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Breeding and Genetics of Soybean Mosaic Breeding and Genetics of Soybean Mosaic Virus Resistance in SoybeanVirus Resistance in Soybean
Ainong Shi
Syngenta SeedsSlater, IA
USA
Pengyin Chen
University of ArkansasFayetteville, ARUSA
Soybean Mosaic Virus (SMV)
• Most destructive viral disease in soybean
• Symptoms: Resistant (R, symptomless), necrotic (N, necrotic lesions, or stem-tip necrosis), and susceptible (S, mosaic)
• Seven strains G1 to G7 identified in U.S.
R N S R R R
PI96983 (Rsv1) L29 (Rsv3) V94-5152 (Rsv4)
G1 G7G7G1G7G1
• Genetic resistance is the primary method of controlling
this disease • Three loci, Rsv1, Rsv3, and Rsv4 have been identified and mapped on the molecular linkage group (MLG) F, B2, and D1b, respectively• Nine alleles (Rsv1, Rsv1-t, Rsv1-y, Rsv1-m, Rsv1-k, Rsv1-s, Rsv1-r, Rsv1-n, and Rsv1-h) at Rsv1 locus have been reported
I. Confirm 2 Genes of Resistance to SMV in J05 Soybean Using SSR Markers
II. A PCR-based Marker for the Rsv1 Locus Conferring Resistance to SMV
III. Gene Pyramiding for SMV Resistance Using Molecular Markers
IV. SNP Markers for SMV Resistance
Topic
• J05, a Chinese soybean cultivar
• Resistant to all seven SMV strains• Containing Rsv1 and Rsv3
(Zheng et al. 2006. J. Hered. 97(5):429-437)
I. Confirm 2 Genes of Resistance to SMV in J05 Soybean Using SSR Markers
* Ainong Shi, Pengyin Chen, Dexiao Li, Cuiming Zheng, Anfu Hou, and Bo Zhang. 2008. Genetic confirmation of two independent genes for resistance to soybean mosaic virus in J05 Soybean using SSR markers. J. Heredity 99(6):598–603
Each genotype: two pots each with 6 plantsSMV strains: G1 - G7Mechanical inoculation at unifoliolate leaf stage
Evaluation of SMV Resistance
Linkage Analysis&
Genetic Mapping
Procedure of this Research
Genotypic Data
Obtaining Segregating Populations
Phenotypic Data
J05 x Essex(Rsv1Rsv1Rsv3Rsv3) (rsv1rsv1rsv3 rsv3)
F1 (Rsv1rsv1Rsv3rsv3)
F2
X
F2 population and F2:3 Lines(R) (S)
SMV strain
G1 G7Rsv1 R NRsv3 S R
G1 3R : 1S (3Rsv1_ : 1rsv1rsv1)
1R : 2Seg : 1S (1Rsv3Rsv3 : 2Rsv3rsv3 : 1rsv3rsv3)
G7 F2:3
J05 x Essex F2 population
SMV strain G1
R S Exp. 2 R-gene-------------------------------------------------- 102 36 3:1 0.087 Rsv1
R = Resistant (symptomless)S = Susceptible (mosaic)
G1-susceptible F2 plants(No Rsv1)
SMV strain
G1 G7Rsv1 R NRsv3 S R
R Seg. S Exp. 2 R-gene-------------------------------------------------------- 10 20 15 1:2:1 0.087 Rsv3
R = Homozygous resistant (Rsv3Rsv3) S = Homozygous susceptible (rsv3rsv3) Seg. = Segregating (Rsv3rsv3)
F2:3 lines G7
Genetic map of the Rsv1 region
Marker LG Pos (cM)
Sat_297 F 59.6
Sat_229 F 62.8
Satt114 F 63.7
Sat_234 F 66.5
SOYHSP176 F 68.4
Sat_154 F 68.9
Satt510 F 71.4
Sat_317 F 73.0
Sat_120 F 76.0
Rsv1
59.60 Sat_297
Sat_229
Satt114
Sat_234
SOYHSP176
Sat_154
Satt510
Sat_317
Sct_03374.13
72.97
71.41
68.91
68.44
66.55
63.69
62.79
MLG F
P1=J05, P2=Essex, R = resistant, and S = susceptible plants of in the F2 (J05 x Essex)
SSR primer Satt114 for Rsv1An example
Rsv1
Sat_154
Satt510
A genetic map for the Rsv1 region on MLG F
0.5 cM
2.3 cM
Satt114
3.8 cM
Genetic maps of the Rsv3 region
Marker LGPos(cM)
Satt063 B2 93.5
Satt560 B2 97.9
Sat_424 B2 100.1
Satt726 B2 100.6
Satt687 B2 113.6
Rsv3
Satt687
Satt063
Satt560
Sat_424
Satt726
MLG B2
93.49
97.92
100.10
100.59
113.61
Polymorphisms among G1-susceptible plants of J05 x Essex F2 amplified from Satt726 for Rsv3
Rsv3
Sat_424
Satt726
A genetic map for the Rsv3 region on MLG B2
1.5 cM
0.5 cM
SUMMARY I
J05 contains two genes, Rsv1 on MLG F and
Rsv3 on MLG B2, for resistance to SMV. Two genetic maps were constructed.
Rsv3
Sat_424
Satt726
Rsv1
Sat_154
Satt510
MLG F MLG B2
1.5 cM
0.5 cM
0.5 cM
2.3 cM
Satt114
3.8 cM
II. A PCR-based Marker for the Rsv1 Locus Conferring Resistance to SWV
Ainong Shi, Pengyin Chen, Cuiming Zheng, Anfu Hou, and Bo Zhang. 2008. A PCR-based marker for the Rsv1 Locus conferring resistance to Soybean Mosaic Virus. Crop Sci. 48:262-268.
DNA fragments amplified from the primer pair Rsv1-f/r (Lane 1 to 8) on 1.3% agrose gel
No. Genotype Gene Rsv1-f/r
1 Essex - -
2 PI 96983 Rsv1 +
3 Marshall Rsv1-m +
4 York Rsv1-y -
5 Raiden Rsv1-r +
6 Kwanggyo Rsv1-k +
7 Ogden Rsv1-t +
8 Sweon 97 Rsv1-h +
M 1 2 3 4 5 6 7 8
341 bp
PCR-based Marker Rsv1-f/r in Rsv1 Locus
Amplification patterns of the marker with the primer pair Rsv1-f/r in the F2 population derived from J05 x Essex on 1.3% agrose gel. R = resistant and S = susceptible plants in the F2
Screen of Segregating Population
R R R R R R R R R R R S R R R R S S S R R R S R R R R R R S S R S R R R
Amplification patterns of the DNA fragments with the SSR primer Satt 114 in the F2 population derived from J05 x Essex on 6% non-denaturing polyacrylamide gel. P1=J05, P2=Essex, R = resistant, and S = susceptible plants in the F2. Lane M is a 100 bp molecular-weight marker.
A genetic map around the 3gG2 gene between Sat_297 at 59.60 cM to Sct_033 at 74.13 cM of the Molecular Linkage Group (MLG) F.
59.60 Sat_297
Sat_229
Satt114
Sat_234
SOYHSP176
Sat_154Rsv1-f/r: 3gG2
Satt510
Sat_317
Sct_03374.13
72.97
71.41
69.11
68.91
68.44
66.55
63.69
62.79
Rsv1
• A pair of DNA primers Rsv1-f/r was developed from the candidate gene 3gG2 at the Rsv1 locus for SMV resistance
• All genotypes contained Rsv1, Rsv1-k, Rsv1-m,, Rsv1-n, Rsv1-r, Rsv1-s, and Rsv1-t produced the Rsv1-f/r fragment, but those carried Rsv1-y did not
• The fragment was neither amplified in only Rsv3 containing genotypes or only Rsv4 containing genotypes, nor in the susceptible genotypes
SUMMARY II
III. Gene Pyramiding for SMV Resistance Using Molecular Markers
J05(Rsv1Rsv1Rsv3Rsv3)
x V94-5152(Rsv4Rsv4)
F1(Rsv1rsv1Rsv3rsv3Rsv4rsv4)
x
F2
F2:3 F2:3 F2:3
F2:4 F2:4F2:4
F2:3
F2:4F2:4
F2:3
(3Rsv1:1rsv1)(3Rsv3:1rsv3)(3Rsv4:1rsv4)
1/64 Rsv1Rsv1Rsv3Rsv3Rsv4Rsv4
F2:5 F2:6F2:7
* Ainong Shi, Pengyin Chen, Dexiao Li, Cuiming Zheng, Anfu Hou, and Bo Zhang. 2009. Gene pyramiding of genes of soybean mosaic virus resistance. Molecular Breeding 23(1):113-124
Gore et al. 2002. Genome 45:592-599Jeong et al. 2002. Crop Sci. 42:265-270Cregan. 2003. (http://bldg6.arsusda.gov/~pooley/soy/cregan/cregan.htm)
Satt510+Sat_317 (lane 1 & 2), Sat_154+Sat_317(lane 3 & 4), Satt063 (lane 5 & 6), Satt560 (lane 7 & 8), Satt266 (lane 9 & 10), and AI856415 (lane 11 & 12). Lane M is a 100 bp DNA molecular ladder.
M 1 2 3 4 5 6 7 8 9 10 11 12
Six Polymorphic Markers between J05(Rsv1+3) and V94-5152(Rsv4)
Marker Linked Band
gene J05 V94-5152 (Rsv1,3) (Rsv4)
Satt510+Sat_317 Rsv1 1+0 2+1Sat_154+Sat_317 Rsv1 1+0 2+1Satt063 Rsv3 1 2Satt560 Rsv3 1 2Satt266 Rsv4 1 2AI856415 Rsv4 1 2
PCR products amplified from 30 F2:4 plants of J05 x V94-5152 and two parents
Satt063 (Rsv3)
Sat_317
Satt510
Rsv1
Satt266 (Rsv4)
Polymorphism of Six SSR Markers in Five F2:3 Plants Derived from J05 x V94-5152
Rsv1 Rsv3 Rsv4
F2:3 plant Satt510+Sat_317
Sat_154+Sat_317
Satt063 Satt560 Satt266 AI856415
JV-06G3-1-1 1+2 1+2 1 1 2 2
JV-06G3-3-1 1+2 1+2 1 1 2 2
JV-06G3-5-1 1 1 1+2 1 2 2
JV-06G3-7-1 1 1 1 1 1+2 2
JV-06G3-8-1 1 1 1 1 2 2
J05 (Rsv1+3) 1 1 1 1 1 1
V94-5152 (Rsv4)
2 2 2 2 2 2
1 = same PCR fragment as in J05, 2 = same PCR fragment as in V94-5152, and 1+2 = both of J05 and V94-5152.
• Two SSR markers (Sat_154 and Satt510) and one gene-specific marker (Rsv1-f/r) were used for selecting plants containing Rsv1; Satt560 and Satt063 for Rsv3; and Satt266, AI856415, and AI856415-g for Rsv4.
• Five F4:5 lines were homozygous for all eight marker alleles and presumably carry all three SMV resistance genes that would potentially provide multiple and durable resistance to SMV.
SUMMARY III
IV. Development of a Multiplex SNP Assay for Soybean Mosaic Virus Resistance
Ainong Shi, Pengyin Chen, Richard Vierling, Innan Cervantes, and Ehsan Shakiba. 2009. Development of multiplex SNP assays for selection of soybean mosaic virus resistance genes in soybean. World Soybean Research Conference VIII, August 10-15, 2009, Beijing, China.
Essex ACCCTGCACTGCACTTCAAATPI 96983 ACCCTGCACTTCACTTCAAATMarshall ACCCTGCACTTCACTTCAAATOgden ACCCTGCACTTCACTTCAAAT
SNP Discovery
Essex
Ogden
PI 96983
Marshall
G
T
T
T
1. Development of allele-specific SNP markers for 3gG2 (Rsv1)
2. Selection of allele-linked SNP markers for Rsv3, and Rsv4
3. Selection of SNP markers from soybean MLGs
Content
3gG2 is the resistance allele at Rsv1 locus and the gene has been cloned and sequenced (Hayes et al. 2004). The sequence of 3gG2 was deposited in GenBank with the accession AY518517.
AAAGAAACATACACTCTAGCAGCAGGCACTAATGAAAATAGAGCACATTTTAAGGGAAGTAAGATGGTAGAGAAAGTAGTGAATGCTAGAAAATCAGGTGTGAATCAAATGGACAGAGAAACCAAAGCAAAAGCTTTCAA............................................................................................................................................AATTAAACGTTTTAAAAAATACTAGTTAAACAATATGTCTAAAAATATTAAAAATTACTAAGAAACTATTAAAAATACTAGTTAAACAATTTATAGTGCCTTAAGATAATTTCTTCCTTTATAACTATTTAAATAACTAATTTTTAATAAATAATAATTTTTGTATACTATTTTTACTCATAAAATTAAAACAAACAAGTGTTGGTTAATATCCCAAGAGGTACCCTCAGCAGAATCATAGCCACGCATTAATTCATGAAATACAGCTGTTAAAGTTTGTCTTGTTGGGAAGACTCAACAGATATTGGTTGACTCAATCTTAATTAAAGCCATCCTTCATAAAATTGCCT............................................................................................................................................ATATTTAACAGATGAATTTTGTTCCCAATTTTTTTTTTATAAAGAATTGTCGTCTAAAAAAGTGTTTATTTCATTTTTCTTTTATTGCAAAGCTTTGAAATTGTTAAATGCGATTTTAGGCCCTAAATTAAATCTAACTAGAAGAGCTC
1. Develop Allele-specific SNP Marker for 3gG2 (Rsv1)
3gG2 – AY518517
3gG2 Gene Sequence
Primer Design
PCR
Sequencing PCR Products
Multiple Sequence Alignment
SNP Discovery
SNP Validation SNP Genotyping
Procedure for 3gG2-specific SNP markers
3gG2-AY518517
Primer design Tool: Primer-BLAST
PCR
PCR ProductSequencing
3gG2-f1: TCAGCAGAATCATAGCCACG3gG2-r1: CCAACACCACTTGCATTTTG (CAAAATGCAAGTGGTGTTGG)
Multiple Sequence Alignment - Tool - BioEdit
SNP Validation and Genotyping by Sequenom An example
Soybeangenotype
SMVallele
3gG2snp1
3gG2snp2
Essex rsv G T A A
PI 96983 Rsv1 T T C C
Kwanggyo Rsv1-k T T C C
Marshall Rsv1-m T T C C
Ogden Rsv1-t T T C C
V262 Rsv1-n T T C C
Suweon 97 Rsv1-h T T C C
Raiden Rsv1-r T T C C
York Rsv1-y G G C C
Davis Rsv1-y G G C C
L29 Rsv3 G T C C
V94-5152 Rsv4 G T A A
V94-9003 Rsv4 G T A A
Viginia Rsv4 G T A A
J05 Rsv1+3 T T C C
Hourei Rsv1+3 T T C C
PI 486355 Rsv1+4 T T C C
Columbia Rsv3+4 G T C C
Zao 18 Rsv1-y+3 G G C C
Rsv1:[TT][CC]
Rsv1-y:[GG][CC]
[GT][CC]
2. Identify Allele-linked SNP Markers for Rsv3 and Rsv4
A519GenBank A519snp1 A519snp2
AF348331 TATCCAAATAATAGTACCTCT ACCCCTAAATGCTCAACCTTA
AF125933 TATCCAAATACTAGTACCTCT ACCCCTAAATGCTCAACCTTA
AF125932 TATCCAAATAATAGTACCTCT ACCCCTAAATGCTCAACCTTA
AF125931 TATCCAAATAATAGTACCTCT ACCCCTAAATGCTCAACCTTA
AF125930 TATCCAAATAATAGTACCTCT ACCCCTAAATGCTCAACCTTA
AF125929 TATCCAAATACTAGTACCTCT ACCCCTAAATCCTCAACCTTA
AF125928 TATCCAAATACTAGTACCTCT ACCCCTAAATCCTCAACCTTA
AF125927 TATCCAAATACTAGTACCTCT ACCCCTAAATCCTCAACCTTA
AF125926 TATCCAAATACTAGTACCTCT ACCCCTAAATCCTCAACCTTA
AF125925 TATCCAAATACTAGTACCTCT ACCCCTAAATCCTCAACCTTA
A519
Rsv3
0.8 CM
AW307114A
Rsv4
3.3 CM
AW307114genbank AW307114snp1 AW307114snp2
J05GGCAAAAAAAACCATCAATTG
ATTCTAACGGTGATTATTATT
V94-5152
GGCAAAAAAAACCATCAATTG
ATTCTAACGGTGATTATTATT
L29GGCAAAAAAA-CCATCAATTG
ATTCTAACGGTGATTATTATT
EssexGGCAAAAAAA-CCATCAATTG
ATTCTAACGGGGATTATTATT
SNP marker LG Loc Linked gene
BARC-010137-00526 F 68.11 Rsv1
BARC-025897-05144 F 68.85 Rsv1
BARC-022043-04271 F 69.85 Rsv1
BARC-029823-06424 F 69.9 Rsv1
BARC-039667-07534 B2 90.78 Rsv3
BARC-012953-00413 B2 90.8 Rsv3
BARC-024363-04860 B2 94.68 Rsv3
BARC-010469-00648 B2 96.75 Rsv3
BARC-012703-00380 B2 96.79 Rsv3
BARC-025955-05182 D1b 45.98 Rsv4
BARC-016079-02060 D1b 48.73 Rsv4
BARC-025183-06457 D1b 51.92 Rsv4
BARC-030679-06925 D1b 54.89 Rsv4
3. Select SNP Markers from Soybean MLGs
SNP markers from the Soybean Linkage Map – 2006 (http://bfgl.anri.barc.usda.gov/soybean/index.html)
SNP genotyping in Soybean by Sequenom An example
Soybean
Linked to Rsv1 Linked to Rsv3 Linked to Rsv4
3gG2-snp1
F015435-01966 N11PF A519d
B2-012953-00413
D1b-011147-00855
D1b-025955-05182 AW307114
Da bai ma G T C C T T T T T T A A A A d d
Essex G T C C T T C C C T A A A A d d
J05 T T C C T T T T T T A A A A A A
Jindou1 G G A A T T T T C T G G A A d d
Jiunong 21 G T A A T T T T C T A A G G d d
Kefeng No. 1 T T A A T T T T T T G G A A A A
Kwanggyo T T C C G G T T C T G G G G A A
L29 G T A A T T 0 0 T T G G G G d d
Marshall G T A A T T C C C T G G G G A A
Ogden G T A A T T C C C T A A G G A A
PI 506809 T T C C G G C C C T A A G G d d
PI 96983 T T C C T T T T C T A A G G A A
Raiden T T C C G G C C C T A A G G d d
Suweon 97 T T C C G G C C C T A A G G d d
V262 T T C C G G C C C T A A A A d d
V94-5152 G T C C T T T T T T A A A A A A
Viginia G T A A T T T T T T G G A A d d
York G G A A T T C C C T G G A A d d
Zho 18 G G C C T T T T C T G G A A d d
SNP Genotyping in J05 x Essex F2 population
• J05 contains two SMV resistance genes Rsv1 and Rsv3
• Rsv1 in J05 is co-segregated with the SNP marker 3gG2snp, unlinked to SNP A519snp and AW307114snp1
• Rsv3 in J05 is co-segregated with SNP A519snp and unlinked to AW307114snp1
Locus Phenotype
χ2A
(3:1)
χ2B
(1:2:1)
χ2AB
(3:6:3:1:2:1)A B RM11 RM12 RM22 SM11 SM12 SM22
Rsv1 3gG2snp 19 55 0 0 0 20 0.181 2.836 265.939**
Rsv1 A519snp 18 40 17 2 12 5 0.181 1.149 3.362
Rsv1 AW307114snp1 24 35 16 5 11 3 0.181 2.17 4.042
Rsv3 3gG2snp 0 0 34 0 0 14 0.341 0.000 (1:0) -
Rsv3 A519snp 11 23 0 0 0 14 0.341 0.337 60.166**
Rsv3 AW307114snp1 13 15 6 4 8 2 0.341 3.458 0.604
• 2 SNP markers were identified from the 3gG2 gene at Rsv1 locus
• 4 SNPs near Rsv1, 5 near Rsv3, and 5 near Rsv4 were validated
• The inheritance mode in two F2 populations
(J05 x Essex and Jindou1 x Essex) were tested by the SNP assay and verified these SNP markers were useful in idenfication and selection of SMV resistance genes
SUMMARY IV
ACKNOWLEDGEMENTS
University of Arkansas:Dr. Pengyin Chen, Dr. Rose Gergerich, Dr. Cuiming Zheng, Dr. Bo Zhang, Dr. Anfu Hou, Dr. Tetsuaki Ishabahi,Dexiao, Ehsan, Innan, Justin, Caroline, Tina,Leandro, Andrew, Liliana, Julieta, Xiaoyu, Luciano,Moldir, Michael, Mioko, Takuro, Yoko, and Bryan.
ICIA and Purdue University:Dr. Rick Vierling, Larry Svajgr, Alan Galbreth, Pam Hogue, Karan Miller, and Rachael Sondeno
Thank you!
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