U.S. patent application number 12/201206 was filed with the patent office on 2009-03-12 for methods and compositions for goss' wilt resistance in corn.
This patent application is currently assigned to MONSANTO TECHNOLOGY LLC. Invention is credited to David Butruille, Kevin Cook, Travis J. Frey, Hongwu Jia, Michael R. Kerns, Laron Peters.
Application Number | 20090070903 12/201206 |
Document ID | / |
Family ID | 39873944 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090070903 |
Kind Code |
A1 |
Kerns; Michael R. ; et
al. |
March 12, 2009 |
Methods and Compositions for Goss' Wilt Resistance in Corn
Abstract
The present invention relates to the field of plant breeding.
More specifically, the present invention includes a method of using
haploid plants for genetic mapping of traits of interest such as
disease resistance. Further, the invention includes a method for
breeding corn plants containing quantitative trait loci (QTL) that
are associated with resistance to Goss' Wilt, a bacterial disease
associated with Clavibacter michiganense spp.
Inventors: |
Kerns; Michael R.; (Ankeny,
IA) ; Jia; Hongwu; (Mystic, CT) ; Butruille;
David; (Urbandale, IA) ; Frey; Travis J.;
(Brentwood, MO) ; Cook; Kevin; (Ankeny, IA)
; Peters; Laron; (Gothenburg, NE) |
Correspondence
Address: |
THOMPSON COBURN, LLP
ONE US BANK PLAZA
ST. LOUIS
MO
63101
US
|
Assignee: |
MONSANTO TECHNOLOGY LLC
St. Louis
MO
|
Family ID: |
39873944 |
Appl. No.: |
12/201206 |
Filed: |
August 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60966706 |
Aug 29, 2007 |
|
|
|
Current U.S.
Class: |
800/320.1 ;
435/6.12; 536/24.3 |
Current CPC
Class: |
A01H 5/10 20130101; C12N
15/8282 20130101; A01H 1/04 20130101; C12Q 1/6895 20130101; C12Q
2600/156 20130101; C12N 15/8281 20130101; C12Q 2600/172 20130101;
C12Q 2600/13 20130101 |
Class at
Publication: |
800/320.1 ;
435/6; 536/24.3 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12Q 1/68 20060101 C12Q001/68; C07H 21/00 20060101
C07H021/00 |
Claims
1. A method of identifying a corn plant comprising at least one
allele associated with Goss' Wilt resistance allele in a corn plant
comprising: a) genotyping at least one corn plant with at least one
nucleic acid marker selected from the group consisting of SEQ ID
NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106,
110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146,
153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202,
203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,
250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,
294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368,
370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422,
423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,
490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589,
593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,
649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,
719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,
792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874,
876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,
957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,
1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,
1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142,
1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,
1204, 1212, 1215, 1229, 1234-1302, and 1303, and b) selecting at
least one corn plant comprising an allele of at least one of said
markers that is associated with resistance to Goss' Wilt.
2. The method according to claim 1, wherein the at least one corn
plant genotyped in step (a) and/or the at least one corn plant
selected in step (b) is a corn plant from a population generated by
a cross.
3. The method of claim 2, wherein said cross is effected by
mechanical emasculation, chemical sterilization, or genetic
sterilization of a pollen acceptor.
4. The method of claim 1, wherein said genotyping is effected in
step (a) by determining the allelic state of at least one of said
corn genomic DNA markers.
5. The method according to claim 1, wherein said selected corn
plant(s) of step (b) exhibit at least partial resistance to a Goss'
Wilt-inducing bacteria or at least substantial resistance to a
Goss' Wilt-inducing bacteria.
6. The method according to claim 1, wherein said nucleic acid
marker is selected from the group consisting of SEQ ID NOs: 27,
121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480,
533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054,
1122, 1186, 1246, 1250, and 1251.
7. The method according to claim 6, wherein said nucleic acid
marker is selected from the group consisting of SEQ ID NOs: 234 and
1250.
8. The method of claim 2, wherein said population is generated by a
cross of at least one Goss' Wilt resistant corn plant with at least
one Goss' Wilt sensitive corn plant.
9. The method of claim 2, wherein said population is a segregating
population.
10. The method of claim 2, wherein said cross is a back cross of at
least one Goss' Wilt resistant corn plant with at least one Goss'
Wilt sensitive corn plant to introgress Goss' Wilt resistance into
a corn germplasm.
11. The method of claim 2, wherein said population is a haploid
breeding population.
12. A method of introgressing a Goss' Wilt resistance QTL into a
corn plant comprising: a) screening a population with at least one
nucleic acid marker to determine if one or more corn plants from
the population contains a Goss' Wilt resistance QTL, wherein the
Goss' Wilt resistance QTL is a QTL selected from the group
consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, and 131 as provided in FIG. 1; and b) selecting
from said population at least one corn plant comprising an allele
of said marker associated with a Grey Leaf Spot (GLS)
resistance.
13. The method according to claim 12, wherein at least one of the
markers is located within 5 cM of said Goss' Wilt resistance
QTL.
14. The method according to claim 13, wherein at least one of the
markers is located within 2 cM of said Goss' Wilt resistance
QTL.
15. The method according to claim 14, wherein at least one of the
markers is located within 1 cM of said Goss' Wilt resistance
QTL.
16. The method according to claim 12, wherein at least one of the
markers exhibits a LOD score of greater than 4.0 with said Goss'
Wilt resistance QTL.
17. The method according to claim 16, wherein said nucleic acid
marker is selected from the group consisting of SEQ ID NOs: 27,
121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480,
533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054,
1122, 1186, 1246, 1250, and 1251.
18. The method according to claim 17, wherein said nucleic acid
marker is selected from the group consisting of SEQ ID NOs: 234 and
1250.
19. The method of claim 12, wherein said population is a
segregating population.
20. A corn plant obtained by the method of claim 1, wherein said
corn plant comprises at least one allele of a nucleic acid marker
selected from the group consisting of SEQ ID NOs: 13, 19, 24, 27,
36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121,
122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164,
166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216,
218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260,
265-267, 271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320,
332-334, 337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382,
392, 395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438,
440, 447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525,
530, 533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618,
621, 623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669,
678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733,
734, 744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825,
835, 844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893,
896, 897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976,
981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053,
1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108,
1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149,
1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229,
1234-1302, and 1303, wherein said allele is associated with Goss'
Wilt resistance.
21. The corn plant according to claim 20, wherein said nucleic acid
marker is selected from the group consisting of SEQ ID NOs: 27,
121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480,
533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054,
1122, 1186, 1246, 1250, and 1251.
22. The corn plant of claim 20, wherein the corn plant exhibits at
least partial resistance to a Goss' Wilt-inducing bacterium or at
least substantial resistance to a Goss' Wilt-inducing
bacterium.
23. The corn plant according to claim 20 a nucleic acid marker
selected from the group consisting of SEQ ID NOs: 234 and 1250.
24. The corn plant of claim 20, wherein said corn plant is a
haploid corn plant.
25. A corn plant obtained by the method of claim 12, wherein said
corn plant comprises a Goss' Wilt resistance QTL selected from the
group consisting of QTL numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, and 131 as provided in FIG. 1.
26. An isolated nucleic acid molecule for detecting a molecular
marker representing a polymorphism in corn DNA, wherein said
nucleic acid molecule comprises at least 15 nucleotides that
include or are immediately adjacent to said polymorphism, wherein
said nucleic acid molecule is at least 90 percent identical to a
sequence of the same number of consecutive nucleotides in either
strand of DNA that include or are immediately adjacent to said
polymorphism, and wherein said molecular marker is selected from
the group consisting of SEQ ID NOs: 27, 121, 141, 175, 177, 220,
224, 234, 248, 252, 440, 479, 480, 533, 582, 585, 639, 721, 727,
733, 746, 768, 773, 940, 1053, 1054, 1122, 1186, 1234-1302, and
1303.
27. The isolated nucleic acid according to claim 26, wherein said
molecular marker is selected from the group consisting of SEQ ID
NOs: 27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440,
479, 480, 533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940,
1053, 1054, 1122, 1186, 1246, 1250, and 1251.
28. The isolated nucleic acid of claim 27, wherein said molecular
marker is selected from the group consisting of SEQ ID NOs: 1250.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/966,706, filed Aug. 29, 2007, and incorporated
herein by reference in its' entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] A sequence listing contained in the file named
"46.sub.--25(54886.sub.--003_US).txt" which is 2432172 bytes
(measured in MS-Windows) and comprising 1,361 nucleotide sequences,
created on Aug. 21, 2008, is electronically filed herewith and is
incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to the field of plant
breeding. More specifically, the present invention includes a
method of using haploid plants for genetic mapping of traits such
as disease resistance. The invention further includes a method for
breeding corn plants containing QTL that are associated with Goss'
Wilt, a bacterial disease associated with Clavibacter michiganense
spp.
BACKGROUND OF INVENTION
[0004] Goss' Wilt, caused by the bacterial pathogen Clavibacter
michiganensis subsp. nebraskensis (CN), is a disease that causes
significant damage to corn crops. Goss' Wilt has been identified
throughout the U.S. Corn Belt, primarily in the western regions.
Symptoms include leaf freckles which are small dark green to black
water soaked spots and vascular wilt which results in loss of
yield. Conservation tillage practices can increase pervasiveness
because the bacterial pathogen Clavibacter michiganensis subsp.
nebraskensis (CN) can overwinter in debris, particularly stalks,
from infected corn plants (Bradbury, J. F. IMI description of Fungi
and Bacteria, (1998)). A mapping study conducted by Rocheford et
al., reported a genomic region on maize Chromosome 4 associated
with Goss' Wilt (Rocheford, et al., Journal of Heredity 80(5),
(1989)). Goss' Wilt is a significant pathogen of corn, and a need
exists for development of disease resistant lines.
[0005] Breeding for corn plants resistant to Goss' Wilt can be
greatly facilitated by the use of marker-assisted selection. Of the
classes of genetic markers, single nucleotide polymorphisms (SNPs)
have characteristics which make them preferential to other genetic
markers in detecting, selecting for, and introgressing disease
resistance in a corn plant. SNPs are preferred because technologies
are available for automated, high-throughput screening of SNP
markers, which can decrease the time to select for and introgress
disease resistance in corn plants. Further, SNP markers are ideal
because the likelihood that a particular SNP allele is derived from
independent origins in the extant population of a particular
species is very low. As such, SNP markers are useful for tracking
and assisting introgression of disease resistance alleles,
particularly in the case of disease resistance haplotypes.
[0006] The present invention further provides and includes a method
for screening and selecting a corn plant comprising QTL for Goss'
Wilt resistance using endemic strains of CN and SNP marker
technology.
SUMMARY OF THE INVENTION
[0007] Methods for identifying corn plants that comprise alleles of
genetic loci associated with Goss' Wilt resistance are provided
herein. In certain embodiments, methods of identifying a corn plant
comprising at least one allele associated with Goss' Wilt
resistance allele in a corn plant comprising: a) genotyping at
least one corn plant with at least one nucleic acid marker selected
from the group consisting of SEQ ID NOs: 13, 19, 24, 27, 36, 50,
53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122,
124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166,
169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218,
220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,
271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334,
337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392,
395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440,
447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530,
533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621,
623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669, 678,
679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,
744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835,
844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896,
897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981,
983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054,
1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110,
1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159,
1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1302,
and 1303, and b) selecting at least one corn plant comprising an
allele of at least one of the markers that is associated with
resistance to Goss' Wilt are provided. In certain embodiments of
the methods, the at least one corn plant genotyped in step (a)
and/or the at least one corn plant selected in step (b) is a corn
plant from a population generated by a cross. In embodiments of the
methods where the corn plant from a population generated by a
cross, the cross can be effected by mechanical emasculation,
chemical sterilization, or genetic sterilization of a pollen
acceptor. In certain embodiments of the methods, genotyping is
effected in step (a) by determining the allelic state of at least
one of the corn genomic DNA markers. In such embodiments of the
methods, an allelic state can be determined by single base
extension (SBE), allele-specific primer extension sequencing
(ASPE), DNA sequencing, RNA sequencing, microarray-based analyses,
universal PCR, allele specific extension, hybridization, mass
spectrometry, ligation, extension-ligation, and/or a Flap
Endonuclease-mediated assay(s). In other embodiments of the
methods, the selected corn plant(s) of step (b) exhibit at least
partial resistance to a Goss' Wilt-inducing bacteria or at least
substantial resistance to a Goss' Wilt-inducing bacteria. In
certain embodiments of the methods, the nucleic acid marker is
selected from the group consisting of SEQ ID NOs: 27, 121, 141,
175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582,
585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122,
1186, 1246, 1250, and 1251. Alternatively, the nucleic acid marker
can be selected from the group consisting of SEQ ID NOs: 234 and
1250. In embodiments where a population is generated by a cross,
the population can be generated by a cross of at least one Goss'
Wilt resistant corn plant with at least one Goss' Wilt sensitive
corn plant. In certain embodiments of the methods where a
population is generated by a cross, the cross can be a back cross
of at least one Goss' Wilt resistant corn plant with at least one
Goss' Wilt sensitive corn plant to introgress Goss' Wilt resistance
into a corn germplasm. In embodiments where the corn plant is from
a population, the population can be a segregating population. In
certain embodiments of the methods, the population can be a haploid
breeding population.
[0008] Also provided herein are corn plants obtained by any of the
aforementioned methods of identifying corn plants that comprise
alleles of genetic loci associated with Goss' Wilt resistance. In
certain embodiments, a corn plant obtained by any of these
aforementioned methods can comprise at least one allele of a
nucleic acid marker selected from the group consisting of SEQ ID
NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106,
110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146,
153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202,
203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,
250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,
294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368,
370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422,
423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,
490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589,
593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,
649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,
719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,
792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874,
876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,
957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,
1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,
1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142,
1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,
1204, 1212, 1215, 1229, 1234-1302, and 1303, wherein the allele is
associated with Goss' Wilt resistance. In certain embodiments, a
corn plant obtained by any of these aforementioned methods can
comprise at least one allele of a nucleic acid marker is selected
from the group consisting of SEQ ID NOs: 27, 121, 141, 175, 177,
220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582, 585, 639,
721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122, 1186, 1246,
1250, and 1251, wherein the allele is associated with Goss' Wilt
resistance. In certain embodiments, a corn plant obtained by any of
these aforementioned methods can comprise at least one allele of a
nucleic acid marker is selected from the group consisting of SEQ ID
NOs: 234 and 1250, wherein the allele is associated with Goss' Wilt
resistance. In certain embodiments, a corn plant obtained by any of
these aforementioned methods exhibits at least partial resistance
to a Goss' Wilt-inducing bacterium. In certain embodiments, a corn
plant obtained by any of these aforementioned methods exhibits at
least substantial resistance to a Goss' Wilt-inducing bacterium. In
still other embodiments, a corn plant obtained by any of these
aforementioned methods can be a haploid corn plant. In certain
embodiments, a corn plant obtained by any of the aforementioned
methods can comprise at least one transgenic trait. In such
embodiments, the transgenic trait can be herbicide tolerance and/or
pest resistance. In embodiments where the corn plant obtained is
herbicide tolerant, herbicide tolerance can be selected from the
group consisting of glyphosate, dicamba, glufosinate, sulfonylurea,
bromoxynil and norflurazon herbicide tolerance. In certain
embodiments, the nucleic acid marker is present as a single copy in
a corn plant obtained by any of these aforementioned methods. In
other embodiments, the nucleic acid marker can be present in two
copies in a corn plant obtained by any of these aforementioned
methods.
[0009] Also provided are methods for introgressing a Goss' Wilt
resistance QTL into a corn plant. In certain embodiments, methods
of introgressing a Goss' Wilt resistance QTL into a corn plant
comprising: a) screening a population with at least one nucleic
acid marker to determine if one or more corn plants from the
population contains a Goss' Wilt resistance QTL, wherein the Goss'
Wilt resistance QTL is a QTL selected from the group consisting of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, and 131 as provided in FIG. 1; and b) selecting from the
population at least one corn plant comprising an allele of the
marker associated with Goss' Wilt resistance are provided. In
certain embodiments of the methods, at least one of the markers is
located within 30 cM, 25 cM, 20 cM, 15 cM, or 10 cM of the Goss'
Wilt resistance QTL. In other embodiments of the methods, at least
one of the markers is located within 5 cM, 2 cM, or 1 cM of the
Goss' Wilt resistance QTL. In certain embodiments of the methods,
at least one of the markers exhibits an LOD score of greater than
2.0, 2.5, or 3.0 with the Goss' Wilt resistance QTL. In other
embodiments of the methods, at least one of the markers exhibits a
LOD score of greater than 4.0 with the Goss' Wilt resistance QTL.
In certain embodiments of these methods, the nucleic acid marker is
selected from the group consisting of SEQ ID NOs: 27, 121, 141,
175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582,
585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122,
1186, 1246, 1250, and 1251, wherein the nucleic acid marker is
selected from the group consisting of SEQ ID NOs: 234 and 1250. In
certain embodiments of the methods, the population is a segregating
population.
[0010] Also provided herein are corn plants obtained by any of the
aforementioned methods of identifying corn plants that comprise a
Goss' Wilt resistance QTL. In certain embodiments, a corn plant
obtained by any of the aforementioned methods is provided, wherein
the corn plant comprises a Goss' Wilt resistance QTL selected from
the group consisting of QTL numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
124, 125, 126, 127, 128, 129, 130, and 131 as provided in FIG. 1.
In certain embodiments, a corn plant obtained by any of these
aforementioned methods and comprising at least one of the QTL
exhibits at least partial resistance to a Goss' Wilt-inducing
bacterium. In certain embodiments, a corn plant obtained by any of
these aforementioned methods exhibits at least substantial
resistance to a Goss' Wilt-inducing bacterium. In still other
embodiments, a corn plant obtained by any of these aforementioned
methods and comprising at least one of the QTL can be a haploid
corn plant. In certain embodiments, a corn plant obtained by any of
the aforementioned methods and comprising at least one of the QTL
can comprise at least one transgenic trait. In such embodiments,
the transgenic trait can be herbicide tolerance and/or pest
resistance. In embodiments where the corn plant obtained is
herbicide tolerant, herbicide tolerance can be selected from the
group consisting of glyphosate, dicamba, glufosinate, sulfonylurea,
bromoxynil and norflurazon herbicide tolerance.
[0011] Also provided herein are isolated nucleic acid markers for
identifying polymorphisms in corn DNA. These isolated nucleic acids
can be used in a variety of applications, including but not limited
to, the identification of corn plants that comprise alleles of
genetic loci associated with Goss' Wilt resistance. In certain
embodiments, an isolated nucleic acid molecule for detecting a
molecular marker representing a polymorphism in corn DNA, wherein
the nucleic acid molecule comprises at least 15 nucleotides that
include or are immediately adjacent to the polymorphism, wherein
the nucleic acid molecule is at least 90 percent identical to a
sequence of the same number of consecutive nucleotides in either
strand of DNA that include or are immediately adjacent to the
polymorphism, and wherein the molecular marker is selected from the
group consisting of SEQ ID NOs: 27, 121, 141, 175, 177, 220, 224,
234, 248, 252, 440, 479, 480, 533, 582, 585, 639, 721, 727, 733,
746, 768, 773, 940, 1053, 1054, 1122, 1186, 1234-1302, and 1303 is
provided. In other embodiments, the molecular marker is selected
from the group consisting of SEQ ID NOs: 27, 121, 141, 175, 177,
220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582, 585, 639,
721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122, 1186, 1246,
1250, and 1251. In still other embodiments, the molecular marker is
selected from the group consisting of SEQ ID NOs: 234 and 1250. In
certain embodiments, the isolated nucleic acid further comprises a
detectable label or provides for incorporation of a detectable
label. In such embodiments that comprise or provide for
incorporation of a detectable label, the detectable label is
selected from the group consisting of an isotope, a fluorophore, an
oxidant, a reductant, a nucleotide and a hapten. In certain
embodiments, the detectable label is added to the nucleic acid by a
chemical reaction or is incorporated by an enzymatic reaction. In
certain embodiments, the isolated nucleic acid molecule comprises
at least 16 or 17 nucleotides that include or are immediately
adjacent to the polymorphism. In other embodiments, the nucleic
acid molecule comprises at least 18 nucleotides that include or are
immediately adjacent to the polymorphism or comprises at least 20
nucleotides that include or are immediately adjacent to the
polymorphism. In certain embodiments, the isolated nucleic acid
molecule hybridizes to at least one allele of the molecular marker
under stringent hybridization conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate the embodiments of the
present invention and together with the description, serve to
explain the principles of the invention.
[0013] In the drawings:
[0014] FIG. 1. Displays markers associated with resistance to Goss'
Wilt. The symbol "*" represents a single nucleotide deletion.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0015] The definitions and methods provided herein define the
present invention and guide those of ordinary skill in the art in
the practice of the present invention. Unless otherwise noted,
terms are to be understood according to conventional usage by those
of ordinary skill in the relevant art. Definitions of common terms
in molecular biology may also be found in Alberts et al., Molecular
Biology of The Cell, 3rd Edition, Garland Publishing, Inc.: New
York, 1994; Rieger et al., Glossary of Genetics: Classical and
Molecular, 5th Edition, Springer-Verlag: New York, 1991; and Lewin,
Genes V, Oxford University Press New York, 1994. The nomenclature
for DNA bases as set forth at 37 CFR .sctn. 1.822 is used.
[0016] As used herein, a "locus" is a fixed position on a
chromosome and may represent a single nucleotide, a few nucleotides
or a large number of nucleotides in a genomic region.
[0017] As used herein, "polymorphism" means the presence of one or
more variations of a nucleic acid sequence at one or more loci in a
population of one or more individuals. The variation may comprise
but is not limited to, one or more base changes, the insertion of
one or more nucleotides or the deletion of one or more nucleotides.
A polymorphism includes a single nucleotide polymorphism (SNP), a
simple sequence repeat (SSR) and indels, which are insertions and
deletions. A polymorphism may arise from random processes in
nucleic acid replication, through mutagenesis, as a result of
mobile genomic elements, from copy number variation and during the
process of meiosis, such as unequal crossing over, genome
duplication and chromosome breaks and fusions. The variation can be
commonly found or may exist at low frequency within a population,
the former having greater utility in general plant breeding and the
later may be associated with rare but important phenotypic
variation.
[0018] As used herein, "marker" means a detectable characteristic
that can be used to discriminate between organisms. Examples of
such characteristics may include genetic markers, protein
composition, protein levels, oil composition, oil levels,
carbohydrate composition, carbohydrate levels, fatty acid
composition, fatty acid levels, amino acid composition, amino acid
levels, biopolymers, pharmaceuticals, starch composition, starch
levels, fermentable starch, fermentation yield, fermentation
efficiency, energy yield, secondary compounds, metabolites,
morphological characteristics, and agronomic characteristics.
[0019] As used herein, "genetic marker" means polymorphic nucleic
acid sequence or nucleic acid feature. A "polymorphism" is a
variation among individuals in sequence, particularly in DNA
sequence, or feature, such as a transcriptional profile or
methylation pattern. Useful polymorphisms include single nucleotide
polymorphisms (SNPs), insertions or deletions in DNA sequence
(Indels), simple sequence repeats of DNA sequence (SSRs) a
restriction fragment length polymorphism, a haplotype, and a tag
SNP. A genetic marker, a gene, a DNA-derived sequence, a
RNA-derived sequence, a promoter, a 5' untranslated region of a
gene, a 3' untranslated region of a gene, microRNA, siRNA, a QTL, a
satellite marker, a transgene, mRNA, ds mRNA, a transcriptional
profile, and a methylation pattern may comprise polymorphisms.
[0020] As used herein, "marker assay" means a method for detecting
a polymorphism at a particular locus using a particular method,
e.g. measurement of at least one phenotype (such as seed color,
flower color, or other visually detectable trait), restriction
fragment length polymorphism (RFLP), single base extension,
electrophoresis, sequence alignment, allelic specific
oligonucleotide hybridization (ASO), random amplified polymorphic
DNA (RAPD), microarray-based technologies, and nucleic acid
sequencing technologies, etc.
[0021] As used herein, the phrase "immediately adjacent", when used
to describe a nucleic acid molecule that hybridizes to DNA
containing a polymorphism, refers to a nucleic acid that hybridizes
to DNA sequences that directly abut the polymorphic nucleotide base
position. For example, a nucleic acid molecule that can be used in
a single base extension assay is "immediately adjacent" to the
polymorphism.
[0022] As used herein, "interrogation position" refers to a
physical position on a solid support that can be queried to obtain
genotyping data for one or more predetermined genomic
polymorphisms.
[0023] As used herein, "consensus sequence" refers to a constructed
DNA sequence which identifies SNP and Indel polymorphisms in
alleles at a locus. Consensus sequence can be based on either
strand of DNA at the locus and states the nucleotide base of either
one of each SNP in the locus and the nucleotide bases of all Indels
in the locus. Thus, although a consensus sequence may not be a copy
of an actual DNA sequence, a consensus sequence is useful for
precisely designing primers and probes for actual polymorphisms in
the locus.
[0024] As used herein, the term "single nucleotide polymorphism,"
also referred to by the abbreviation "SNP," means a polymorphism at
a single site wherein said polymorphism constitutes a single base
pair change, an insertion of one or more base pairs, or a deletion
of one or more base pairs.
[0025] As used herein, "genotype" means the genetic component of
the phenotype and it can be indirectly characterized using markers
or directly characterized by nucleic acid sequencing. Suitable
markers include a phenotypic character, a metabolic profile, a
genetic marker, or some other type of marker. A genotype may
constitute an allele for at least one genetic marker locus or a
haplotype for at least one haplotype window. In some embodiments, a
genotype may represent a single locus and in others it may
represent a genome-wide set of loci. In another embodiment, the
genotype can reflect the sequence of a portion of a chromosome, an
entire chromosome, a portion of the genome, and the entire
genome.
[0026] As used herein, the term "haplotype" means a chromosomal
region within a haplotype window defined by at least one
polymorphic molecular marker. The unique marker fingerprint
combinations in each haplotype window define individual haplotypes
for that window. Further, changes in a haplotype, brought about by
recombination for example, may result in the modification of a
haplotype so that it comprises only a portion of the original
(parental) haplotype operably linked to the trait, for example, via
physical linkage to a gene, QTL, or transgene. Any such change in a
haplotype would be included in our definition of what constitutes a
haplotype so long as the functional integrity of that genomic
region is unchanged or improved.
[0027] As used herein, the term "haplotype window" means a
chromosomal region that is established by statistical analyses
known to those of skill in the art and is in linkage
disequilibrium. Thus, identity by state between two inbred
individuals (or two gametes) at one or more molecular marker loci
located within this region is taken as evidence of
identity-by-descent of the entire region. Each haplotype window
includes at least one polymorphic molecular marker. Haplotype
windows can be mapped along each chromosome in the genome.
Haplotype windows are not fixed per se and, given the
ever-increasing density of molecular markers, this invention
anticipates the number and size of haplotype windows to evolve,
with the number of windows increasing and their respective sizes
decreasing, thus resulting in an ever-increasing degree confidence
in ascertaining identity by descent based on the identity by state
at the marker loci.
[0028] As used herein, a plant referred to as "haploid" has a
single set (genome) of chromosomes and the reduced number of
chromosomes (n) in the haploid plant is equal to that of the
gamete.
[0029] As used herein, a plant referred to as "doubled haploid" is
developed by doubling the haploid set of chromosomes. A plant or
seed that is obtained from a doubled haploid plant that is selfed
any number of generations may still be identified as a doubled
haploid plant. A doubled haploid plant is considered a homozygous
plant. A plant is considered to be doubled haploid if it is
fertile, even is the entire vegetative part of the plant does not
consist of the cells with the doubled set of chromosomes; that is,
a plant will be considered doubled haploid if it contains viable
gametes, even if it is chimeric.
[0030] As used herein, a plant referred to as "diploid" has two
sets (genomes) of chromosomes and the chromosome number (2n) is
equal to that of the zygote.
[0031] As used herein, the term "plant" includes whole plants,
plant organs (i.e., leaves, stems, roots, etc.), seeds, and plant
cells and progeny of the same. "Plant cell" includes without
limitation seeds, suspension cultures, embryos, meristematic
regions, callus tissue, leaves, shoots, gametophytes, sporophytes,
pollen, and microspores.
[0032] As used herein, a "genetic map" is the ordered list of loci
known for a particular genome.
[0033] As used herein, "phenotype" means the detectable
characteristics of a cell or organism which are a manifestation of
gene expression.
[0034] As used herein, a "phenotypic marker" refers to a marker
that can be used to discriminate phenotypes displayed by
organisms.
[0035] As used herein, "linkage" refers to relative frequency at
which types of gametes are produced in a cross. For example, if
locus A has genes "A" or "a" and locus B has genes "B" or "b" and a
cross between parent I with AABB and parent B with aabb will
produce four possible gametes where the genes are segregated into
AB, Ab, aB and ab. The null expectation is that there will be
independent equal segregation into each of the four possible
genotypes, i.e. with no linkage 1/4 of the gametes will of each
genotype. Segregation of gametes into a genotypes differing from
1/4 are attributed to linkage.
[0036] As used herein, "linkage disequilibrium" is defined in the
context of the relative frequency of gamete types in a population
of many individuals in a single generation. If the frequency of
allele A is p, a is p', B is q and b is q', then the expected
frequency (with no linkage disequilibrium) of genotype AB is pq, Ab
is pq', aB is p'q and ab is p'q'. Any deviation from the expected
frequency is called linkage disequilibrium. Two loci are said to be
"genetically linked" when they are in linkage disequilibrium.
[0037] As used herein, "quantitative trait locus (QTL)" means a
locus that controls to some degree numerically representable traits
that are usually continuously distributed.
[0038] As used herein, the term "transgene" means nucleic acid
molecules in form of DNA, such as cDNA or genomic DNA, and RNA,
such as mRNA or microRNA, which may be single or double
stranded.
[0039] As used herein, the term "inbred" means a line that has been
bred for genetic homogeneity.
[0040] As used herein, the term "hybrid" means a progeny of mating
between at least two genetically dissimilar parents. Without
limitation, examples of mating schemes include single crosses,
modified single cross, double modified single cross, three-way
cross, modified three-way cross, and double cross wherein at least
one parent in a modified cross is the progeny of a cross between
sister lines.
[0041] As used herein, the term "tester" means a line used in a
testcross with another line wherein the tester and the lines tested
are from different germplasm pools. A tester may be isogenic or
nonisogenic.
[0042] As used herein, "resistance allele" means the isolated
nucleic acid sequence that includes the polymorphic allele
associated with resistance to the disease or condition of
concern.
[0043] As used herein, the term "corn" means Zea mays or maize and
includes all plant varieties that can be bred with corn, including
wild maize species.
[0044] As used herein, the term "comprising" means "including but
not limited to".
[0045] As used herein, an "elite line" is any line that has
resulted from breeding and selection for superior agronomic
performance.
[0046] As used herein, an "inducer" is a line which when crossed
with another line promotes the formation of haploid embryos.
[0047] As used herein, "haplotype effect estimate" means a
predicted effect estimate for a haplotype reflecting association
with one or more phenotypic traits, wherein the associations can be
made de novo or by leveraging historical haplotype-trait
association data.
[0048] As used herein, "breeding value" means a calculation based
on nucleic acid sequence effect estimates and nucleic acid sequence
frequency values, the breeding value of a specific nucleic acid
sequence relative to other nucleic acid sequences at the same locus
(i.e., haplotype window), or across loci (i.e., haplotype windows),
can also be determined. In other words, the change in population
mean by fixing said nucleic acid sequence is determined. In
addition, in the context of evaluating the effect of substituting a
specific region in the genome, either by introgression or a
transgenic event, breeding values provide the basis for comparing
specific nucleic acid sequences for substitution effects. Also, in
hybrid crops, the breeding value of nucleic acid sequences can be
calculated in the context of the nucleic acid sequence in the
tester used to produce the hybrid.
[0049] To the extent to which any of the preceding definitions is
inconsistent with definitions provided in any patent or non-patent
reference incorporated herein or in any reference found elsewhere,
it is understood that the preceding definition will be used
herein.
Methods and Compositions for Goss' Wilt Resistance in Corn
[0050] The present invention provides a method of using haploid
plants to identify genotypes associated with phenotypes of interest
wherein the haploid plant is assayed with at least one marker and
associating the at least one marker with at least one phenotypic
trait. The genotype of interest can then be used to make decisions
in a plant breeding program. Such decisions include, but are not
limited to, selecting among new breeding populations which
population has the highest frequency of favorable nucleic acid
sequences based on historical genotype and agronomic trait
associations, selecting favorable nucleic acid sequences among
progeny in breeding populations, selecting among parental lines
based on prediction of progeny performance, and advancing lines in
germplasm improvement activities based on presence of favorable
nucleic acid sequences. Non-limiting examples of germplasm
improvement activities include line development, hybrid
development, transgenic event selection, making breeding crosses,
testing and advancing a plant through self fertilization, using
plants for transformation, using plants for candidates for
expression constructs, and using plants for mutagenesis.
[0051] Non-limiting examples of breeding decisions include progeny
selection, parent selection, and recurrent selection for at least
one haplotype. In another aspect, breeding decisions relating to
development of plants for commercial release comprise advancing
plants for testing, advancing plants for purity, purification of
sublines during development, inbred development, variety
development, and hybrid development. In yet other aspects, breeding
decisions and germplasm improvement activities comprise transgenic
event selection, making breeding crosses, testing and advancing a
plant through self-fertilization, using plants for transformation,
using plants for candidates for expression constructs, and using
plants for mutagenesis.
[0052] In still another embodiment, the present invention
acknowledges that preferred haplotypes and QTL identified by the
methods presented herein may be advanced as candidate genes for
inclusion in expression constructs, i.e., transgenes. Nucleic acids
underlying haplotypes or QTL of interest may be expressed in plant
cells by operably linking them to a promoter functional in plants.
In another aspect, nucleic acids underlying haplotypes or QTL of
interest may have their expression modified by double-stranded
RNA-mediated gene suppression, also known as RNA interference
("RNAi"), which includes suppression mediated by small interfering
RNAs ("siRNA"), trans-acting small interfering RNAs ("ta-siRNA"),
or microRNAs ("miRNA"). Examples of RNAi methodology suitable for
use in plants are described in detail in U.S. patent application
publications 2006/0200878 and 2007/0011775.
[0053] Methods are known in the art for assembling and introducing
constructs into a cell in such a manner that the nucleic acid
molecule for a trait is transcribed into a functional mRNA molecule
that is translated and expressed as a protein product. For the
practice of the present invention, conventional compositions and
methods for preparing and using constructs and host cells are well
known to one skilled in the art, see for example, Molecular
Cloning: A Laboratory Manual, 3rd Edition Volumes 1, 2, and 3
(2000) J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring
Harbor Laboratory Press. Methods for making transformation
constructs particularly suited to plant transformation include,
without limitation, those described in U.S. Pat. Nos. 4,971,908,
4,940,835, 4,769,061 and 4,757,011, all of which are herein
incorporated by reference in their entirety. Transformation methods
for the introduction of expression units into plants are known in
the art and include electroporation as illustrated in U.S. Pat. No.
5,384,253; microprojectile bombardment as illustrated in U.S. Pat.
Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and
6,403,865; protoplast transformation as illustrated in U.S. Pat.
No. 5,508,184; and Agrobacterium-mediated transformation as
illustrated in U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616;
5,981,840; and 6,384,301.
[0054] The present invention provides Goss' Wilt resistance loci
that are located in public bins in the maize genome that were not
previously associated with Goss' Wilt resistance.
[0055] The present invention provides 130 Goss' Wilt resistance
loci that are located in public bins in the maize genome that were
not previously associated with Goss' Wilt resistance. QTL were
assigned by dividing maize chromosomal regions into 10 cM windows.
A total of 131 QTL were identified, with 130 not having been
previously reported. SNP markers are also provided for monitoring
the introgression of the 131 QTL associated with Goss' Wilt
resistance.
[0056] In the present invention, Goss' Wilt resistance loci 1-53
and 55-131 have not been previously associated with Goss' Wilt and
are provided. SNP markers are also provided for monitoring the
introgression of Goss' Wilt resistance. In the present invention,
Goss' Wilt resistance loci 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, and 20 are located on Chromosome 1. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 1 include those selected from the group consisting of SEQ ID
NOs: 13 and 1274. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 2 included those selected from the
group consisting of SEQ ID NOs: 1234 and 19. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 3 include
those selected from the group consisting of SEQ ID NOs: 27 and 24.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 4 include SEQ ID NO: 36. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 5 included
those selected from the group consisting of SEQ ID NOs: 50 and 53.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 6 include SEQ ID NO: 90. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 7 include
those selected from the group consisting of SEQ ID NOs: 94, 95, 97,
1235, 1236, 99, 101, 102, 1237, 106, 1238, 110, 111, and 1239. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 8 include those selected from the group consisting of SEQ ID
NOs: 1240, 119, 121, 122, and 124. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 9 include those
selected from the group consisting of SEQ ID NOs: 128, 130, 131,
and 132. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 10 include those selected from the group
consisting of SEQ ID NOs: 136, 138, and 1275. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 11 include
SEQ ID NOs: 141. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 12 include SEQ ID NOs: 146. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 13
include those selected from the group consisting of SEQ ID NOs:
153, 1241, 159, 160, 162, and 158. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 14 include those
selected from the group consisting of SEQ ID NOs: 164, 166, 169,
and 172. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 15 include those selected from the group
consisting of SEQ ID NOs: 175 and 177. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 16 include those
selected from the group consisting of SEQ ID NOs: 1242 and 186. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 17 include SEQ ID NO: 200. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 18 include those
selected from the group consisting of SEQ ID NOs: 202 and 203. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 19 include those selected from the group consisting of SEQ ID
NOs: 207 and 208. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 20 include SEQ ID NO: 1243.
[0057] In the present invention Goss' Wilt resistance loci 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 129 are
located on Chromosome 2. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 21 include SEQ ID NO:
215. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 22 include those selected from the group
consisting of SEQ ID NOs: 216, 1244, 220, 218, and 1229. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 23 include those selected from the group consisting of SEQ ID
NOs: 224. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 24 include those selected from the group
consisting of SEQ ID NO: 228, 231, and 1276. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 25 include
those selected from the group consisting of SEQ ID NOs: 232, 233,
234, 235, and 236. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 26 include those selected from the
group consisting of SEQ ID NOs: 244, 248, and 1277. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 27
include those selected from the group consisting of SEQ ID NOs:
250, 252, 256, 257, 260, 1295, and 1278. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 28 include
those selected from the group consisting of SEQ ID NOs: 265, 266,
and 267. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 29 include those selected from the group
consisting of SEQ ID NOs: 271, 273, 1245, 274, 278, 279, 282, 287,
289, and 272. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 30 include those selected from the
group consisting of SEQ ID NOs: 294, 295, 296, and 299. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 31
include those selected from the group consisting of SEQ ID NOs:
1246, 317, and 320. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 32 include those selected from the
group consisting of SEQ ID NOs: 332, 333, and 334. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 33
include SEQ ID NO: 337. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 34 include SEQ ID NO:
347. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 35 include SEQ ID NO: 355. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 129
include SEQ ID NO: 1294.
[0058] In the present invention Goss' Wilt resistance loci 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 122, and 123
are located on Chromosome 3. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 36 include those
selected from the group consisting of SEQ ID NOs: 362 and 363. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 37 include SEQ ID NO: 1247. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 38 include SEQ ID NO:
366. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 39 include those selected from the group
consisting of SEQ ID NO: 367, 368, and 1279. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 40 include
those selected from the group consisting of SEQ ID NO: 370 and 371.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 41 include those selected from the group
consisting of SEQ ID NOs: 381, 382, 392, and 395. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 42
include those selected from the group consisting of SEQ ID NOs:
409, 411, and 412. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 43 include those selected from the
group consisting of SEQ ID NOs: 419, 422, 423, and 1280. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 44 include those selected from the group consisting of SEQ ID
NOs: 429, 430, 433 and 1281. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 45 include those
selected from the group consisting of SEQ ID NOs: 438, 440, 1248,
and 447. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 46 include SEQ ID NO: 1249. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 47
include those selected from the group consisting of SEQ ID NOs:
474, 476, 479, 480, and 482. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 48 include those
selected from the group SEQ ID NO: 486. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 49 include SEQ ID
NO: 490. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 50 include SEQ ID NO: 493. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 122
include those selected from the group consisting of SEQ ID NOs: 375
and 1296. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 123 include those selected from the group
consisting of SEQ ID NOs: 401 and 408.
[0059] In the present invention Goss' Wilt resistance loci 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 124, 125, and 126 are located
on Chromosome 4. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 51 include SEQ ID NO: 500. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 52
include those selected from the group consisting of SEQ ID NOs:
1250, 525, and 530. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 53 include SEQ ID NOs: 533. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 54 include those selected from the group consisting of SEQ ID
NOs: 556 and 566. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 55 include those selected from the
group consisting of SEQ ID NOs: 582, 585, 1251, and 1283. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 56 include those selected from the group consisting of SEQ ID
NOs: 589 and 587. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 57 include those selected from the
group consisting of SEQ ID NOs: 593, 594, and 599. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 58
include SEQ ID NO: 611, 1297, 1298, and 1284. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 59 include
those selected from the group consisting of SEQ ID NOs: 1252, 618,
621, and 623. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 60 include those selected from the
group consisting of SEQ ID NOs: 630, 632, 637, 639, and 629. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 61 include those selected from the group consisting of SEQ ID
NOs: 646, 649, and 650. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 124 include SEQ ID NO:
498. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 125 include SEQ ID NO: 1282.
[0060] In the present invention Goss' Wilt resistance loci 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and 130 are located
on Chromosome 5. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 62 include SEQ ID NO: 657. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 63
include those selected from the group consisting of SEQ ID NOs:
665, 1286, and 1299. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 64 include SEQ ID NO: 669. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 65 include those selected from SEQ ID NO: 1253. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 66
include those selected from the group consisting of SEQ ID NOs:
678, 1254, and 1255. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 67 include those selected from the
group consisting of SEQ ID NOs: 679, 688, and 690. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 68
include those selected from the group consisting of SEQ ID NOs:
1256, 704, 709, and 1300. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 69 include those
selected from the group consisting of SEQ ID NOs: 710, 717, 719,
720, 1257, and 721. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 70 include those selected from the
group SEQ ID NOs: 726, 727, and 1258. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 71 include those
selected from the group consisting of SEQ ID NOs: 733 and 734. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 72 include those selected from the group consisting of SEQ ID
NOs: 746 and 744. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 73 include those selected from the
group consisting of SEQ ID NOs: 758 and 760. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 74 include
those selected from the group consisting of SEQ ID NOs: 764, 768,
and 1287. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 75 include SEQ ID NO: 773. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 130
include SEQ ID NO: 1301.
[0061] In the present invention Goss' Wilt resistance loci 76, 77,
78, 79, 80, 81, 82, 83, and 84 are located on Chromosome 6. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 76 include those selected from the group consisting of SEQ ID
NOs: 1259, 792, 793, and 812. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 77 include those
selected from the group consisting of SEQ ID NOs: 821 and 825. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 78 include those selected from the group consisting of SEQ ID
NOs: 835, 1260, and 844. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 79 include those
selected from the group consisting of SEQ ID NOs: 846, 850, and
854. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 80 include those selected from the group
consisting of SEQ ID NOs: 856, 857, and 858. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 81 include
SEQ ID NO: 1261. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 82 include SEQ ID NO: 874. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 83
include those selected from the group consisting of SEQ ID NOs:
876, 880, and 882. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 84 include SEQ ID NO: 885.
[0062] In the present invention Goss' Wilt resistance loci 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 127 are located on
Chromosome 7. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 85 include SEQ ID NO: 893. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 86
include those selected from the group consisting of SEQ ID NOs: 897
and 896. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 87 include SEQ ID NO: 1262. SNP markers used
to monitor the introgression of Goss' Wilt resistance locus 88
include those selected from the group consisting of SEQ ID NOs:
915, 926, and 1288. SNP markers used to monitor the introgression
of Goss' Wilt resistance locus 89 include those selected from the
group consisting of SEQ ID NOs: 940 and 942. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 90 include
those selected from the group consisting of SEQ ID NOs: 949 and
951. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 91 include SEQ ID NO: 957 and 963. SNP markers
used to monitor the introgression of Goss' Wilt resistance locus 92
include those selected from the group consisting of SEQ ID NO: 964
and 1289. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 93 include those selected from the group
consisting of SEQ ID NO: 974 and 976. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 94 include SEQ ID
NO: 1263. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 95 include those selected from the group
consisting of SEQ ID NO: 981 and 1291. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 96 include SEQ ID
NOs: 983 and 990. SNP markers used to monitor the introgression of
Goss' Wilt resistance locus 127 include SEQ ID NO: 1290.
[0063] In the present invention Goss' Wilt resistance loci 97, 98,
99, 100, 101, 102, 103, and 131 are located on Chromosome 8. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 97 include those selected from the group consisting of SEQ ID
NOs: 997, 999, and 1000. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 98 include those
selected from the group consisting of SEQ ID NOs: 1016 and 1264.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 99 include those selected from the group
consisting of SEQ ID NOs: 1027, 1265, and 1303. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 100
include SEQ ID NO: 1043. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 101 include SEQ ID NO:
1049. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 102 include SEQ ID NO: 1056. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 103
include SEQ ID NO: 1075. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 131 include SEQ ID NO:
1015.
[0064] In the present invention Goss' Wilt resistance loci 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, and 115 are
located on Chromosome 9. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 104 include those
selected from the group consisting of SEQ ID NOs: 1266 and 1081.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 105 include SEQ ID NO: 1087. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 106
include SEQ ID NO: 1088. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 107 include SEQ ID NO:
1098. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 108 include those selected from the group
consisting of SEQ ID NOs: 1099, 1100, 1104, 1105, 1108, 1110, and
1292. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 109 include those selected from the group
consisting of SEQ ID NOs: 1267 and 1115. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 110
include those selected from the group consisting of SEQ ID NOs:
1122, 1268, 1131, and 1133. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 111 include those
selected from the group consisting of SEQ ID NOs: 1269 and 1142.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 112 include those selected from the group
consisting of SEQ ID NOs: 1143, 1145, 1146, 1148, and 1149. SNP
markers used to monitor the introgression of Goss' Wilt resistance
locus 113 include SEQ ID NO: 1270. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 114 include SEQ ID NO:
1159.
[0065] In the present invention Goss' Wilt resistance loci 115,
116, 117, 118, 119, 120, 121, and 122 are located on Chromosome 10.
SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 115 include SEQ ID NO: 1168. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 116
include SEQ ID NO: 1174. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 117 include those
selected from the group consisting of SEQ ID NOs: 1271, 1184, and
1186. SNP markers used to monitor the introgression of Goss' Wilt
resistance locus 118 include those selected from the group
consisting of SEQ ID NO: 1272 and 1196. SNP markers used to monitor
the introgression of Goss' Wilt resistance locus 119 include SEQ ID
NO: 1204. SNP markers used to monitor the introgression of Goss'
Wilt resistance locus 120 include those selected from the group
consisting of SEQ ID NOs: 1212 and 1215. SNP markers used to
monitor the introgression of Goss' Wilt resistance locus 121
include SEQ ID NO: 1273. SNP markers used to monitor the
introgression of Goss' Wilt resistance locus 128 include SEQ ID NO:
1293.
[0066] Exemplary marker assays for screening for Goss' Wilt
resistance loci are provided in Tables 3, 4, and 5. Illustrative
Goss' Wilt resistance locus 87 SNP marker DNA sequence SEQ ID NO:
896 can be amplified using the primers indicated as SEQ ID NOs:
1332 through 1333 and detected with probes indicated as SEQ ID NOs:
1334 through 1335. Illustrative Goss' Wilt resistance locus 91 SNP
marker DNA sequence SEQ ID NO: 951 can be amplified using the
primers indicated as SEQ ID NOs: 1336 through 1337 and detected
with probes indicated as SEQ ID NOs: 1338 through 1339.
Illustrative Goss' Wilt resistance locus 72 SNP marker DNA sequence
SEQ ID NO: 733 can be amplified using the primers indicated as SEQ
ID NOs: 1340 through 1341 and detected with probes indicated as SEQ
ID NOs: 1342 through 1343. Illustrative Goss' Wilt resistance locus
109 SNP marker DNA sequence SEQ ID NO: 1098 can be amplified using
the primers indicated as SEQ ID NOs: 1344 through 1345 and detected
with probes indicated as SEQ ID NOs: 1346 through 1347.
Illustrative oligonucleotide hybridization probes for Goss' Wilt
resistance locus 87 SNP marker DNA sequence SEQ ID NO: 896 are
provided as SEQ ID NO: 1348 and SEQ ID NO 1349. Illustrative
oligonucleotide hybridization probes for Goss' Wilt resistance
locus 91 SNP marker DNA sequence SEQ ID NO: 951 are provided as SEQ
ID NO: 1350 and SEQ ID NO 1351. Illustrative oligonucleotide
hybridization probes for Goss' Wilt resistance locus 72 SNP marker
DNA sequence SEQ ID NO: 733 are provided as SEQ ID NO: 1352 and SEQ
ID NO 1353. Illustrative oligonucleotide hybridization probes for
Goss' Wilt resistance locus 109 SNP marker DNA sequence SEQ ID NO:
1098 are provided as SEQ ID NO: 1354 and SEQ ID NO 1355. An
illustrative probe for single base extension assays for Goss' Wilt
resistance locus 87 SNP marker DNA sequence SEQ ID NO: 896 is
provided as SEQ ID NO: 1356. An illustrative probe for single base
extension assays for Goss' Wilt resistance locus 91 SNP marker DNA
sequence SEQ ID NO: 951 is provided as SEQ ID NO: 1357. An
illustrative probe for single base extension assays for Goss' Wilt
resistance locus 72 SNP marker DNA sequence SEQ ID NO: 733 is
provided as SEQ ID NO: 1358. An illustrative probe for single base
extension assays for Goss' Wilt resistance locus 109 SNP marker DNA
sequence SEQ ID NO: 1098 is provided as SEQ ID NO: 1359.
[0067] As used herein, Goss' Wilt refers to any Goss' Wilt variant
or isolate. A corn plant of the present invention can be resistant
to one or more bacteria capable of causing or inducing Goss' Wilt.
In one aspect, the present invention provides plants resistant to
Goss' Wilt as well as methods and compositions for screening corn
plants for resistance or susceptibility to Goss' Wilt, caused by
the genus Clavibacter. In a preferred aspect, the present invention
provides methods and compositions for screening corn plants for
resistance or susceptibility to Clavibacter michiganense spp.
[0068] In an aspect, the plant is selected from the genus Zea. In
another aspect, the plant is selected from the species Zea mays. In
a further aspect, the plant is selected from the subspecies Zea
mays L. ssp. mays. In an additional aspect, the plant is selected
from the group Zea mays L. subsp. mays Indentata, otherwise known
as dent corn. In another aspect, the plant is selected from the
group Zea mays L. subsp. mays Indurata, otherwise known as flint
corn. In an aspect, the plant is selected from the group Zea mays
L. subsp. mays Saccharata, otherwise known as sweet corn. In
another aspect, the plant is selected from the group Zea mays L.
subsp. mays Amylacea, otherwise known as flour corn. In a further
aspect, the plant is selected from the group Zea mays L. subsp.
mays Everta, otherwise known as pop corn. Zea plants include
hybrids, inbreds, partial inbreds, or members of defined or
undefined populations.
[0069] Plants of the present invention can be a corn plant that is
very resistant, resistant, substantially resistant, mid-resistant,
comparatively resistant, partially resistant, mid-susceptible, or
susceptible.
[0070] In a preferred aspect, the present invention provides a corn
plant to be assayed for resistance or susceptibility to Goss' Wilt
by any method to determine whether a corn plant is very resistant,
resistant, substantially resistant, mid-resistant, comparatively
resistant, partially resistant, mid-susceptible, or
susceptible.
[0071] Phenotyping for Goss' Wilt is based on visually screening
plants to determine percentage of infected leaf area. The
percentage of leaf area infected is used to rate plants on a scale
of 1 (very resistant) to 9 (susceptible).
[0072] A disease resistance QTL of the present invention may be
introduced into an elite corn inbred line.
[0073] In another aspect, the corn plant can show a comparative
resistance compared to a non-resistant control corn plant. In this
aspect, a control corn plant will preferably be genetically similar
except for the Goss' Wilt resistant allele or alleles in question.
Such plants can be grown under similar conditions with equivalent
or near equivalent exposure to the pathogen. In this aspect, the
resistant plant or plants has less than 25%, 15%, 10%, 5%, 2% or 1%
of leaf area infected.
[0074] A disease resistance QTL of the present invention may be
introduced into an elite corn inbred line. An "elite line" is any
line that has resulted from breeding and selection for superior
agronomic performance.
[0075] A Goss' Wilt resistance QTL of the present invention may
also be introduced into an elite corn plant comprising one or more
transgenes conferring herbicide tolerance, increased yield, insect
control, fungal disease resistance, virus resistance, nematode
resistance, bacterial disease resistance, mycoplasma disease
resistance, modified oils production, high oil production, high
protein production, germination and seedling growth control,
enhanced animal and human nutrition, low raffinose, environmental
stress resistant, increased digestibility, industrial enzymes,
pharmaceutical proteins, peptides and small molecules, improved
processing traits, improved flavor, nitrogen fixation, hybrid seed
production, reduced allergenicity, biopolymers, and biofuels among
others. In one aspect, the herbicide tolerance is selected from the
group consisting of glyphosate, dicamba, glufosinate, sulfonylurea,
bromoxynil and norflurazon herbicides. These traits can be provided
by methods of plant biotechnology as transgenes in corn.
[0076] A disease resistant QTL allele or alleles can be introduced
from any plant that contains that allele (donor) to any recipient
corn plant. In one aspect, the recipient corn plant can contain
additional Goss' Wilt resistant loci. In another aspect, the
recipient corn plant can contain a transgene. In another aspect,
while maintaining the introduced QTL, the genetic contribution of
the plant providing the disease resistant QTL can be reduced by
back-crossing or other suitable approaches. In one aspect, the
nuclear genetic material derived from the donor material in the
corn plant can be less than or about 50%, less than or about 25%,
less than or about 13%, less than or about 5%, 3%, 2% or 1%, but
that genetic material contains the Goss' Wilt resistant locus or
loci of interest.
[0077] It is further understood that a corn plant of the present
invention may exhibit the characteristics of any relative maturity
group. In an aspect, the maturity group is selected from the group
consisting of RM90-95, RM 95-100, RM 100-105, RM 105-110, RM
110-115, and RM 115-120.
[0078] The present invention also includes a method of
introgressing an allele into a corn plant comprising: (A) crossing
at least one Goss' Wilt resistant corn plant with at least one
Goss' Wilt sensitive corn plant in order to form a segregating
population; (B) screening the segregating population with one or
more nucleic acid markers to determine if one or more corn plants
from the segregating population contains a Goss' Wilt resistant
allele, wherein the Goss' Wilt resistant allele is an allele
selected from the group consisting of Goss' Wilt resistant locus 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 132, 124, 125, 126, 127, 128, 129,
130, and Goss' Wilt resistant locus 131.
[0079] The present invention includes isolated nucleic acid
molecules. Such molecules include those nucleic acid molecules
capable of detecting a polymorphism genetically or physically
linked to a Goss' Wilt locus. Such molecules can be referred to as
markers. Additional markers can be obtained that are linked to
Goss' Wilt resistance locus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, and Goss' Wilt resistant locus 131 by
available techniques. In one aspect, the nucleic acid molecule is
capable of detecting the presence or absence of a marker located
less than 30, 20, 10, 5, 2, or 1 centimorgans from a Goss' Wilt
resistance locus. In another aspect, a marker exhibits a LOD score
of 2 or greater, 3 or greater, or 4 or greater with Goss' Wilt,
measuring using Qgene Version 2.23 (1996) and default parameters.
In another aspect, the nucleic acid molecule is capable of
detecting a marker in a locus selected from the group Goss' Wilt
resistance locus 1 through resistance locus 131. In a further
aspect, a nucleic acid molecule is selected from the group
consisting of SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95,
97, 99, 101, 102, 106, 110, 111, 119, 121, 122, 124, 128, 130-132,
136, 138, 141, 146, 153, 158-160, 162, 164, 166, 169, 172, 175,
177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220, 224, 228,
231-236, 244, 248, 250, 252, 256, 257, 260, 265-267, 271-274, 278,
279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337, 347, 355,
362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401, 408,
409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474, 476,
479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556, 566,
582, 585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630,
632, 637, 639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690,
704, 709, 710, 717, 719-721, 726, 727, 733, 734, 744, 746, 758,
760, 764, 768, 773, 792, 793, 812, 821, 825, 835, 844, 846, 850,
854, 856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915, 926,
940, 942, 949, 951, 957, 963, 964, 974, 976, 981, 983, 990, 997,
999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054, 1056, 1075,
1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115, 1122,
1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174,
1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303, 1332-1359
fragments thereof, complements thereof, and nucleic acid molecules
capable of specifically hybridizing to one or more of these nucleic
acid molecules.
[0080] In a preferred aspect, a nucleic acid molecule of the
present invention includes those that will specifically hybridize
to one or more of the nucleic acid molecules set forth in SEQ ID
NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106,
110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146,
153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202,
203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,
250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,
294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368,
370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422,
423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,
490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589,
593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,
649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,
719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,
792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874,
876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,
957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,
1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,
1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142,
1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,
1204, 1212, 1215, 1229, 1234-1303, 1332-1359 or complements thereof
or fragments of either under moderately stringent conditions, for
example at about 2.0.times.SSC and about 65.degree. C. In a
particularly preferred aspect, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth in SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53,
90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122, 124,
128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166, 169,
172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220,
224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,
271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334,
337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392,
395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440,
447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530,
533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621,
623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669, 678,
679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,
744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835,
844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896,
897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981,
983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054,
1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110,
1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159,
1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,
1332-1359 or complements or fragments of either under high
stringency conditions. In one aspect of the present invention, a
preferred marker nucleic acid molecule of the present invention has
the nucleic acid sequence set forth in SEQ ID NOs: 13, 19, 24, 27,
36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121,
122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164,
166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216,
218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260,
265-267, 271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320,
332-334, 337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382,
392, 395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438,
440, 447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525,
530, 533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618,
621, 623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669,
678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733,
734, 744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825,
835, 844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893,
896, 897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976,
981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053,
1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108,
1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149,
1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229,
1234-1303, 1332-1359 or complements thereof or fragments of either.
In another aspect of the present invention, a preferred marker
nucleic acid molecule of the present invention shares between 80%
and 100% or 90% and 100% sequence identity with the nucleic acid
sequences set forth in SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53, 90,
94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122, 124, 128,
130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166, 169, 172,
175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220, 224,
228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267, 271-274,
278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337, 347,
355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401,
408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474,
476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556,
566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629,
630, 632, 637, 639, 646, 649, 650, 657, 665, 669, 678, 679, 688,
690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734, 744, 746,
758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835, 844, 846,
850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915,
926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981, 983, 990,
997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054, 1056,
1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115,
1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168,
1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,
1332-1359 or complements thereof or fragments of either. In a
further aspect of the present invention, a preferred marker nucleic
acid molecule of the present invention shares between 95% and 100%
sequence identity with the sequences set forth in SEQ ID NOs: 13,
19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110,
111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153,
158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202, 203,
207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248, 250,
252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,
294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368,
370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422,
423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,
490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589,
593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,
649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,
719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,
792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874,
876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,
957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,
1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,
1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142,
1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,
1204, 1212, 1215, 1229, 1234-1303, 1332-1359 or complements thereof
or fragments of either. In a more preferred aspect of the present
invention, a preferred marker nucleic acid molecule of the present
invention shares between 98% and 100% sequence identity with the
nucleic acid sequence set forth in SEQ ID NOs: 13, 19, 24, 27, 36,
50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122,
124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166,
169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218,
220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,
271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334,
337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392,
395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440,
447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530,
533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621,
623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669, 678,
679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,
744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835,
844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896,
897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981,
983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054,
1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110,
1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159,
1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,
1332-1359 or complement thereof or fragments of either.
[0081] Nucleic acid molecules or fragments thereof are capable of
specifically hybridizing to other nucleic acid molecules under
certain circumstances. As used herein, two nucleic acid molecules
are capable of specifically hybridizing to one another if the two
molecules are capable of forming an anti-parallel, double-stranded
nucleic acid structure. A nucleic acid molecule is the "complement"
of another nucleic acid molecule if they exhibit complete
complementarity. As used herein, molecules are exhibit "complete
complementarity" when every nucleotide of one of the molecules is
complementary to a nucleotide of the other. Two molecules are
"minimally complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under at least conventional "low-stringency" conditions.
Similarly, the molecules are "complementary" if they can hybridize
to one another with sufficient stability to permit them to remain
annealed to one another under conventional "high-stringency"
conditions. Conventional stringency conditions are described by
Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
and by Haymes et al., In: Nucleic Acid Hybridization, A Practical
Approach, IRL Press, Washington, D.C. (1985). Departures from
complete complementarity are therefore permissible, as long as such
departures do not completely preclude the capacity of the molecules
to form a double-stranded structure. In order for a nucleic acid
molecule to serve as a primer or probe it need only be sufficiently
complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0082] As used herein, a substantially homologous sequence is a
nucleic acid sequence that will specifically hybridize to the
complement of the nucleic acid sequence to which it is being
compared under high stringency conditions. The nucleic-acid probes
and primers of the present invention can hybridize under stringent
conditions to a target DNA sequence. The term "stringent
hybridization conditions" is defined as conditions under which a
probe or primer hybridizes specifically with a target sequence(s)
and not with non-target sequences, as can be determined
empirically. The term "stringent conditions" is functionally
defined with regard to the hybridization of a nucleic-acid probe to
a target nucleic acid (i.e., to a particular nucleic-acid sequence
of interest) by the specific hybridization procedure discussed in
Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al.,
1989 at 9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res.
12:203-213; and Wetmur et al., 1968 J. Mol. Biol. 31:349-370.
Appropriate stringency conditions that promote DNA hybridization
are, for example, 6.0.times. sodium chloride/sodium citrate (SSC)
at about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C., are known to those skilled in the art or can be
found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y., 1989, 6.3.1-6.3.6. For example, the salt concentration
in the wash step can be selected from a low stringency of about
2.0.times.SSC at 50.degree. C. to a high stringency of about
0.2.times.SSC at 50.degree. C. In addition, the temperature in the
wash step can be increased from low stringency conditions at room
temperature, about 22.degree. C., to high stringency conditions at
about 65.degree. C. Both temperature and salt may be varied, or
either the temperature or the salt concentration may be held
constant while the other variable is changed.
[0083] For example, hybridization using DNA or RNA probes or
primers can be performed at 65.degree. C. in 6.times.SSC, 0.5% SDS,
5.times.Denhardt's, 100 .mu.g/mL nonspecific DNA (e.g., sonicated
salmon sperm DNA) with washing at 0.5.times.SSC, 0.5% SDS at
65.degree. C., for high stringency.
[0084] It is contemplated that lower stringency hybridization
conditions such as lower hybridization and/or washing temperatures
can be used to identify related sequences having a lower degree of
sequence similarity if specificity of binding of the probe or
primer to target sequence(s) is preserved. Accordingly, the
nucleotide sequences of the present invention can be used for their
ability to selectively form duplex molecules with complementary
stretches of DNA, RNA, or cDNA fragments.
[0085] A fragment of a nucleic acid molecule provided herein can be
of any size. Fragments provided herein include, but are not limited
to, fragments of nucleic acid sequences set forth in SEQ ID NOs:
13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110,
111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153,
158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202, 203,
207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248, 250,
252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,
294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368,
370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422,
423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,
490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589,
593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,
649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,
719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,
792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874,
876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,
957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,
1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,
1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142,
1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,
1204, 1212, 1215, 1229, 1234-1303, 1332-1359 and complements
thereof. In one aspect, a fragment of a nucleic acid molecule can
be 15 to 25, 15 to 30, 15 to 40, 15 to 50, 15 to 100, 20 to 25, 20
to 30, to 40, 20 to 50, 20 to 100, 25 to 30, 25 to 40, 25 to 50, 25
to 100, 30 to 40, 30 to 50, or to 100 nucleotides in length. In
another aspect, the fragment can be greater than 10, 15, 20, 25,
30, 35, 40, 50, 100, or 250 nucleotides in length.
[0086] Additional genetic markers can be used to select plants with
an allele of a QTL associated with Goss' Wilt resistance. Examples
of public marker databases include, but are not limited to, the
Maize Genome Database located on the world wide web at
www.maizegdb.org, the MaizeSeq database located on the world wide
web at www.www.maizeseq.org, the Panzea maize marker and map
database located on the world wide web at www.panzea.org, and the
MAGI database located on the world wide web at
www.plantgenomics.iastate.edu/maize.
Marker Technology
[0087] Genetic markers of the present invention include "dominant"
or "codominant" markers. "Codominant markers" reveal the presence
of two or more alleles (two per diploid individual). "Dominant
markers" reveal the presence of only a single allele. The presence
of the dominant marker phenotype (e.g., a band of DNA) is an
indication that one allele is present in either the homozygous or
heterozygous condition. The absence of the dominant marker
phenotype (e.g., absence of a DNA band) is merely evidence that
"some other" undefined allele is present. In the case of
populations where individuals are predominantly homozygous and loci
are predominantly dimorphic, dominant and codominant markers can be
equally valuable. As populations become more heterozygous and
multiallelic, codominant markers often become more informative of
the genotype than dominant markers.
[0088] In another embodiment, markers, such as single sequence
repeat markers (SSR), AFLP markers, RFLP markers, RAPD markers,
phenotypic markers, isozyme markers, single nucleotide
polymorphisms (SNPs), insertions or deletions (Indels), single
feature polymorphisms (SFPs, for example, as described in Borevitz
et al. 2003 Gen. Res. 13:513-523), microarray transcription
profiles, DNA-derived sequences, and RNA-derived sequences that are
genetically linked to or correlated with alleles of a QTL of the
present invention can be utilized.
[0089] In one embodiment, nucleic acid-based analyses for the
presence or absence of the genetic polymorphism can be used for the
selection of seeds in a breeding population. A wide variety of
genetic markers for the analysis of genetic polymorphisms are
available and known to those of skill in the art. The analysis may
be used to select for genes, QTL, alleles, or genomic regions
(haplotypes) that comprise or are linked to a genetic marker.
[0090] Herein, nucleic acid analysis methods are known in the art
and include, but are not limited to, PCR-based detection methods
(for example, TaqMan assays), microarray methods, and nucleic acid
sequencing methods. In one embodiment, the detection of polymorphic
sites in a sample of DNA, RNA, or cDNA may be facilitated through
the use of nucleic acid amplification methods. Such methods
specifically increase the concentration of polynucleotides that
span the polymorphic site, or include that site and sequences
located either distal or proximal to it. Such amplified molecules
can be readily detected by gel electrophoresis, fluorescence
detection methods, or other means.
[0091] A method of achieving such amplification employs the
polymerase chain reaction (PCR) (Mullis et al., 1986 Cold Spring
Harbor Symp. Quant. Biol. 51:263-273; European Patent 50,424;
European Patent 84,796; European Patent 258,017; European Patent
237,362; European Patent 201,184; U.S. Pat. No. 4,683,202; U.S.
Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194), using primer
pairs that are capable of hybridizing to the proximal sequences
that define a polymorphism in its double-stranded form.
[0092] Polymorphisms in DNA sequences can be detected or typed by a
variety of effective methods well known in the art including, but
not limited to, those disclosed in U.S. Pat. Nos. 5,468,613 and
5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431;
5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944;
and 5,616,464, all of which are incorporated herein by reference in
their entireties. However, the compositions and methods of this
invention can be used in conjunction with any polymorphism typing
method to type polymorphisms in corn genomic DNA samples. These
corn genomic DNA samples used include but are not limited to, corn
genomic DNA isolated directly from a corn plant, cloned corn
genomic DNA, or amplified corn genomic DNA.
[0093] For instance, polymorphisms in DNA sequences can be detected
by hybridization to allele-specific oligonucleotide (ASO) probes as
disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.
5,468,613 discloses allele specific oligonucleotide hybridizations
where single or multiple nucleotide variations in nucleic acid
sequence can be detected in nucleic acids by a process in which the
sequence containing the nucleotide variation is amplified, spotted
on a membrane and treated with a labeled sequence-specific
oligonucleotide probe.
[0094] Target nucleic acid sequence can also be detected by probe
ligation methods as disclosed in U.S. Pat. No. 5,800,944 where
sequence of interest is amplified and hybridized to probes followed
by ligation to detect a labeled part of the probe.
[0095] Microarrays can also be used for polymorphism detection,
wherein oligonucleotide probe sets are assembled in an overlapping
fashion to represent a single sequence such that a difference in
the target sequence at one point would result in partial probe
hybridization (Borevitz et al., Genome Res. 13:513-523 (2003); Cui
et al., Bioinformatics 21:3852-3858 (2005). On any one microarray,
it is expected there will be a plurality of target sequences, which
may represent genes and/or noncoding regions wherein each target
sequence is represented by a series of overlapping
oligonucleotides, rather than by a single probe. This platform
provides for high throughput screening a plurality of
polymorphisms. A single-feature polymorphism (SFP) is a
polymorphism detected by a single probe in an oligonucleotide
array, wherein a feature is a probe in the array. Typing of target
sequences by microarray-based methods is disclosed in U.S. Pat.
Nos. 6,799,122; 6,913,879; and 6,996,476.
[0096] Target nucleic acid sequence can also be detected by probe
linking methods as disclosed in U.S. Pat. No. 5,616,464 employing
at least one pair of probes having sequences homologous to adjacent
portions of the target nucleic acid sequence and having side chains
which non-covalently bind to form a stem upon base pairing of said
probes to said target nucleic acid sequence. At least one of the
side chains has a photoactivatable group which can form a covalent
cross-link with the other side chain member of the stem.
[0097] Other methods for detecting SNPs and Indels include single
base extension (SBE) methods. Examples of SBE methods include, but
are not limited, to those disclosed in U.S. Pat. Nos. 6,004,744;
6,013,431; 5,595,890; 5,762,876; and 5,945,283. SBE methods are
based on extension of a nucleotide primer that is immediately
adjacent to a polymorphism to incorporate a detectable nucleotide
residue upon extension of the primer. In certain embodiments, the
SBE method uses three synthetic oligonucleotides. Two of the
oligonucleotides serve as PCR primers and are complementary to
sequence of the locus of corn genomic DNA which flanks a region
containing the polymorphism to be assayed. Following amplification
of the region of the corn genome containing the polymorphism, the
PCR product is mixed with the third oligonucleotide (called an
extension primer) which is designed to hybridize to the amplified
DNA immediately adjacent to the polymorphism in the presence of DNA
polymerase and two differentially labeled
dideoxynucleosidetriphosphates. If the polymorphism is present on
the template, one of the labeled dideoxynucleosidetriphosphates can
be added to the primer in a single base chain extension. The allele
present is then inferred by determining which of the two
differential labels was added to the extension primer. Homozygous
samples will result in only one of the two labeled bases being
incorporated and thus only one of the two labels will be detected,
Heterozygous samples have both alleles present, and will thus
direct incorporation of both labels (into different molecules of
the extension primer) and thus both labels will be detected.
[0098] In a preferred method for detecting polymorphisms, SNPs and
Indels can be detected by methods disclosed in U.S. Pat. Nos.
5,210,015; 5,876,930; and 6,030,787 in which an oligonucleotide
probe having a 5'fluorescent reporter dye and a 3'quencher dye
covalently linked to the 5' and 3' ends of the probe. When the
probe is intact, the proximity of the reporter dye to the quencher
dye results in the suppression of the reporter dye fluorescence,
e.g. by Forster-type energy transfer. During PCR forward and
reverse primers hybridize to a specific sequence of the target DNA
flanking a polymorphism while the hybridization probe hybridizes to
polymorphism-containing sequence within the amplified PCR product.
In the subsequent PCR cycle DNA polymerase with 5'.fwdarw.3'
exonuclease activity cleaves the probe and separates the reporter
dye from the quencher dye resulting in increased fluorescence of
the reporter.
Marker-Trait Associations
[0099] For the purpose of QTL mapping, the markers included should
be diagnostic of origin in order for inferences to be made about
subsequent populations. SNP markers are ideal for mapping because
the likelihood that a particular SNP allele is derived from
independent origins in the extant populations of a particular
species is very low. As such, SNP markers are useful for tracking
and assisting introgression of QTLs, particularly in the case of
haplotypes.
[0100] The genetic linkage of additional marker molecules can be
established by a gene mapping model such as, without limitation,
the flanking marker model reported by Lander et al., (Lander et
al., 1989 Genetics, 121:185-199), and the interval mapping, based
on maximum likelihood methods described therein, and implemented in
the software package MAPMAKER/QTL (Lincoln and Lander, Mapping
Genes Controlling Quantitative Traits Using MAPMAKER/QTL, Whitehead
Institute for Biomedical Research, Massachusetts, (1990).
Additional software includes Qgene, Version 2.23 (1996), Department
of Plant Breeding and Biometry, 266 Emerson Hall, XXell University,
Ithaca, N.Y.). Use of Qgene software is a particularly preferred
approach.
[0101] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10(MLE for the presence of a
QTL/MLE given no linked QTL). The LOD score essentially indicates
how much more likely the data are to have arisen assuming the
presence of a QTL versus in its absence. The LOD threshold value
for avoiding a false positive with a given confidence, say 95%,
depends on the number of markers and the length of the genome.
Graphs indicating LOD thresholds are set forth in Lander et al.,
(1989), and further described by Ar s and Moreno-Gonzalez, Plant
Breeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall,
London, pp. 314-331 (1993).
[0102] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use of non-parametric methods (Kruglyak et al., 1995
Genetics, 139:1421-1428). Multiple regression methods or models can
also be used, in which the trait is regressed on a large number of
markers (Jansen, Biometrics in Plant Breed, van Oijen, Jansen
(eds.) Proceedings of the Ninth Meeting of the Eucarpia Section
Biometrics in Plant Breeding, The Netherlands, pp. 116-124 (1994);
Weber and Wricke, Advances in Plant Breeding, Blackwell, Berlin, 16
(1994)). Procedures combining interval mapping with regression
analysis, whereby the phenotype is regressed onto a single putative
QTL at a given marker interval, and at the same time onto a number
of markers that serve as `cofactors,` have been reported by Jansen
et al. (Jansen et al., 1994 Genetics, 136:1447-1455) and Zeng (Zeng
1994 Genetics 136:1457-1468). Generally, the use of cofactors
reduces the bias and sampling error of the estimated QTL positions
(Utz and Melchinger, Biometrics in Plant Breeding, van Oijen,
Jansen (eds.) Proceedings of the Ninth Meeting of the Eucarpia
Section Biometrics in Plant Breeding, The Netherlands, pp. 195-204
(1994), thereby improving the precision and efficiency of QTL
mapping (Zeng 1994). These models can be extended to
multi-environment experiments to analyze genotype-environment
interactions (Jansen et al., 1995 Theor. Appl. Genet. 91:33-3).
[0103] Selection of appropriate mapping populations is important to
map construction. The choice of an appropriate mapping population
depends on the type of marker systems employed (Tanksley et al.,
Molecular mapping in plant chromosomes. chromosome structure and
function: Impact of new concepts J. P. Gustafson and R. Appels
(eds.). Plenum Press, New York, pp. 157-173 (1988)). Consideration
must be given to the source of parents (adapted vs. exotic) used in
the mapping population. Chromosome pairing and recombination rates
can be severely disturbed (suppressed) in wide crosses
(adapted.times.exotic) and generally yield greatly reduced linkage
distances. Wide crosses will usually provide segregating
populations with a relatively large array of polymorphisms when
compared to progeny in a narrow cross (adapted.times.adapted).
[0104] An F.sub.2 population is the first generation of selfing.
Usually a single F.sub.1 plant is selfed to generate a population
segregating for all the genes in Mendelian (1:2:1) fashion. Maximum
genetic information is obtained from a completely classified
F.sub.2 population using a codominant marker system (Mather,
Measurement of Linkage in Heredity: Methuen and Co., (1938)). In
the case of dominant markers, progeny tests (e.g. F.sub.3,
BCF.sub.2) are required to identify the heterozygotes, thus making
it equivalent to a completely classified F.sub.2 population.
However, this procedure is often prohibitive because of the cost
and time involved in progeny testing. Progeny testing of F.sub.2
individuals is often used in map construction where phenotypes do
not consistently reflect genotype (e.g. disease resistance) or
where trait expression is controlled by a QTL. Segregation data
from progeny test populations (e.g. F.sub.3 or BCF.sub.2) can be
used in map construction. Marker-assisted selection can then be
applied to cross progeny based on marker-trait map associations
(F.sub.2, F.sub.3), where linkage groups have not been completely
disassociated by recombination events (i.e., maximum
disequilibrium).
[0105] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter et al., 1992 Proc. Natl. Acad. Sci.
(USA) 89:1477-1481). However, as the distance between markers
becomes larger (i.e., loci become more independent), the
information in RIL populations decreases dramatically.
[0106] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
1992). Information obtained from backcross populations using either
codominant or dominant markers is less than that obtained from
F.sub.2 populations because one, rather than two, recombinant
gametes are sampled per plant. Backcross populations, however, are
more informative (at low marker saturation) when compared to RILs
as the distance between linked loci increases in RIL populations
(i.e. about 0.15% recombination). Increased recombination can be
beneficial for resolution of tight linkages, but may be undesirable
in the construction of maps with low marker saturation.
[0107] Near-isogenic lines (NIL) created by many backcrosses to
produce an array of individuals that are nearly identical in
genetic composition except for the trait or genomic region under
interrogation can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci are expected to map to
a selected region.
[0108] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore et al., 1991 Proc. Natl. Acad. Sci. (U.S.A.)
88:9828-9832). In BSA, two bulked DNA samples are drawn from a
segregating population originating from a single cross. These bulks
contain individuals that are identical for a particular trait
(resistant or susceptible to particular disease) or genomic region
but arbitrary at unlinked regions (i.e. heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
Marker-Assisted Breeding
[0109] Further, the present invention contemplates that preferred
haploid plants comprising at least one genotype of interest are
identified using the methods disclosed in U.S. Patent Application
Ser. No. 60/837,864, which is incorporated herein by reference in
its entirety, wherein a genotype of interest may correspond to a
QTL or haplotype and is associated with at least one phenotype of
interest. The methods include association of at least one haplotype
with at least one phenotype, wherein the association is represented
by a numerical value and the numerical value is used in the
decision-making of a breeding program. Non-limiting examples of
numerical values include haplotype effect estimates, haplotype
frequencies, and breeding values. In the present invention, it is
particularly useful to identify haploid plants of interest based on
at least one genotype, such that only those lines undergo doubling,
which saves resources. Resulting doubled haploid plants comprising
at least one genotype of interest are then advanced in a breeding
program for use in activities related to germplasm improvement.
[0110] In the present invention, haplotypes are defined on the
basis of one or more polymorphic markers within a given haplotype
window, with haplotype windows being distributed throughout the
crop's genome. In another aspect, de novo and/or historical
marker-phenotype association data are leveraged to infer haplotype
effect estimates for one or more phenotypes for one or more of the
haplotypes for a crop. Haplotype effect estimates enable one
skilled in the art to make breeding decisions by comparing
haplotype effect estimates for two or more haplotypes. Polymorphic
markers, and respective map positions, of the present invention are
provided in U.S. Patent Applications 2005/0204780, 2005/0216545,
2005/0218305, and Ser. No. 11/504,538, which are incorporated
herein by reference in their entirety.
[0111] In yet another aspect, haplotype effect estimates are
coupled with haplotype frequency values to calculate a haplotype
breeding value of a specific haplotype relative to other haplotypes
at the same haplotype window, or across haplotype windows, for one
or more phenotypic traits. In other words, the change in population
mean by fixing the haplotype is determined. In still another
aspect, in the context of evaluating the effect of substituting a
specific region in the genome, either by introgression or a
transgenic event, haplotype breeding values are used as a basis in
comparing haplotypes for substitution effects. Further, in hybrid
crops, the breeding value of haplotypes is calculated in the
context of at least one haplotype in a tester used to produce a
hybrid. Once the value of haplotypes at a given haplotype window
are determined and high density fingerprinting information is
available on specific varieties or lines, selection can be applied
to these genomic regions using at least one marker in the at least
one haplotype.
[0112] In the present invention, selection can be applied at one or
more stages of a breeding program:
[0113] a) Among genetically distinct populations, herein defined as
"breeding populations," as a pre-selection method to increase the
selection index and drive the frequency of favorable haplotypes
among breeding populations, wherein pre-selection is defined as
selection among populations based on at least one haplotype for use
as parents in breeding crosses, and leveraging of marker-trait
association identified in previous breeding crosses.
[0114] b) Among segregating progeny from a breeding population, to
increase the frequency of the favorable haplotypes for the purpose
of line or variety development.
[0115] c) Among segregating progeny from a breeding population, to
increase the frequency of the favorable haplotypes prior to QTL
mapping within this breeding population.
[0116] d) For hybrid crops, among parental lines from different
heterotic groups to predict the performance potential of different
hybrids.
[0117] In the present invention, it is contemplated that methods of
determine associations between genotype and phenotype in haploid
plants can be performed based on haplotypes, versus markers alone
(Fan et al., 2006 Genetics). A haplotype is a segment of DNA in the
genome of an organism that is assumed to be identical by descent
for different individuals when the knowledge of identity by state
at one or more loci is the same in the different individuals, and
that the regional amount of linkage disequilibrium in the vicinity
of that segment on the physical or genetic map is high. A haplotype
can be tracked through populations and its statistical association
with a given trait can be analyzed. By searching the target space
for a QTL association across multiple QTL mapping populations that
have parental lines with genomic regions that are identical by
descent, the effective population size associated with QTL mapping
is increased. The increased sample size results in more recombinant
progeny which increases the precision of estimating the QTL
position.
[0118] Thus, a haplotype association study allows one to define the
frequency and the type of the ancestral carrier haplotype. An
"association study" is a genetic experiment where one tests the
level of departure from randomness between the segregation of
alleles at one or more marker loci and the value of individual
phenotype for one or more traits. Association studies can be done
on quantitative or categorical traits, accounting or not for
population structure and/or stratification. In the present
invention, associations between haplotypes and phenotypes for the
determination of "haplotype effect estimates" can be conducted de
novo, using mapping populations for the evaluation of one or more
phenotypes, or using historical genotype and phenotype data.
[0119] A haplotype analysis is important in that it increases the
statistical power of an analysis involving individual biallelic
markers. In a first stage of a haplotype frequency analysis, the
frequency of the possible haplotypes based on various combinations
of the identified biallelic markers of the invention is determined.
The haplotype frequency is then compared for distinct populations
and a reference population. In general, any method known in the art
to test whether a trait and a genotype show a statistically
significant correlation may be used.
[0120] Methods for determining the statistical significance of a
correlation between a phenotype and a genotype, in this case a
haplotype, may be determined by any statistical test known in the
art and with any accepted threshold of statistical significance
being required. The application of particular methods and
thresholds of significance are well within the skill of the
ordinary practitioner of the art.
[0121] To estimate the frequency of a haplotype, the base reference
germplasm has to be defined (collection of elite inbred lines,
population of random mating individuals, etc.) and a representative
sample (or the entire population) has to be genotyped. For example,
in one aspect, haplotype frequency is determined by simple counting
if considering a set of inbred individuals. In another aspect,
estimation methods that employ computing techniques like the
Expectation/Maximization (EM) algorithm are required if individuals
genotyped are heterozygous at more than one locus in the segment
and linkage phase is unknown (Excoffier et al., 1995 Mol. Biol.
Evol. 12: 921-927; Li et al., 2002 Biostatistics). Preferably, a
method based on the EM algorithm (Dempster et al., 1977 J. R. Stat.
Soc. Ser. B 39:1-38) leading to maximum-likelihood estimates of
haplotype frequencies under the assumption of Hardy-Weinberg
proportions (random mating) is used (Excoffier et al., 1995 Mol.
Biol. Evol. 12: 921-927). Alternative approaches are known in the
art that for association studies: genome-wide association studies,
candidate region association studies and candidate gene association
studies (Li et al., 2006 BMC Bioinformatics 7:258). The polymorphic
markers of the present invention may be incorporated in any map of
genetic markers of a plant genome in order to perform genome-wide
association studies.
[0122] The present invention comprises methods to detect an
association between at least one haplotype in a haploid crop plant
and a preferred trait, including a transgene, or a multiple trait
index and calculate a haplotype effect estimate based on this
association. In one aspect, the calculated haplotype effect
estimates are used to make decisions in a breeding program. In
another aspect, the calculated haplotype effect estimates are used
in conjunction with the frequency of the at least one haplotype to
calculate a haplotype breeding value that will be used to make
decisions in a breeding program. A multiple trait index (MTI) is a
numerical entity that is calculated through the combination of
single trait values in a formula. Most often calculated as a linear
combination of traits or normalized derivations of traits, it can
also be the result of more sophisticated calculations (for example,
use of ratios between traits). This MTI is used in genetic analysis
as if it were a trait.
[0123] Any given chromosome segment can be represented in a given
population by a number of haplotypes that can vary from 1 (region
is fixed), to the size of the population times the ploidy level of
that species (2 in a diploid species), in a population in which
every chromosome has a different haplotype. Identity-by-descent
among haplotype carried by multiple individuals in a non-fixed
population will result in an intermediate number of haplotype and
possibly a differing frequency among the different haplotypes. New
haplotypes may arise through recombination at meiosis between
existing haplotypes in heterozygous progenitors. The frequency of
each haplotype may be estimated by several means known to one
versed in the art (e.g. by direct counting, or by using an EM
algorithm). Let us assume that "k" different haplotypes, identified
as "h.sub.i" (i=1, . . . , k), are known, that their frequency in
the population is "f.sub.i" (i=1, . . . , k), and for each of these
haplotypes we have an effect estimate "Est.sub.i" (i=1, . . . , k).
If we call the "haplotype breeding value" (BV.sub.i) the effect on
that population of fixing that haplotype, then this breeding value
corresponds to the change in mean for the trait(s) of interest of
that population between its original state of haplotype
distribution at the window and a final state at which haplotype
"h.sub.i" encounters itself at a frequency of 100%.
[0124] The haplotype breeding value of h.sub.i in this population
is calculated as:
BV i = Est i - i = 1 k Est i f i ##EQU00001##
[0125] One skilled in the art will recognize that haplotypes that
are rare in the population in which effects are estimated tend to
be less precisely estimated, this difference of confidence may lead
to adjustment in the calculation. For example one can ignore the
effects of rare haplotypes, by calculating breeding value of better
known haplotype after adjusting the frequency of these (by dividing
it by the sum of frequency of the better known haplotypes). One
could also provide confidence intervals for the breeding value of
each haplotypes.
[0126] The present invention anticipates that any particular
haplotype breeding value will change according to the population
for which it is calculated, as a function of difference of
haplotype frequencies. The term "population" will thus assume
different meanings, below are two examples of special cases. In one
aspect, a population is a single inbred in which one intends to
replace its current haplotype h.sub.j by a new haplotype h.sub.i,
in this case BV.sub.i=Est.sub.i-Est.sub.j. In another aspect, a
"population" is a F2 population in which the two parental haplotype
h.sub.i and h.sub.j are originally present in equal frequency
(50%), in which case BV.sub.i=1/2(Est.sub.i-Est.sub.j).
[0127] These statistical approaches enable haplotype effect
estimates to inform breeding decisions in multiple contexts. Other
statistical approaches to calculate breeding values are known to
those skilled in the art and can be used in substitution without
departing from the spirit and scope of this invention.
[0128] In cases where conserved genetic segments, or haplotype
windows, are coincident with segments in which QTL have been
identified it is possible to deduce with high probability that QTL
inferences can be extrapolated to other germplasm having an
identical haplotype in that haplotype window. This a priori
information provides the basis to select for favorable QTLs prior
to QTL mapping within a given population.
[0129] For example, plant breeding decisions could comprise:
[0130] a) Selection among haploid breeding populations to determine
which populations have the highest frequency of favorable
haplotypes, wherein haplotypes are designated as favorable based on
coincidence with previous QTL mapping and preferred populations
undergo doubling; or
[0131] b) Selection of haploid progeny containing the favorable
haplotypes in breeding populations prior to, or in substitution
for, QTL mapping within that population, wherein selection could be
done at any stage of breeding and at any generation of a selection
and can be followed by doubling; or
[0132] c) Prediction of progeny performance for specific breeding
crosses; or
[0133] d) Selection of haploid plants for doubling for subsequent
use in germplasm improvement activities based on the favorable
haplotypes, including line development, hybrid development,
selection among transgenic events based on the breeding value of
the haplotype that the transgene was inserted into, making breeding
crosses, testing and advancing a plant through self fertilization,
using plant or parts thereof for transformation, using plants or
parts thereof for candidates for expression constructs, and using
plant or parts thereof for mutagenesis.
[0134] In cases where haplotype windows are coincident with
segments in which genes have been identified it is possible to
deduce with high probability that gene inferences can be
extrapolated to other germplasm having an identical genotype, or
haplotype, in that haplotype window. This a priori information
provides the basis to select for favorable genes or gene alleles on
the basis of haplotype identification within a given population.
For example, plant breeding decisions could comprise:
[0135] a) Selection among haploid breeding populations to determine
which populations have the highest frequency of favorable
haplotypes, wherein haplotypes are designated as favorable based on
coincidence with previous gene mapping and preferred populations
undergo doubling; or
[0136] b) Selection of haploid progeny containing the favorable
haplotypes in breeding populations, wherein selection is
effectively enabled at the gene level, wherein selection could be
done at any stage of breeding and at any generation of a selection
and can be followed by doubling; or
[0137] c) Prediction of progeny performance for specific breeding
crosses; or
[0138] d) Selection of haploid plants for doubling for subsequent
use in germplasm improvement activities based on the favorable
haplotypes, including line development, hybrid development,
selection among transgenic events based on the breeding value of
the haplotype that the transgene was inserted into, making breeding
crosses, testing and advancing a plant through self fertilization,
using plant or parts thereof for transformation, using plants or
parts thereof for candidates for expression constructs, and using
plant or parts thereof for mutagenesis.
[0139] A preferred haplotype provides a preferred property to a
parent plant and to the progeny of the parent when selected by a
marker means or phenotypic means. The method of the present
invention provides for selection of preferred haplotypes, or
haplotypes of interest, and the accumulation of these haplotypes in
a breeding population.
[0140] In the present invention, haplotypes and associations of
haplotypes to one or more phenotypic traits provide the basis for
making breeding decisions and germplasm improvement activities.
Non-limiting examples of breeding decisions include progeny
selection, parent selection, and recurrent selection for at least
one haplotype. In another aspect, breeding decisions relating to
development of plants for commercial release comprise advancing
plants for testing, advancing plants for purity, purification of
sublines during development, inbred development, variety
development, and hybrid development. In yet other aspects, breeding
decisions and germplasm improvement activities comprise transgenic
event selection, making breeding crosses, testing and advancing a
plant through self-fertilization, using plants or parts thereof for
transformation, using plants or parts thereof for candidates for
expression constructs, and using plants or parts thereof for
mutagenesis.
[0141] In another embodiment, this invention enables indirect
selection through selection decisions for at least one phenotype
based on at least one numerical value that is correlated, either
positively or negatively, with one or more other phenotypic traits.
For example, a selection decision for any given haplotype
effectively results in selection for multiple phenotypic traits
that are associated with the haplotype.
[0142] In still another embodiment, the present invention
acknowledges that preferred haplotypes identified by the methods
presented herein may be advanced as candidate genes for inclusion
in expression constructs, i.e., transgenes. Nucleic acids
underlying haplotypes of interest may be expressed in plant cells
by operably linking them to a promoter functional in plants. In
another aspect, nucleic acids underlying haplotypes of interest may
have their expression modified by double-stranded RNA-mediated gene
suppression, also known as RNA interference ("RNAi"), which
includes suppression mediated by small interfering RNAs ("siRNA"),
trans-acting small interfering RNAs ("ta-siRNA"), or microRNAs
("miRNA"). Examples of RNAi methodology suitable for use in plants
are described in detail in U.S. Patent Application Publications
2006/0200878 and 2007/0011775.
[0143] Methods are known in the art for assembling and introducing
constructs into a cell in such a manner that the nucleic acid
molecule for a trait is transcribed into a functional mRNA molecule
that is translated and expressed as a protein product. For the
practice of the present invention, conventional compositions and
methods for preparing and using constructs and host cells are well
known to one skilled in the art, see for example, Molecular
Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3
(2000) J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring
Harbor Laboratory Press. Methods for making transformation
constructs particularly suited to plant transformation include,
without limitation, those described in U.S. Pat. Nos. 4,971,908,
4,940,835, 4,769,061 and 4,757,011, all of which are herein
incorporated by reference in their entirety. Transformation methods
for the introduction of expression units into plants are known in
the art and include electroporation as illustrated in U.S. Pat. No.
5,384,253; microprojectile bombardment as illustrated in U.S. Pat.
Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and
6,403,865; protoplast transformation as illustrated in U.S. Pat.
No. 5,508,184; and Agrobacterium-mediated transformation as
illustrated in U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616;
5,981,840; and 6,384,301.
[0144] Another preferred embodiment of the present invention is to
build additional value by selecting a composition of haplotypes
wherein each haplotype has a haplotype effect estimate that is not
negative with respect to yield, or is not positive with respect to
maturity, or is null with respect to maturity, or amongst the best
50 percent with respect to a phenotypic trait, transgene, and/or a
multiple trait index when compared to any other haplotype at the
same chromosome segment in a set of germplasm, or amongst the best
50 percent with respect to a phenotypic trait, transgene, and/or a
multiple trait index when compared to any other haplotype across
the entire genome in a set of germplasm, or the haplotype being
present with a frequency of 75 percent or more in a breeding
population or a set of germplasm provides evidence of its high
value, or any combination of these.
[0145] This invention anticipates a stacking of haplotypes from
multiple windows into plants or lines by crossing parent plants or
lines containing different haplotype regions. The value of the
plant or line comprising in its genome stacked haplotype regions is
estimated by a composite breeding value, which depends on a
combination of the value of the traits and the value of the
haplotype(s) to which the traits are linked. The present invention
further anticipates that the composite breeding value of a plant or
line is improved by modifying the components of one or each of the
haplotypes. Additionally, the present invention anticipates that
additional value can be built into the composite breeding value of
a plant or line by selection of at least one recipient haplotype
with a preferred haplotype effect estimate or, in conjunction with
the haplotype frequency, breeding value to which one or any of the
other haplotypes are linked, or by selection of plants or lines for
stacking haplotypes by breeding.
[0146] Another embodiment of this invention is a method for
enhancing breeding populations by accumulation of one or more
preferred haplotypes in a set of germplasm. Genomic regions defined
as haplotype windows include genetic information that contribute to
one or more phenotypic traits of the plant. Variations in the
genetic information at one or more loci can result in variation of
one or more phenotypic traits, wherein the value of the phenotype
can be measured. The genetic mapping of the haplotype windows
allows for a determination of linkage across haplotypes. A
haplotype of interest has a DNA sequence that is novel in the
genome of the progeny plant and can in itself serve as a genetic
marker for the haplotype of interest. Notably, this marker can also
be used as an identifier for a gene or QTL. For example, in the
event of multiple traits or trait effects associated with the
haplotype, only one marker would be necessary for selection
purposes. Additionally, the haplotype of interest may provide a
means to select for plants that have the linked haplotype region.
Selection can be performed by screening for tolerance to an applied
phytotoxic chemical, such as an herbicide or antibiotic, or to
pathogen resistance. Selection may be performed using phenotypic
selection means, such as, a morphological phenotype that is easy to
observe such as seed color, seed germination characteristic,
seedling growth characteristic, leaf appearance, plant
architecture, plant height, and flower and fruit morphology.
[0147] The present invention also provides for the screening of
progeny haploid plants for haplotypes of interest and using
haplotype effect estimates as the basis for selection for use in a
breeding program to enhance the accumulation of preferred
haplotypes. The method includes: a) providing a breeding population
comprising at least two haploid plants wherein the genome of the
breeding population comprises a plurality of haplotype windows and
each of the plurality of haplotype windows comprises at least one
haplotype; and b) associating a haplotype effect estimate for one
or more traits for two or more haplotypes from one or more of the
plurality of haplotype windows, wherein the haplotype effect
estimate can then be used to calculate a breeding value that is a
function of the estimated effect for any given phenotypic trait and
the frequency of each of the at least two haplotypes; and c)
ranking one or more of the haplotypes on the basis of a value,
wherein the value is a haplotype effect estimate, a haplotype
frequency, or a breeding value and wherein the value is the basis
for determining whether a haplotype is a preferred haplotype, or
haplotype of interest; and d) utilizing the ranking as the basis
for decision-making in a breeding program; and e) at least one
progeny haploid plant is selected for doubling on the basis of the
presence of the respective markers associated with the haplotypes
of interest, wherein the progeny haploid plant comprises in its
genome at least a portion of the haplotype or haplotypes of
interest of the first plant and at least one preferred haplotype of
the second plant; and f) using resulting doubled haploid plants in
activities related to germplasm improvement wherein the activities
are selected from the group consisting of line and variety
development, hybrid development, transgenic event selection, making
breeding crosses, testing and advancing a plant through self
fertilization, using plant or parts thereof for transformation,
using plants or parts thereof for candidates for expression
constructs, and using plant or parts thereof for mutagenesis.
[0148] Using this method, the present invention contemplates that
haplotypes of interest are selected from a large population of
plants, and the selected haplotypes can have a synergistic breeding
value in the germplasm of a crop plant. Additionally, this
invention provides for using the selected haplotypes in the
described breeding methods to accumulate other beneficial and
preferred haplotype regions and to be maintained in a breeding
population to enhance the overall germplasm of the crop plant.
[0149] The marker assisted breeding methods and/or methods of
associating markers with traits provided herein can be used with
one or more individuals, including SSD, from any generation of
plant population. Non-limiting examples of plant populations
include to F1, F2, BC1, BC2F1, F3:F4, F2:F3, and so on, including
subsequent filial generations, as well as experimental populations
such as RILs and NILs. It is further anticipated that the degree of
segregation within the one or more plant populations of the present
invention can vary depending on the nature of the trait and
germplasm under evaluation.
Plant Breeding
[0150] Plants of the present invention can be part of or generated
from a breeding program. The choice of breeding method depends on
the mode of plant reproduction, the heritability of the trait(s)
being improved, and the type of cultivar used commercially (e.g.,
F.sub.1 hybrid cultivar, pureline cultivar, etc). A cultivar is a
race or variety of a plant species that has been created or
selected intentionally and maintained through cultivation.
[0151] Selected, non-limiting approaches for breeding the plants of
the present invention are set forth below. A breeding program can
be enhanced using marker assisted selection (MAS) on the progeny of
any cross. It is understood that nucleic acid markers of the
present invention can be used in a MAS (breeding) program. It is
further understood that any commercial and non-commercial cultivars
can be utilized in a breeding program. Factors such as, for
example, emergence vigor, vegetative vigor, stress tolerance,
disease resistance, branching, flowering, seed set, seed size, seed
density, standability, and threshability etc. will generally
dictate the choice.
[0152] Genotyping can be further economized by high throughput,
non-destructive seed sampling. In one embodiment, plants can be
screened for one or more markers, such as genetic markers, using
high throughput, non-destructive seed sampling. In a preferred
aspect, haploid seed is sampled in this manner and only seed with
at least one marker genotype of interest is advanced for doubling.
Apparatus and methods for the high-throughput, non-destructive
sampling of seeds have been described which would overcome the
obstacles of statistical samples by allowing for individual seed
analysis. For example, U.S. patent application Ser. No. 11/213,430
(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,431
(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,432
(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,434
(filed Aug. 26, 2005); and U.S. patent application Ser. No.
11/213,435 (filed Aug. 26, 2005), U.S. patent application Ser. No.
11/680,611 (filed Mar. 2, 2007), which are incorporated herein by
reference in their entirety, disclose apparatus and systems for the
automated sampling of seeds as well as methods of sampling, testing
and bulking seeds.
[0153] For highly heritable traits, a choice of superior individual
plants evaluated at a single location will be effective, whereas
for traits with low heritability, selection should be based on mean
values obtained from replicated evaluations of families of related
plants. Popular selection methods commonly include pedigree
selection, modified pedigree selection, mass selection, and
recurrent selection. In a preferred aspect, a backcross or
recurrent breeding program is undertaken.
[0154] The complexity of inheritance influences choice of the
breeding method. Backcross breeding can be used to transfer one or
a few favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes.
[0155] Breeding lines can be tested and compared to appropriate
standards in environments representative of the commercial target
area(s) for two or more generations. The best lines are candidates
for new commercial cultivars; those still deficient in traits may
be used as parents to produce new populations for further
selection.
[0156] The development of new elite corn hybrids requires the
development and selection of elite inbred lines, the crossing of
these lines and selection of superior hybrid crosses. The hybrid
seed can be produced by manual crosses between selected
male-fertile parents or by using male sterility systems. Additional
data on parental lines, as well as the phenotype of the hybrid,
influence the breeder's decision whether to continue with the
specific hybrid cross.
[0157] Pedigree breeding and recurrent selection breeding methods
can be used to develop cultivars from breeding populations.
Breeding programs combine desirable traits from two or more
cultivars or various broad-based sources into breeding pools from
which cultivars are developed by selfing and selection of desired
phenotypes. New cultivars can be evaluated to determine which have
commercial potential.
[0158] Backcross breeding has been used to transfer genes for a
simply inherited, highly heritable trait into a desirable
homozygous cultivar or inbred line, which is the recurrent parent.
The source of the trait to be transferred is called the donor
parent. After the initial cross, individuals possessing the
phenotype of the donor parent are selected and repeatedly crossed
(backcrossed) to the recurrent parent. The resulting plant is
expected to have most attributes of the recurrent parent (e.g.,
cultivar) and, in addition, the desirable trait transferred from
the donor parent.
[0159] The single-seed descent procedure in the strict sense refers
to planting a segregating population, harvesting a sample of one
seed per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F.sub.2
to the desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2 individuals. The
number of plants in a population declines each generation due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
[0160] Descriptions of other breeding methods that are commonly
used for different traits and crops can be found in one of several
reference books (Allard, "Principles of Plant Breeding," John Wiley
& Sons, NY, U. of CA, Davis, Calif., 50-98, 1960; Simmonds,
"Principles of Crop Improvement," Longman, Inc., NY, 369-399, 1979;
Sneep and Hendriksen, "Plant Breeding Perspectives," Wageningen
(ed), Center for Agricultural Publishing and Documentation, 1979;
Fehr, In: Soybeans: Improvement, Production and Uses, 2nd Edition,
Manograph., 16:249, 1987; Fehr, "Principles of Variety
Development," Theory and Technique, (Vol. 1) and Crop Species
Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,
360-376, 1987).
[0161] An alternative to traditional QTL mapping involves achieving
higher resolution by mapping haplotypes, versus individual markers
(Fan et al., 2006 Genetics 172:663-686). This approach tracks
blocks of DNA known as haplotypes, as defined by polymorphic
markers, which are assumed to be identical by descent in the
mapping population. This assumption results in a larger effective
sample size, offering greater resolution of QTL. Methods for
determining the statistical significance of a correlation between a
phenotype and a genotype, in this case a haplotype, may be
determined by any statistical test known in the art and with any
accepted threshold of statistical significance being required. The
application of particular methods and thresholds of significance
are well with in the skill of the ordinary practitioner of the
art.
[0162] It is further understood, that the present invention
provides bacterial, viral, microbial, insect, mammalian and plant
cells comprising the nucleic acid molecules of the present
invention.
[0163] As used herein, a "nucleic acid molecule," be it a naturally
occurring molecule or otherwise may be "substantially purified", if
desired, referring to a molecule separated from substantially all
other molecules normally associated with it in its native state.
More preferably a substantially purified molecule is the
predominant species present in a preparation. A substantially
purified molecule may be greater than 60% free, preferably 75%
free, more preferably 90% free, and most preferably 95% free from
the other molecules (exclusive of solvent) present in the natural
mixture. The term "substantially purified" is not intended to
encompass molecules present in their native state.
[0164] The agents of the present invention will preferably be
"biologically active" with respect to either a structural
attribute, such as the capacity of a nucleic acid to hybridize to
another nucleic acid molecule, or the ability of a protein to be
bound by an antibody (or to compete with another molecule for such
binding). Alternatively, such an attribute may be catalytic, and
thus involve the capacity of the agent to mediate a chemical
reaction or response.
[0165] The agents of the present invention may also be recombinant.
As used herein, the term recombinant means any agent (e.g. DNA,
peptide etc.), that is, or results, however indirect, from human
manipulation of a nucleic acid molecule.
[0166] The agents of the present invention may be labeled with
reagents that facilitate detection of the agent (e.g. fluorescent
labels (Prober et al., 1987 Science 238:336-340; Albarella et al.,
European Patent 144914), chemical labels (Sheldon et al., U.S. Pat.
No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified
bases (Miyoshi et al., European Patent 119448).
[0167] The plant breeding methods provided herein can be used with
one or more individuals, including SSD, from any generation of
plant population. Non-limiting examples of plant populations
include to F1, F2, BC1, BC2F1, F3:F4, F2:F3, and so on, including
subsequent filial generations, as well as experimental populations
such as RILs and NILs. It is further anticipated that the degree of
segregation within the one or more plant populations of the present
invention can vary depending on the nature of the trait and
germplasm under evaluation.
[0168] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
Example 1
Phenotyping for Goss' Wilt
[0169] In order to detect QTL associated with resistance to Goss'
Wilt, plants were phenotyped to determine Goss' Wilt reaction. The
following rating scale was used in order to assess resistance or
susceptibility to Goss' Wilt. Phenotypic evaluations of Goss' Wilt
reaction is based on percentage of infected leaf area and rated
according to a 1 (very resistant) to 9 (susceptible) scale as
provided in Table 1. Plants are artificially inoculated and
visually rated approximately 3 to 4 weeks after pollination.
TABLE-US-00001 TABLE 1 Disease rating scale for Goss' Wilt.
Description Rating Symptoms Very Resistant 1 0% of leaf area
infected; no visible lesions Very Resistant 2 ILA < 1%; few
lesions, dispersed through lower leaves Resistant 3 1% .ltoreq. ILA
.ltoreq. 20% Resistant 4 20% .ltoreq. ILA .ltoreq. 40%
Mid-resistant 5 40% .ltoreq. ILA .ltoreq. 50% Mid-Susceptible 6 50%
.ltoreq. ILA .ltoreq. 60%; lesions Susceptible 7 60% .ltoreq. ILA
.ltoreq. 75% Susceptible 8 75% .ltoreq. ILA .ltoreq. 90%
Susceptible 9 >90% of foliar area infected ILA = Infected Leaf
Area
Example 2
Goss' Wilt Resistance Mapping Study 1
[0170] To examine associations between SNP markers and Goss' Wilt
resistance, analyzed data from a number of studies was combined. An
association study was conducted to evaluate whether significant
associations between one or marker genotypes and Goss' Wilt
resistance are present in one or more populations. In this
association study, data from 10 mapping populations were combined.
The number of individuals in the populations ranged from 186 to
369. The number of SNP markers used for screening ranged from 104
to 134. The populations were either F3 or BC1F2. A total of 172
significant associations between SNP markers and Goss' Wilt
resistance were identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8,
9, and 10. The SNP markers provided can be used to monitor the
introgression of Goss' Wilt resistance into a breeding population.
Significant marker-Goss' Wilt associations are reported in FIG.
1.
Example 3
Goss' Wilt Resistance Mapping Study 2
[0171] An association study was conducted to evaluate whether
significant associations between one or marker genotypes and Goss'
Wilt resistance are present in one or more populations. In this
association study, 988 inbred lines were screened with 1051 SNP
markers. A total of 53 significant associations between SNP markers
and Goss' Wilt resistance were identified on Chromosomes 1, 2, 3,
4, 5, 6, 8, 9, and 10. The SNP markers provided can be used to
monitor the introgression of Goss' Wilt resistance into a breeding
population. SNP markers associated with Goss' Wilt resistance,
level of significance, and favorable alleles are reported in FIG.
1.
Example 4
Goss' Wilt Resistance Mapping Study 3
[0172] An association study was conducted to evaluate whether
significant associations between one or more marker genotypes and
Goss' Wilt resistance are present in one or more populations. In
this study, a rating scale of 1 to 4 was used with 1 being
resistant, 2 moderately resistant, 3 moderately susceptible, and 4
susceptible. In this association study, two F3 populations of 154
and 212 individuals were screened with 104 SNP markers. A total of
35 significant associations between SNP markers and Goss' Wilt
resistance were identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8,
9, and 10. The SNP markers provided can be used to monitor the
introgression of Goss' Wilt resistance into a breeding population.
SNP markers associated with Goss' Wilt resistance, level of
significance, and favorable alleles are reported in FIG. 1.
Example 5
Goss' Wilt Resistance Mapping Study 4
[0173] An association study was conducted to evaluate whether
significant associations between one or more marker genotypes and
Goss' Wilt resistance are present in one or more populations. A
population was screened with 518 SNP markers. A total of 80
significant associations between SNP markers and Goss' Wilt
resistance were identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8,
9, and 10. The SNP markers provided can be used to monitor the
introgression of Goss' Wilt resistance into a breeding population.
SNP markers associated with Goss' Wilt resistance, level of
significance, and favorable alleles are reported in FIG. 1.
Example 6
Exemplary Marker Assays for Detecting Goss' Wilt Resistance
[0174] In one embodiment, the detection of polymorphic sites in a
sample of DNA, RNA, or cDNA may be facilitated through the use of
nucleic acid amplification methods. Such methods specifically
increase the concentration of polynucleotides that span the
polymorphic site, or include that site and sequences located either
distal or proximal to it. Such amplified molecules can be readily
detected by gel electrophoresis, fluorescence detection methods, or
other means. Exemplary primers and probes for amplifying and
detecting genomic regions associated with Goss' Wilt resistance are
given in Table 2.
TABLE-US-00002 TABLE 2 Exemplary assays for detecting Goss' Wilt
resistance loci. SEQ ID SEQ ID Marker SNP Forward Reverse SEQ ID
SEQ ID Marker SEQ ID Position Primer Primer Probe 1 Probe 2
NC0027347 896 128 1332 1333 1334 1335 NC0071001 951 359 1336 1337
1338 1339 NC0017678 733 171 1340 1341 1342 1343 NC0028095 1098 116
1344 1345 1346 1347
Example 7
Oligonucleotide Hybridization Probes Useful for Detecting Corn
Plants with Goss' Wilt Resistance Loci
[0175] Oligonucleotides can also be used to detect or type the
polymorphisms associated with Goss' Wilt resistance disclosed
herein by hybridization-based SNP detection methods.
Oligonucleotides capable of hybridizing to isolated nucleic acid
sequences which include the polymorphism are provided. It is within
the skill of the art to design assays with experimentally
determined stringency to discriminate between the allelic state of
the polymorphisms presented herein. Exemplary assays include
Southern blots, Northern blots, microarrays, in situ hybridization,
and other methods of polymorphism detection based on hybridization.
Exemplary oligonucleotides for use in hybridization-based SNP
detection are provided in Table 3. These oligonucleotides can be
detectably labeled with radioactive labels, fluorophores, or other
chemiluminescent means to facilitate detection of hybridization to
samples of genomic or amplified nucleic acids derived from one or
more corn plants using methods known in the art.
TABLE-US-00003 TABLE 3 Exemplary Oligonucleotide Hybridization
Probes*. Marker SEQ SEQ SNP ID Marker ID Position Probe Probe
NC0027347 896 128 GCTACTAGGAAAATGG 1348 NC0027347 896 128
GCTACTAGAAAAATGG 1349 NC0071001 951 359 CAACTACCTAGCATTT 1350
NC0071001 951 359 CAACTACCAAGCATTT 1351 NC0017678 733 171
AGTCAAAGATACTGCA 1352 NC0017678 733 171 AGTCAAAGCTACTGCA 1353
NC0028095 1098 116 TGCCCACATTTGTTAT 1354 NC0028095 1098 116
TGCCCACACTTGTTAT 1355 *SNP nucleotides in bold and underlined.
Example 8
Oligonucleotide Probes Useful for Detecting Corn Plants with Goss'
Wilt Resistance Loci by Single Base Extension Methods
[0176] Oligonucleotides can also be used to detect or type the
polymorphisms associated with Goss' Wilt resistance disclosed
herein by single base extension (SBE)-based SNP detection methods.
Exemplary oligonucleotides for use in SBE-based SNP detection are
provided in Table 4. SBE methods are based on extension of a
nucleotide primer that is hybridized to sequences immediately
adjacent to a polymorphism to incorporate a detectable nucleotide
residue upon extension of the primer. It is also anticipated that
the SBE method can use three synthetic oligonucleotides. Two of the
oligonucleotides serve as PCR primers and are complementary to the
sequence of the locus which flanks a region containing the
polymorphism to be assayed. Exemplary PCR primers that can be used
to type certain polymorphisms disclosed in this invention are
provided in Table 3 in the columns labeled "Forward Primer SEQ ID"
and "Reverse Primer SEQ ID". Following amplification of the region
containing the polymorphism, the PCR product is hybridized with an
extension primer which anneals to the amplified DNA immediately
adjacent to the polymorphism. DNA polymerase and two differentially
labeled dideoxynucleoside triphosphates are then provided. If the
polymorphism is present on the template, one of the labeled
dideoxynucleoside triphosphates can be added to the primer in a
single base chain extension. The allele present is then inferred by
determining which of the two differential labels was added to the
extension primer. Homozygous samples will result in only one of the
two labeled bases being incorporated and thus only one of the two
labels will be detected. Heterozygous samples have both alleles
present, and will thus direct incorporation of both labels (into
different molecules of the extension primer) and thus both labels
will be detected.
TABLE-US-00004 TABLE 4 Probes (extension primers) for Single Base
Extension (SBE) assays. Marker SEQ SEQ SNP ID Marker ID Position
Probe Probe NC0027347 896 128 TTTTGTACTGCTACTAG 1356 NC0071001 951
359 TACGGAATGCAACTACC 1357 NC0017678 733 171 GTCATGGCGAGTCAAAG 1358
NC0028095 1098 116 TGGATGCTTTGCCCACA 1359
Example 9
Haploid Mapping Study for Goss' Wilt with I208993/LH287
Population
[0177] The utility of haploid plants in genetic mapping of traits
of interest is further demonstrated in the following example. A
mapping population was developed for using haploid plants to map
QTL associated with resistance to Goss' Wilt. The population was
from the cross of inbred corn lines I208993 by LH287. F1 plants
were induced to produce haploid seed. From the I208993/LH287
population, 1384 haploid plants were inoculated with the Goss' Wilt
pathogen and phenotyped using a truncated rating scale of 1, 5, or
9. Ratings are done approximately 3 to 4 weeks after pollination.
Plants rated either 1 or 9 were used in the QTL mapping. By using
only the extreme values (1 or 9), environmental variation that is
inherent with disease phenotyping was reduced and a bulk segregate
analysis was created from which to detect major QTL. Genotyping was
done using 114 SNP markers. Composite interval mapping was
conducted to examine significant associations between Goss' Wilt
and SNP markers. Table 5 provides markers useful for detecting QTL
associated with resistance to Goss' Wilt in the I208993/LH287
haploid mapping population. The chromosome (Chr.) location,
chromosome position (Chr. pos), and favorable (Fav.) allele are
also provided in Table 5.
[0178] It is appreciated by one skilled in the art that the methods
of the present invention can be used with one or more individuals,
including SSD, from any generation of plant population.
Non-limiting examples of plant populations include to F1, F2, BC1,
BC2F1, F3:F4, F2:F3, and so on, including subsequent filial
generations, as well as experimental populations such as RILs and
NILs. It is further anticipated that the degree of segregation
within the one or more plant populations of the present invention
can vary depending on the nature of the trait and germplasm under
evaluation.
TABLE-US-00005 TABLE 5 Markers useful for detecting QTL associated
with Goss' Wilt resistance in the I208993/LH287 haploid mapping
population. Goss' Chr Wilt Likelyhood Additive Fav. SEQ SNP Marker
Chr pos. QTL ratio LOD effect allele ID Position* NC0202383 2 19 22
100.304 21.78074 0.737618 T 1229 34 NC0199732 2 37 24 113.9429
24.74239 0.779994 T 1276 138 NC0048553 2 46.8 25 103.8964 22.56081
0.758496 A 234 485 NC0201646 2 55.4 129 96.43437 20.94046 0.746649
T 1294 416 NC0201821 2 71.4 27 40.13758 8.715765 0.202738 T 1295
331 NC0019110 2 75.1 27 28.41102 6.169374 0.173568 C 1278 153
NC0004821 3 54.4 40 47.57959 10.33178 0.451741 C 371 294 NC0200643
3 70.3 122 47.48045 10.31025 0.424893 C 1296 106 NC0040461 4 51.2
125 80.02493 17.37719 0.620383 A 1282 366 NC0034462 4 67.8 52
76.55974 16.62474 0.574876 T 1250 301 NC0200535 4 132 58 29.47242
6.399855 0.142544 T 1297 411 NC0029435 4 138 58 29.25183 6.351953
0.139488 G 1298 551 NC0011194 5 29.3 63 27.51088 5.973912 -0.227689
C 1299 218 NC0016527 5 49 66 29.15712 6.331388 -0.219392 T 1255 351
NC0202055 5 76.4 68 26.18668 5.686366 -0.252002 T 1300 505
NC0147719 5 160 130 47.9265 10.40711 0.492815 G 1301 48 NC0012417 5
175 74 48.68852 10.57258 0.505586 T 768 137 NC0113381 6 83.8 79
28.96126 6.288858 -0.21407 A 850 303 NC0022200 6 93.7 80 31.16025
6.766361 -0.201408 G 1302 153 NC0010347 8 69.2 131 27.38218
5.945966 -0.144382 T 1015 160 NC0199582 8 86.3 99 26.24576 5.699195
-0.169537 A 1303 201 *SNP position: refers to the position of the
SNP polymorphism in the indicated SEQ ID NO.
Example 10
Haploid Mapping Study for Goss' Wilt with I208993/LH295
Population
[0179] The utility of haploid plants in genetic mapping of traits
of interest is further demonstrated in the following example. A
mapping population was developed for using haploid plants to map
QTL associated with resistance to Goss' Wilt. The population was
from the cross of LH295 by I208993. F1 plants were induced to
produce haploid seed.
[0180] From the I1208993/LH295 haploid mapping population, 980
individuals were naturally exposed to the Goss' Wilt pathogen and
phenotyped using a modified rating scale of 1, 5, or 9. Plants were
rated approximately 3 to 4 weeks after pollination. Plants rated
either 1 or 9 were used in the QTL mapping. By using only the
extreme values (1 or 9), environmental variation that is inherent
with disease phenotyping was reduced and a bulk segregate analysis
was created from which to detect major QTL. Genotyping was done
with 980 SNP markers. Table 6 provides markers useful for detecting
QTL associated with Goss' Wilt in the I208993/LH295 haploid mapping
population.
[0181] It is appreciated by one skilled in the art that the methods
of the present invention can be used with one or more individuals,
including SSD, from any generation of plant population.
Non-limiting examples of plant populations include F1, F2, BC1,
BC2F1, F3:F4, F2:F3, and so on, including subsequent filial
generations, as well as experimental populations such as RILs and
NILs. It is further anticipated that the degree of segregation, as
well as heterozygosity, within the one or more plant populations of
the present invention can vary depending on the nature of the trait
and germplasm under evaluation.
TABLE-US-00006 TABLE 6 Markers useful for detecting QTL associated
with Goss' Wilt in the I208993/LH295 haploid mapping population
Goss' Chr. Wilt Additive Favorable SEQ SNP Marker Chr. pos QTL
Likelihood LOD Effect Allele ID Position* NC0199051 1 19.3 1
28.02118 6.084721 -0.22604 G 1274 141 NC0105051 1 31.4 3 28.79147
6.251987 -0.236914 C 24 426 NC0032288 1 133.6 10 31.20763 6.77665
0.252864 C 1275 413 NC0070305 1 166.5 13 29.73574 6.457033 0.216902
A 158 532 NC0143411 2 15.4 22 31.80736 6.90688 -0.372898 C 218 401
NC0199732 2 37 24 51.17309 11.11209 -0.506613 T 1276 138 NC0013275
2 49.7 25 56.78186 12.33002 -0.677671 T 236 430 NC0199350 2 67.8 26
57.35414 12.45429 -0.577154 G 1277 226 NC0019110 2 75.1 27 51.54673
11.19323 -0.633508 C 1278 153 NC0027319 2 93.2 29 41.90672 9.099928
-0.572435 T 272 54 NC0104528 3 24.6 37 29.36476 6.376476 -0.189689
G 1247 117 NC0019963 3 40.6 39 32.03588 6.956503 -0.139199 C 368
1173 NC0077220 3 43.2 39 27.90631 6.059777 -0.133108 A 1279 149
NC0108727 3 77.4 122 32.5836 7.075438 -0.031362 C 375 241 NC0039785
3 94.5 123 30.35128 6.590696 -0.083537 T 401 512 NC0031720 3 99.7
123 46.9907 10.2039 0.199348 G 408 434 NC0200377 3 116.9 43
47.01889 10.21002 0.181809 A 1280 352 NC0199741 3 125.7 44 28.60384
6.211245 -0.315998 A 1281 294 NC0041040 3 145.4 45 36.85657
8.003303 -0.551354 A 440 497 NC0055502 4 1.8 124 36.00788 7.819012
-0.390433 C 498 105 NC0040461 4 51.2 125 42.90587 9.316891
-0.469569 A 1282 366 NC0199420 4 102.9 55 43.93528 9.540424
-0.452635 G 1283 356 NC0036240 4 112 56 38.3635 8.330528 -0.381557
A 587 441 NC0028933 4 127.6 57 29.32225 6.367245 0.144007 C 599 355
NC0147712 4 136.7 58 33.6318 7.303051 0.185174 A 1284 74 NC0028579
4 155.7 60 37.46012 8.134361 0.109588 A 629 242 NC0029487 4 171.1
126 38.35712 8.329143 0.101598 G 1285 159 NC0200359 5 11.7 63
27.52949 5.977952 -0.167336 A 1286 196 NC0040571 5 88.4 69 59.435
12.90615 -0.58299 C 721 154 NC0017678 5 103.8 71 69.69769 15.13466
-0.722151 A 733 171 NC0083876 5 124 72 29.09207 6.317263 -0.392793
T 744 513 NC0200323 5 174.8 74 27.01332 5.865868 -0.253474 A 1287
181 NC0027347 7 43.8 86 57.87354 12.56708 -0.542521 A 896 128
NC0201872 7 64.4 88 58.07534 12.6109 -0.54188 C 1288 208 NC0145922
7 80.5 89 26.87412 5.835642 -0.271008 G 940 451 NC0071001 7 99.4 90
26.59882 5.775861 -0.262452 T 951 359 NC0199879 7 112.1 92 34.51543
7.494931 -0.28773 A 1289 228 NC0200055 7 122.3 127 36.14355
7.848472 -0.277751 T 1290 116 NC0110771 7 138.5 93 32.98577
7.162769 -0.163457 A 976 490 NC0200495 7 155.9 95 27.69812 6.014571
-0.118782 G 1291 302 NC0028095 9 59.4 107 29.92602 6.498353
0.142796 C 1098 116 NC0144850 9 67 108 30.50354 6.62376 0.146897 G
1292 244 NC0030134 10 79.4 120 27.87616 6.05323 -0.317779 TCCACTAT
1215 94 NC0200312 10 85.7 128 31.10615 6.754615 -0.355789 A 1293 89
*SNP position: refers to the position of the SNP polymorphism in
the indicated SEQ ID NO.
Example 11
Introgression of Goss' Wilt Resistance Using SNP Markers
[0182] Loci associated with resistance to Goss' Wilt can be
introgressed into corn plants by methods known to those skilled in
the art of plant breeding. A plant breeder can use SNP markers to
monitor the introgression of Goss' Wilt resistance loci and to
select for lines carrying the favorable allele for one or more of
said SNP markers. In this example, the inbred line LH287 is used as
a source of Goss' Wilt resistance. SNP markers used to monitor
introgression of Goss' Wilt resistance loci on Chromosome 2 include
NC0202383, NC0199732, NC0048553, and NC0201646 (SEQ ID NOs: 1122,
1276, 1294, and 234). SNP used to monitor introgression of Goss'
Wilt resistance loci on Chromosome 3 include NC0019963 and
NC0004821 (SEQ ID NOs: 368 and 371). SNP markers used to monitor
the introgression of Goss' Wilt resistance loci on Chromosome 4
include NC0040461 and NC0034462 (SEQ ID NOs: 1282 and 1250). SNP
markers used to monitor the introgression of Goss' Wilt resistance
loci on Chromosome 5 include NC0147719 and NC0012417 (SEQ ID NOs:
1301 and 768). The favorable allele is the allele associated with
the resistant donor parent.
[0183] In a further illustration, the inbred line LH295 is used as
a source of Goss' Wilt resistance. SNP markers used to monitor the
introgression of Goss' Wilt resistance loci on Chromosome 2 include
NC0013275, NC0199350, and NC0019110 (SEQ ID NOs: 236, 1277, and
1278). SNP markers used to monitor the introgression of Goss' Wilt
resistance loci on Chromosome 3 include NC0199741 and NC0041040
(SEQ ID NOs: 1281 and 440). SNP markers used to monitor the
introgression of Goss' Wilt resistance loci on Chromosome 4 include
NC0040461, NC0199420, and NC0036240 (SEQ ID NOs: 1282, 1283, and
587). SNP markers used to monitor the introgression of Goss' Wilt
resistance loci on Chromosome 5 include NC0040571 and NC0017678
(SEQ ID NOs: 721 and 733). SNP markers used to monitor the
introgression of Goss' Wilt resistance loci on Chromosome 7 include
NC0201872 and NC0145922 (SEQ ID NOs: 1288 and 940). SNP markers
used to monitor the introgression of Goss' Wilt resistance loci on
Chromosome 10 include NC0200312 (SEQ ID NO: 1293). A plant breeder
can use SNP markers to monitor the introgression of Goss' Wilt
resistance loci and to select for lines carrying the favorable
allele for one or more of said SNP markers.
[0184] The introgression of one or more resistance loci is achieved
via repeated backcrossing to a recurrent parent accompanied by
selection to retain one or more Goss' Wilt resistance loci from the
donor parent using the above-described markers. This backcross
procedure is implemented at any stage in line development and
occurs in conjunction with breeding for superior agronomic
characteristics or one or more traits of interest, including
transgenic and nontransgenic traits.
[0185] Alternatively, a forward breeding approach is employed
wherein one or more Goss' Wilt resistance loci can be monitored for
successful introgression following a cross with a susceptible
parent with subsequent generations genotyped for one or more Goss'
Wilt resistance loci and for one or more additional traits of
interest, including transgenic and nontransgenic traits.
Example 12
Application of Markers Associated with Goss' Wilt in a Corn
Breeding Program
[0186] From the studies presented in FIG. 1, it is apparent that a
chromosomal region can have multiple SNP markers associated with
Goss' Wilt resistance. Following are non-limiting examples of
targeting at least one marker from at least on locus associated
with Goss' Wilt resistance for the purpose of breeding corn
resistant to Goss' Wilt. Specifically the markers of the present
invention have utility for generating corn inbreds and hybrids
resistant to Goss Wilt. The markers of the present invention are
useful in parent selection, progeny selection, and marker-assisted
introgression and backcrossing. Exemplary markers from Chromosome 1
are NC0004909 and NC0005098 (SEQ ID NOs: 175 and 177). Exemplary
markers from Chromosome 3 are NC0146497 and NC0155987 (SEQ ID NOs:
479 and 480). Exemplary markers from Chromosome 4 are NC0077408,
NC0003274, and NC0009280 (SEQ ID NOs: 582, 585, and 1251).
Exemplary markers from Chromosome 8 are NC0010392, NC0012656, and
NC0008831 (SEQ ID NOs: 1053, 1054, and 1056).
[0187] In view of the foregoing, it will be seen that the several
advantages of the invention are achieved and attained.
[0188] The embodiments were chosen and described in order to best
explain the principles of the invention and its practical
application to thereby enable others skilled in the art to best
utilize the invention in various embodiments and with various
modifications as are suited to the particular use contemplated.
[0189] Various patent and non-patent publications are cited herein,
the disclosures of each of which are, to the extent necessary,
incorporated herein by reference in their entireties.
[0190] As various modifications could be made in the constructions
and methods herein described and illustrated without departing from
the scope of the invention, it is intended that all matter
contained in the foregoing description or shown in the accompanying
drawings shall be interpreted as illustrative rather than limiting.
The breadth and scope of the present invention should not be
limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims
appended hereto and their equivalents.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090070903A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090070903A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References