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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