of similar plants that by
structural features and performance can be identified from other varieties or
cultivars within the same species.
Line. This term refers to a group of individuals from a common ancestry;
a more narrowly defined group than a variety.
Synthetic. This term refers to a genetically heterogeneous collection of
plants of known ancestry created by the intermating of any combination of
inbreds, hybrids, varieties, populations, races or other synthetics.
Inbred. This term refers to a substantially homozygous individual, variety
or line.
Recombinant Inbreds. A population of independently derived lines
developed by repeated selfing each generation until complete homozygosity is
approached. Each recombinant inbred is derived from a single F2 plant using a
breeding method commonly referred to as single seed descent.
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Breeding. The art and science of improving a species of plant or animal
through controlled genetic manipulation.
Marker-Assisted Selection. The use of genetic markers to identify and
select plants with superior phenotypic potential. Genetic markers) determined
previously to be associated with a trait locus or trait loci are used to
uncover the
genotype at trait loci by virtue of linkage between the marker locus and the
trait
locus. Plants containing desired trait alleles are chosen based upon their
genotypes at linked marker loci.
Alexho Synthetic. Recurrently selected, high oil corn germplasm
developed by Denton Alexander at the University of Illinois. Alexho synthetic
high oil corn germplasm is composed of multiple synthetic populations defined
by
their cycle of advancement in the recurrent selection breeding program.
Breeding Population. A genetically heterogeneous collection of plants
created for the purpose of identifying one or more individuals with desired
phenotypic characteristics.
Phenotype. The observed expression of one or more plant characteristics.
Phenotypic Value. A measure of the expected expression of an allele at a
trait locus. The phenotypic value of an allele at a trait locus is dependent
upon its
expressive strength in comparison to alternative alleles. The phenotypic value
of
an individual, and hence its phenotypic potential, is based upon its total
genotypic
composition at all loci for a given trait.
Transgressive Segregants. Individuals whose phenotype exceeds the
phenotypic variation predicted by the parents.
Genetic Marker. Any morphological, biochemical, or nucleic acid based
phenotypic difference which reveals a DNA polymorphism. Examples of genetic
markers includes but is not limited to RFLPs, RAPDs, allozymes, SSRs, and
AFLPs.
Marker locus. The genetically defined location of DNA polymorphisms as
revealed by a genetic marker.
Trait Locus. A genetically defined location for a collection of one or more
genes (alleles) which contribute to an observed characteristic.
Genotype. The allelic composition of an individual at genetic loci under
study.
Restriction Fragment Length Polymorphism (RFLP). A DNA-based
genetic marker in which size differences in restriction endonuclease generated
DNA fragments are observed via hybridization (Botstein, D. et al. 1980. Am. J.
Hum. Genet. 3Z: 314-331.
Random Amplified Polymorphic DNA (RAPD}. A DNA amplification-
based genetic marker in which short, sequence arbitrary primers are used and
the
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resulting amplification products are size separated and differences in
amplification
patterns observed (Williams J.G.K. et al. 1990. Nucleic Acids Res. 18:631-
6535).
Simple Sequence Repeat (SSR). A DNA amplification-based genetic
marker in which short stretches of tandemly repeated sequence motifs are
amplified and the resulting amplification products are size separated and
differences in length of the nucleotide repeat are observed (Tautz D. 1989.
Nucleic
Acids Res. 112:4127-4138).
AFLP. A DNA amplification-based genetic marker in which restriction
endonuclease generated DNA fragments are ligated to short DNA fragments
which facilitate the amplification of the restricted DNA fragments (Vos, P. et
al.
1995. Nucleic Acids Res. 23:4407-4414). The amplified fragments are size
separated and differences in amplification patterns observed.
Allozymes. Enzyme variants which are electrophoretically separated and
detected via staining for enzymatic activity (Stuber, C.W. and M.M. Goodman.
1983. USDA Agric. Res. Results, Southern Ser., No. 16).
The present invention relates to the discovery of trait loci controlling
kernel oil concentration through the use of genetic markers. In populations in
which variation for both kernel oil concentration and genetic marker alleles
exist,
oil measurements and marker-based genotypes were generated for members of the
populations. Using least squares methods, the locations of oil concentration
loci
were determined in relation to markers genetically linked to these trait Loci.
Indirect selection of preferred oil alleles may now be practiced using the
information at one or more linked genetic markers. Selected corn plants
comprise
one or more alleles encoding a high oil phenotype.
It is recognized that several different populations and population types
could be used to locate trait loci of interest. Some of the population types
include
but are not limited to recombinant inbreds, backcrosses, F2's or their self
pollinated or intermated derivatives, and synthetics. Further, it is
understood that
an alternative to measuring phenotypic and genotypic variation within
populations
is the measurement of genotypes and phenotypes between populations. In this
alternative the second population is a selected derivative of the first
population,
selection being either on the trait of interest (phenotypic selection) or on
specif c
marker alleles (genotypic selection). It is also recognized by those skilled
in the
art that alternative statistical approaches may be used to determine a linkage
relationship between marker loci and trait loci.
EXAMPLES
The present invention is further defined in the following Examples. It
should be understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration only. From the
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above discussion and these Examples. one skilled in the art can ascertain the
essential characteristics of this invention, and without departing from the
spirit
and scope thereof, can make various changes and modifications of the invention
to
adapt it to various usages and conditions.
EXAMPLE 1
LOCATION OF LOCI CONFERRING INCREASED
KERNEL OIL CONCENTRATION
Population development and trait measurement
LH119wx and LH51, two inbred corn lines developed by Holden's
Foundation Seed Co., Williamsburg, IA were independently intermated with
individual plants from the synthetic population ASKC28wx (deposited at the
American Type Culture Collection, Rockville, MD; Accession No. ATCC 75105)
(waxy kernels are highly represented in ASKC28 and as such I have designated
the ASKC28 as being waxy). The F1 plants were selfed and resulting F2
populations were grown. Individual F2 plants were selfed and derived kernels
were advanced using single seed decent through six generations of selfing (S6)
to
produce recombinant inbred lines. Up to twenty kernels from the S6 generation
were grown and selfed producing a family of S7 ears representing each
recombinant inbred line. Oil values were determined for each ear within a
family
using near infrared transmitance (Williams, P.C. (1987) In: Near Infrared
Technology in the Agricultural and Food Industries; P.C. Williams and C.
Norris,
eds. American Association of Cereal Chemists).
Geno is determination
Ten seeds from single ears representing each of one hundred ninety-four
(LH1 l9wx x ASKC28wx) or two hundred and four (LH51 x ASKC28wx)
recombinant inbred lines were germinated on moistened filter paper. Root
segments were excised from germinated seeds, pooled for each ear and extracted
using an automated DNA extraction machine. The instrument uses a modification
of the Murray and Thompson CTAB procedure (Murray, M.G. and Thompson,
W.F. (1980) Nucl. Acids Res. 8:4321-4325). DNA samples were quantified via
fluorescence using YoPro-1T"" iodide (Molecular Probes, Inc., Eugene, OR) and
diluted to 4 pg/ml.
SSR regions for each DNA sample were analyzed using the following
protocol:
1. Ten pI of amplification cocktail (see Table 2) was added to ~ pl
(20 ng) of extracted DNA;
2. The DNA fragment flanked by sequences complementary to the
primers present in the amplification cocktail was amplified by PCR (U.S.
Patent
No. 4,683,202 and U.S. Patent No. 4,683,195) using the following protocol:
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1 ) ~5 cycles of 50 sec at 95°C, 50 sec at 54°C and 80 sec at
72°C and 2) 1 cycle of
300 sec at 72°C;
3. Approximately 8 pl of each sample was loaded onto agarose gels
composed of 2% Metaphor (FMC Corp., Rockland, ME), 1X TBE, and 0.5 ~g/ml
S ethidium bromide, and electrophoresed for 2 h at 6.1 V/cm in horizontal
electrophoresis units to which IX TBE buffer and 0.5 ~Cg/ml ethidium bromide
was added; and
4. DNA bands were visualized by LJV fluorescence.
Table 2
Amplification Cocktail
Reagent Stock Concentration Final Concentration
Buffer* 1 OX 1.SX
dNTPs 2 mM 0.3 mM
Forward Primer 40 ~M 0.45 ~M
Reverse Primer 40 ~M 0.45 ~M
AmpliTaq PolymeraseTM 5 U/~1 0.05 U/p.l
* 1 OX Buffer is a pH 9.0 solution composed of 800 mM Tris-OH, 200 mM
~4)2SD4~ ~d 25 mM MgCl2.
Localization of oil loci
One hundred thirty three polymorphic SSR marker loci were used to
genotype the recombinant inbreds from the LH 1 l9wx x ASKC28wx cross and
one hundred and three polymorphic SSR marker loci were used to genotype the
LH51 x ASKC28wx-derived population. In addition, twenty publicly available
polymorphic SSR loci with previously established chromosome locations and
covering all ten maize chromosomes (available from Research Genetics,
Huntsville, AL) were also mapped in both populations.
Genetic linkage and distance between marker loci was determined
independently for each population using MAPMAKER 3.0 (Lincoln S.E., et al.
( 1993) Whitehead Inst. Biomed. Res., Cambridge, MA). This resulted in the
establishment of ten linkage groups for each population corresponding to the
ten
chromosomes of maize. Each linkage group was assigned to a chromosome based
upon linkage to the public SSR markers. Twenty-three and ten markers in the
LHI l9wx x ASKC28wx and LH51 x ASKC28wx populations, respectively, were
not assigned chromosome positions because genetic linkage could not be clearly
established.
Analysis of variance was used to identify marker loci in linkage with trait
loci conferring increased oil concentration. Oil concentration was used as a
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dependent variable and separate ANOVAs were calculated with SAS Proc GLM
(SAS Inst., Cary, NC) using each marker locus as a single independent variable
(Edwards, M.D., et aI. ( 1987) Genetics 116:113-I25). Therefore, for each
ANOVA test the mean oil values of marker allele classes were compared. Marker
loci were declared signif cant if p < 0.05.
Linkage data for significant marker loci was examined to determine both
the number of trait Ioci present and their probable location. Significant
marker
loci on the same linkage group are either detecting the same trait locus or
alternatively different trait loci. By careful examination of the phenotypic
variation explained by each marker locus along the chromosome, a determination
of the number trait loci on a linkage group was made. Significant marker loci,
on
the same linkage group and uninterrupted by non-significant marker loci, were
declared to be detecting the same trait locus on the chromosome. If
significant
marker loci on the same chromosome were interrupted by non-significant marker
loci then each significant region was declared to contain a trait locus
resulting in
multiple trait loci on the same chromosome.
To confirm the number of trait loci, marker data assigned to linkage
groups and oil data were also analyzed with Mapmaker/QTL 1.0 (Lincoln, S.E. et
al. (1990) Whitehead Inst. Biomed. Res., Cambridge, MA). Results with
Mapmaker/QTL were in agreement with the initial analysis for the number trait
loci on each chromosome.
Eleven and twelve loci controlling kernel oil concentration were located in
the LH1 l9wx x ASKC28wx and LH51 x ASKC28wx recombinant inbred
populations, respectively. Each oil locus is defined by one or more linked
marker
loci.
In instances where the same marker loci were used in both populations,
alignment of linkage groups is possible. It was found that in most instances
both
populations localized the same oil loci. By considering common marker loci, a
total of seventeen loci controlling kernel oil concentration were found. Each
oil
locus was assigned an arbitrary letter designation (Table 3}.
Table 3
Marker loci genetically linked to and predictive of the location of trait loci
conferring increased kernel oil concentration
Oil locus Chromosome Marker loci
A I s1922
B I s 1478, s I 853, s I 949
C I S I 860, s 1925, s 1931, s2150
D 2 s2175
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E 3 s1394
F 4 s 1476, s 1772, s 1816, s2122,
s 1836
G 4 s1939,s1946
H 4 s1870
I 5 s1529
J ~ s2054, s 1647, s 1500, s 1 X45,
s 1774, s2097
K 6 s1457, s2055, s1757, s2125,
s1780, s1375,
s 1797, s 1416, s 1432, s 1921
L 7 s1630, s1422, s2156
M 8 s1817,s2057
N 9 s1544, s1633, s1384, s1813,
s1767, s2058,
s1933,s1513,s1484
O 10 s1756
P 10 s1480 (positive oil allele in
LH51)
Q N.A.* s1750
*N.A. - chromosome location not known
In instances where comparisons could be made, oil loci which were
identified in one population were identified at the same location in the
second
population. In two exceptions, an oil locus was found in one population, but
not
in the second population. In the first case, the allele with a positive oil
effect was
found in LH51 and thus it would be unexpected to identify the same locus in
the
LH 119wx x ASKC28wx population. In the second case, it was found that
different ASKC28wx-derived marker alleles were segregating in the populations;
therefore, each population was measuring the oil effect of a different
ASKC28wx
allele at the trait locus. The most abundant ASKC28wx oil allele segregating
in
LH 119wx x ASKC28wx had a positive oil effect versus the alternative LH 119-
derived allele, whereas in the LH51 x ASKC28wx population, the abundant
ASKC28wx allele had no positive oil effect. With the exception of the oil
locus
linked to marker s1480, all alleles with positive effects on oil concentration
were
derived from ASKC28wx.
EXAMPLE 2
MARKER-ASSISTED SELECTION OF BREEDING LINES USING GENETIC
MARKERS FOR INCREASE KERNEL OIL CONCENTRATION
Genetic marker loci in linkage with oil trait loci are highly predictive of
oil
concentration and as such may be used as an indirect measurement of kernel oil
in
a marker-assisted selection program. Accordingly, genotypic information from
linked marker loci would facilitate the selection of breeding lines with
increased
oil concentration. Direct oii measurements cannot differentiate between
various
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genotypic trait locus compositions with equivalent phenotypic effects. This is
especially problematic in early generation segregating breeding populations
where
only limited fixation of oil loci has occurred.
By way of example, an objective of a corn breeding program could be the
creation of new elite inbred lines which contain trait alleles conferring
increased
kernel oil concentration. These trait alleles would be introduced by the
intermating of high oil germplasm with one or more elite corn inbreds. The
resultant hybrid could be self pollinated to produce an F2 population for the
purposes of initiating a conventional pedigree breeding program (Allard, R.W.
( 1960) Principles of Plant Breeding. John Wiley & Sons, Inc. New York.
Pp 115-128}.
In order to identify those F2 individuals with the desired genotypes, plant
tissue would be collected from each F2 individual in the population and
genotyped
with the SSR marker loci listed in Table 1. Those F2 individuals with the
highest
frequency of SSR marker alleles derived from the high oil source would be
selected and further culled based upon their agronomic fitness. With continued
inbreeding and segregation, those oil loci in a heterozygous state could
become
fixed for either the high oil or low oil allele. It is therefore likely that
genotyping
and selection of later generation materials would be practiced in order to
further
segregate breeding Iines based upon their marker allele and hence oil allele
composition.
Depending upon population size and serendipity, the resulting inbreds
from the pedigree breeding program may not demonstrate su~cient agronomic
competitiveness or sufficient kernel oil expression because an inadequate
number
of oil alleles was recovered. These new inbreds could therefore be used as
parental material and new breeding projects initiated. The SSR markers could
again be used for further selection of oil as described.
It is obvious to those skilled in the art that many variants to selection
methodology may by envisioned. Selection would be based upon the allelic
composition of one or more marker loci which identify trait oil loci present
in a
population. Further selection would be performed by examination and selection
of genotypes from individual plants, families, or their progeny. Various
predictive
models could be developed using genotypic information, which could generate
various selection indices. These models would permit weighting the effect
predicted by marker loci. This is because the predictive value of an
individual
marker locus is dependent upon its genetic distance from the corresponding
trait
locus as well as the expressivity of the trait locus. Selection strategies
which
combine phenotype-based and genotype-based selection may also be envisioned.
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The marker loci presented here are predictive of oil loci in Alexho
synthetic populations. Because ASKC28wx represents the ?8th oil breeding cycle
of a genetically closed population, earlier breeding cycles are composed of
the
same oil Loci. It is expected that cycles differ simply in their allelic
frequency at
the identified oil loci. Therefore, in breeding populations derived from
earlier
Alexho cycles, the marker loci described in this invention will be useful in
identification of oil loci and in prediction of oil concentration.
EXAMPLE 3
IDENTIFICATION OF CORN PLANTS FOR USE AS PARENTS
FOR THE PRODUCTION OF TRANSGRESSIVE SEGREGANTS FOR
KERNEL OIL CONCENTRATION
It is important to identify corn plants and lines which, when used as
parents, have the greatest probability of producing offspring with superior
performance. Transgressive segregant offspring of such parents would result
from
the crossing of parents with complementary sets of alleles conferring the high-
oil
phenotype. Using the information provided herein, marker alleles which predict
desired trait performance (i.e., high oil) at a given marker locus are known.
By
genotyping lines at those marker loci, the value of those lines as parents is
revealed. For example, if one wanted to create an individual containing
superior
alleles at 5 separate oil loci (A-E), one could identify and cross a parent
composed
of desired alleles for locus A, B, and C with a parent composed of desired
alleles
at B, D, and E. These parents are complementary because they permit the
recovery of progeny containing desired alleles at all 5 loci. Ideally, parents
would
be chosen which when combined ensure maximum complementation of loci, so
that a high frequency of desired recombinants are recovered.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
(B) STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY: USA
(F) ZIP: 19898
(G) TELEPHONE: 302-992-9926
(H) TELEFAX: 302-773-0169
(I) TELEX: 6717325
(ii) TITLE OF INVENTION: A METHOD TO IDENTIFY AND BREED
CORN WITH INCREASED KERNEL OIL
CONCENTRATION
{iii) NUMBER OF SEQUENCES: 104
(iv} COMPUTER READABLE FORM:
(A) MEDIUM TYPE DISKETTE, 3.50 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT WINDOWS 95
(D) SOFTWARE: MICROSOFT WORD VERSION 7.OA
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B} FILING DATE:
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/041,515
(B) FILING DATE: MARCH 24, 1997
(C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MAJARIAN, WILLIAM R.
(B) REGISTRATION NUMBER: P-41,173
(C) REFERENCE/DOCKET NUMBER: BB-1076
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CTGCTTATTG CTTTCGTCAT
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21
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TGCTGCACTA CTTGAACCTA
G
21
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ACACAGAGAT GACAAAAGCA A
21
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GCAGGCGTGC TATGAGAG
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AGCGGTGAAA CCCTTATG 18
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CTGTGGCTGG TTCCTCTC 18
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GCTCTTGATA AAAAGGCAAG T 21
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GCTCGTAGTA GGGGTTACG
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GGC
23
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21
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GAGACTTTTG
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ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: . other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:40:
SEQ ID
TGATTAACTTCTCCCTTTGG T
21
27
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(2) INFORMATION
FOR SEQ
ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:41:
SEQ ID
TCGGCACAACATATGAGTTA C 21
(2) INFORMATION
FOR SEQ
ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:42:
SEQ ID
CCCCCATAGAGAGAGATAGA G 21
(2) INFORMATION
FOR SEQ
ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:43:
SEQ ID
AAGCACGGCCCAATAGAAT 19
(2) INFORMATION
FOR SEQ
ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:49:
SEQ ID
AGGATGTCCCTAGCTTTATT G 21
28
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(2) INFORMATION
FOR
SEQ
ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
tC) STRANDEDNESS: single
(D) TOPOLOGY: linear
tii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:45:
SEQ ID
TCATTGCCCA ~
AAGTGTTG
18
(2) INFORMATION
FOR
SEQ
ID N0:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:96:
SEQ ID
CTCATCACCCCTCCAGAG
18
(2) INFORMATION FOR SEQ ID N0:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
tB) TYPE: nucleic acid
(C) STRANDEDNESS: single
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:47:
SEQ ID
GATCCACGCCATTTAAAC
18
(2) INFORMATION
FOR
SEQ
ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) .SEQUENCE DESCRIPTION: N0:98:
SEQ ID
TGATACTCTGGTGCATGTTC
20
29
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(2) INFORMATION FOR SEQ ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: iinear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:99:
SEQ ID
GATCGCTCCGATCTATCC lg
(2) INFORMATION
FOR SEQ
ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
fii} MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:50:
SEQ ID
AGCGGCATCTATGTTCTATG 20
(2) INFORMATION
FOR SEQ
ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:51:
SEQ ID
CCCAGTGCGAAGAGACTC 18
(2) INFORMATION
FOR SEQ
ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:52:
SEQ ID
ACACCTGCTCTGCACCAC 18
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(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
CTAACCCACG ACGACCCT
18
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
GCATGAGTGC ATGTGCAT 18
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
CTGCCACATG CTTTTCTG 18
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRT_PTION: SEQ ID N0:56:
CTGTAAAGAA GCTGGTCTGG A 21
31
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(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:57:
SEQ ID
TTCTCCTCATGGATGCGT 18
(2) INFORMATION
FOR
SEQ
ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:58:
SEQ ID
CTATTTGGAAGTATGGGCTT CA 22
(2) INFORMATION
FOR
SEQ
ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:59:
SEQ ID
GAGGGCATCTATGTGCAAC 19
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:60:
SEQ ID
GCTCAGAAGTTGCGTTTATG 20
32
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(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
TTCCTTCACG TTTCTCTGTT AA
22
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
CACATAAACC TAATGGGGTA CA 22
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
CCCAAAGGCG ATACCTATT
19
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
CCCACTTTCT CACTCTTTTC T 21
33
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(2) INFORMATION FOR SEQ ICJ N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
GAGGTGAGTA CTATGCAAAT GC 22
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
CAGGCTTACC TAGCCTTCTC 20
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1B base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
CTATGGATGG CTGCTTGC lg
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
GTCAGGCAGC AGAATGTG lg
34
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(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
AAACCGTCCA GCGACTAC
18
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
GGAAGAACCA ATCCCATATC T
21
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
AACATCCTGT CGGAAACAG
19
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
TCATCACGTC TCTCTTTCAA C
21
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(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
TTGTGGCAGA ATCTCAAATT A 21
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
CGACTGGTGA CATGTGAAG 19
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
AGTGAGGAAA GAATATGCTG G 21
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
TGGACTGAGA AACTGATTTG A 21
36
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(2) INFORMATION
FOR SEQ
ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:77;
SEQ ID
CACAAATGTG
AAGGTAAACA
CT
22
(2) INFORMATION
FOR SEQ
ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:78:
SEQ ID
AATGGTACGGTTCAGGATG
19
(2) INFO RMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:79:
SEQ ID
AGATGACGCACGGAACAC
18
(2) INFORMATION
FOR SEQ
ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:80:
SEQ ID
AGCATCATGTAGCAGGAGG
19
37
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(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:81:
SEQ ID
TTGCAGCACTGTCGTAGTC 19
(2) INFORMATION
FOR SEQ
ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:82:
SEQ ID
GCGCGAGTGGAGTAGTAAG 19
(2) INFORMATION
FOR SEQ
ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:83:
SEQ ID
AAGATTATGCAGATGAGACA CC 22
(2) INFORMATION
FOR SEQ
ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:84:
SEQ ID
GTTCCATGCTTTCCTTGG 18
38
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(2) INFORMATION FOR SEQ ID N0:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:85:
CTCTGGGCTC TGTGTTAGAG T
21
(2) INFORMATION FOR SEQ ID N0:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:86:
CTCTGGGCTC TGTGTTAGAG T 21
(2) INFORMATION FOR SEQ ID N0:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:87:
CTGCTTTCTC TGTTCCAGC 1g
(2) INFORMATION FOR SEQ ID N0:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:88:
AATCGCTTAC TTGTAACCCA C 21
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(2) INFORMATION FOR SEQ ID N0:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base
pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:89:
SEQ ID
AAGAACGTACGTCCCATAAA G 21
(2) INFORMATION
FOR SEQ
ID N0:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:90:
SEQ ID
CAAGGTAAAGTGACAAAGCA G 21
(2) INFORMATION
FOR SEQ
ID N0:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:91:
SEQ ID
GTTCAGGATGAGGCGGAA 18
(2) INFORMATION
FOR SEQ
ID N0:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:92:
SEQ ID
GTGATCATCGCAGGAGACC 19
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(2) INFORMATION
FOR
SEQ
ID N0:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:93:
SEQ ID
GGAGCCTGGA
GTGAGAAC
18
(2) INFORMATION
FOR
SEQ
ID N0:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:94:
SEQ ID
CATGCTCACCTAACGTGG
18
(2) INFORMATION
FOR
SEQ
ID N0:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:95:
SEQ ID
ATCTGAACACTTGAGCAACA A
21
(2) INFORMATION
FOR
SEQ
ID N0:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:96:
SEQ ID
ATAGACCGGACCCATCAC
18
41
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(2) INFORMATION FOR SEQ ID N0:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:97:
SEQ ID
CGAACAGCGGGTACACCT 18
(2) INFORMATION
FOR SEQ
ID N0:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:98:
SEQ ID
GAGGTCAGCTTCCTCGATCT 20
(2) INFORMATION
FOR SEQ
ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: N0:99:
SEQ ID
GGAATCGTTCCTCCACAC 18
(2) INFORMATION
FOR SEQ
ID N0:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other acid
nucleic
(xi) SEQUENCE DESCRIPTION: NO:100:
SEQ ID
CTTCCTCGGTGTCAGACG 18
42
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(2) INFORMATION
FOR SEQ
ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:101:
ATGGAAACAT
CAAAGTGGAT
T
21
(2) INFORMATION
FOR SEQ
ID N0:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
TGCTACCCTGATGACCTGAT
20
(2} INFORMATION
FOR SEQ
ID N0:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:103:
ACCACTAGTCTCATATGAAG GG
22
(2) INFORMATION
FOR SEQ
ID N0:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:104:
GGTAGGTGGGTAGGGGTT
18
43
