1. Development of SNP Multiplex Assays for Disease Resistance Genes in Tomato Ainong Shi and Richard Vierling Indiana Crop Improvement Association (Purdue University) Richard Grazzini GardenGenetics LLC

2. Genetic resistance is the primary method of controlling plant diseases Marker assisted selection (MAS) has become important and useful in the selection of disease resistance genes Single nucleotide polymorphism (SNP), because of its abundance and high- throughput scoring potential is becoming a powerful tool in genome mapping, association studies, diversity analysis, and tagging of important genes in plant genomics

3. Linked maker Gene-specific marker Allele-specific marker Sequence-derived SNP Gene sequence-derived SNP (I-2 locus) Allele sequence-derived SNP (I-2 and i-2) Molecular Marker Evolution Single gene Multiple genes in one disease Multiple genes in multiple diseases Molecular Assisted Selection QTLs

4. Single Nucleotide Polymorphism (SNP) An example for Mi Motella LA3130 Mogeor NY07-464 G C G C Motella/LA2823MiAAGTAGACGAGGTTAGTAAAAT Mogeor/LA3471MiAAGTAGACGAGGTTAGTAAAAT NY07-464miAAGTAGACGACGTTAGTAAAAT LA3130miAAGTAGACGACGTTAGTAAAAT

5. A. Fusarium wilt (Fusarium oxysporum f. sp. Lycopersici) (image from apsnet) B. Verticillium wilt (V. dahliae and V. albo-atrum) (image from Cornell) C. Root-knot nematode (Meloidogyne incognita, M. javanica) (image from NCSU) D. Tomato mosaic virus (ToMV) (image from apsnet) E. Tomato spotted wilt virus (TSWV) (image from NCSU) Five Tomato Diseases ABC D E

6. Objective To identify SNP markers for I-2 of Fusarium wilt resistance, Mi of root-knot nematode resistance, Sw-5 of TSWV resistance, Tm-2 of ToMV resistance, and Ve of Verticillium wilt resistance To develop a multiplex SNP assay for marker assisted selection (MAS) of the five genes

7. Sw-5 is the most broadly deployed TSWV resistance gene utilized in tomato breeding because of its durability and stable resistance (Boiteux and Giordano, 1992; Stevens et al., 1992, 1994, 1995). Sw-5 gene has been cloned and its sequence was deposited in GenBank with the accession AY007366. AAGCTTGGTGCAATGACATTATACAATCAACAAAGAATAGAAAGGAGATAAAAAGCACTATCCAAATCCC --------------------------------------------------------------------- ---------------------------------------------------------------------- TCCATTTTCCTTTTTTTGATTATTCCAGCTACCAAGACAACCACTAGGGGCAGTCCTTTGCATTTTTCTG CTACTGCTTGACTCACATCTTGTAGTTCAAGCGGGCAATCTTCCTTTTGAAACACTTTTTTCTGCAACAA TTGGCAACTCTCTTCTGTTGTGAGGAATGGAAGAGAATAAGGATCAGTATGGTACTTGACTTGCTTACCC ACTTCTTCAAGTCGAGTTGTTACGACTATTCTGCTTCTAATTCCAACATCTGGAAAAGAAAGCCTTAAGT CATCCCATACCATACAATCCCACATATCATCCAATACAATGAGATATCTCTTTCCCATTAATTTTCTCCT CAACATGTCGGCAAGAACATCAACCGTAGCTCCATTGTCGTTGAAACCTGTAACTTGACTGAAAATATCT TGTAATAACTCTATCCGGTTATACGTTTGAGAAATGATGCACCATGCTCGAACATCAAAGCGAGAAACAA TAATGTCATTATTGTACAACTTTCTAGCAATTGTCGTTTTCCCTTGTCCTCCCATGCCTACAATTGGGAC AACGTCTAGCTCATTTGTACCTCTAGTCAGATAATCAATTAGTTCTTCTGCTTGTTTCTCAAAACCCACT ACCTCCTCATCAGTTACAAGATTGCTATGTCGATCCGGCAGATGTTTTAATGGAGCAGCCACATAGTGAG ---------------------------------------------------------------------- TTGGCTTAAAATAAGTTTTTTTTAACTTAAAATATGTTATTTTGAGTATTGACAAACAACTAAATAAGTC AAAAATCAGCTTTTAAGTCAGTTTGACCACGCTCTCTTAT Develop Allele-specific SNP Marker for Sw-5 (for example) Sw-5 – AY007366

8. Sw-5 Gene Sequence Primer Design PCR Sequencing PCR Products Multiple Sequence Alignment SNP Discovery SNP ValidationSNP Genotyping Procedure for development of Sw-5 specific SNP markers

9. AAGCTTGGTGCAATGACATTATACAATCAACAAAGAATAGAAAGGAGATAAAAAGCACTATCCAAATCCC --------------------------------------------------------------------- ---------------------------------------------------------------------- TCCATTTTCCTTTTTTTGATTATTCCAGCTACCAAGACAACCACTAGGGGCAGTCCTTTGCATTTTTCTG CTACTGCTTGACTCACATCTTGTAGTTCAAGCGGGCAATCTTCCTTTTGAAACACTTTTTTCTGCAACAA TTGGCAACTCTCTTCTGTTGTGAGGAATGGAAGAGAATAAGGATCAGTATGGTACTTGACTTGCTTACCC ACTTCTTCAAGTCGAGTTGTTACGACTATTCTGCTTCTAATTCCAACATCTGGAAAAGAAAGCCTTAAGT CATCCCATACCATACAATCCCACATATCATCCAATACAATGAGATATCTCTTTCCCATTAATTTTCTCCT CAACATGTCGGCAAGAACATCAACCGTAGCTCCATTGTCGTTGAAACCTGTAACTTGACTGAAAATATCT TGTAATAACTCTATCCGGTTATACGTTTGAGAAATGATGCACCATGCTCGAACATCAAAGCGAGAAACAA TAATGTCATTATTGTACAACTTTCTAGCAATTGTCGTTTTCCCTTGTCCTCCCATGCCTACAATTGGGAC AACGTCTAGCTCATTTGTACCTCTAGTCAGATAATCAATTAGTTCTTCTGCTTGTTTCTCAAAACCCACT ACCTCCTCATCAGTTACAAGATTGCTATGTCGATCCGGCAGATGTTTTAATGGAGCAGCCACATAGTGAG ---------------------------------------------------------------------- TTGGCTTAAAATAAGTTTTTTTTAACTTAAAATATGTTATTTTGAGTATTGACAAACAACTAAATAAGTC AAAAATCAGCTTTTAAGTCAGTTTGACCACGCTCTCTTAT Sw-5: AY007366 Primer design Tool: Primer-BLAST PCR PCR Product Sequencing Sw5-f1AACCACTAGGGGCAGTCCTT Sw5-r1CTCACTATGTGGCTGCTCCA (TGGAGCAGCCACATAGTGAG)

10. Multiple Sequence Alignment - Tool - BioEdit AY007366-sw5bAACCACTAGGGGCAGTCCTTTGCATTTTTCTGCTACTGCTTGACTCACATCTTGTAGTTCAGGCGGGCAATCTTCCTTTTGAAACACTTTTTTCTGCAAC 100 LA3667---------------------------------------------------------------------------------------------------- 100 NY07-464---------------------------------------------------------------------------------------------------- 100 LA2823---------------------------------------------------------------------------------------------------- 100 AY007366-Sw5aAACCACTAGGGGCAGTCCTTTGCATTTTTCTGCTACTGCTTGACTCACATCTTGTAGTTCAAGCGGGCAATCTTCCTTTTGAAACACTTTTTTCTGCAAC 100 AY007366-sw5bAATTGGCAACTCTCTTCTGTTGTGAGGAATGGAAGAGAATAAGGATCAGTATGGTACTTGACTTGCTTACCCACTTCTTCAAGTCGAGTTGTTACGACTA 200 LA3667---------------------------------------------------------------------------------------------------- 200 NY07-464---------------------------------------------------------------------------------------------------- 200 LA2823---------------------------------------------------------------------------------------------------- 200 AY007366-sw5bTTCTGCTTCTAATTCCATCATCTGGAAAAGAAAGCCTTAAGTCATCCCATACCATACAATCCCACATATCATCCAATACAATGAGATATCTCTTTCCCAT 300 LA3667-----------------W---------------------------------------------------------------------------------- 300 NY07-464-----------------A---------------------------------------------------------------------------------- 300 LA2823-----------------A---------------------------------------------------------------------------------- 300 AY007366-sw5bTAATTTTCTCCTCAACATGTCGGCAAGAACATCAACCGTAGCTCCATTGTCGTCGGAACCTGTAACTTGACTGAAAATATCTTGTAATAACTCTCTCCGA 400 LA3667------------------------------A----------------------Y-R----------------G---------------------M--C-R 400 NY07-464------------------------------G----------------------T-A----------------A------------------------T-- 400 LA2823------------------------------G----------------------T-A----------------A------------------------T-- 400 AY007366-sw5bTTATACGTTTGAGAAATGATGCACCATGCTCGAACATCAAAGCGAGAAACAATAATGTCATTATTGTACAACTTTCTAGCAATTGTCGTTTTCCCTTGTC 500 LA3667---------------------------------------------------------------------------------------------------- 500 NY07-464---------------------------------------------------------------------------------------------------- 500 LA2823---------------------------------------------------------------------------------------------------- 500 AY007366-sw5bCTCCCATGCCTACAATTGGGACAACGTCTAGCTCATTTGTACCTCTAATCAGATAATCAATTAGTTCTTCTGCTTTATTCTCAAAACCCACTACCTCCTC 600 LA3667-----------------------------------------------R---------------------------KW----------------------- 600 NY07-464---------------------------------------------------------------------------------------------------- 600 LA2823---------------------------------------------------------------------------------------------------- 600 AY007366-sw5bATCAGTTACAAGATTGCTATGTCGATCCGGCAGATGTTTTAATGGAGCAGCCACATAGTGAG662 LA3667--------------------------------------------------------------662 NY07-464--------------------------------------------------------------662 LA2823--------------------------------------------------------------662

11. AccessionGene GenBank accessionSw5-snp1sw5-snpSsnp3snp4snp5snp6 StevensSw-5bAY00736631279T31392A31415C31417G31434G31459C LA3667Sw-5FJ686041218W331A354Y356R373G398C LA3433sw-5FJ686040218A331G354T356A373A398T Riesentraubesw-5FJ686039218A331G354T356A373A398T LA2823sw-5FJ686042218A331G354T356A373A398T StevensSw-5aAY00736622586A22699A22722T22724A22741G22766C SNP Discovery * Sw-5 consisted of Sw-a and Sw-b. Sw5-a is located at 20824-24561 base and Sw5-b at 29513-33253 base of the GenBank accession AY0073366 that was cloned from the tomato cultivar “Stevens” (Folkertsma et al 1999). Sw-b is the functional allele for conferring resistance to TSWV (Spassova et al., 2001). ‘W’ = ‘A/T’, ‘Y’ = ‘C/T’, and ‘R’ = ‘A/G’

12. SNP Validation and Genotyping by Sequenom Cultivar/Accession Gene at Sw-5 locus Sw5- f1/r1 Sw5-f2/r2 Sw5- snp1 Sw5-snp2 TalladegaSw5-b z & Sw5-a + +A TA SophyaSw5-b & Sw5-a + +A TA BHN-444Sw5-b & Sw5-a + +A TA LA3667Sw5-b & Sw5-a + +A TA Anahu (LA0655)sw5-b + -A G LA0656 sw5-b+ -A G VFNT Cherry (LA1221) sw5-b+ -A G LA1802 sw5-b+ -A G Royal Red (LA2088) sw5-b+ -A G Mobox (LA2821) sw5-b+ -A G Motella (LA2823) sw5-b+ -A G UC-204C (LA3130) sw5-b+ -A G LA3432 sw5-b+ -A G LA3433 sw5-b+ -A G Mogeor (LA3471) sw5-b+ -A G LA3473 sw5-b+ -A G Peto 95-43 (LA3528) sw5-b+ -A G Riesentraube sw5-b+ -A G Bush Celebrity sw5-b+ -A G NY07-461 sw5-b+ -A G NY07-464 sw5-b+ -A G

13. One 9-SNP multiplex assay was developed: one for I-2, one for Mi, two for Sw-5, two for Tm-2 locus, and three for Ve1 Summary I-2G i-2C MiG miC Variety Gene at Ve locus GenBank accession Ve1-snp1Ve1-snp2Ve1-snp3 LocationBaseLocationBaseLocationBase CraigellaVe1AF2723672199T2752G3041C MogeorVe1FJ80992750T603G892C Peto 95-43Ve1FJ80992850T603G892C NY07-464ve1FJ80992550A603A892G Riesentraubeve1FJ68604550A603A892G Tm2RS-snp1Tm2R-snp2 Tm-2TC tm-2CT sw5-SNP1sw5-SNP2 Sw-5TA sw-5AG

14. Linked SNP Gene sequence-derived SNP Allele sequence-derived SNP Single gene Multiple genes in one disease Multiple genes in multiple diseases Further Research More SNPs, more genes, more diseases QTLs

15. ACKNOWLEDGEMENTS Cornell University: G. Moriarty, M. Glos and M. Jahn Restoring our Seed: C. R. Lawn TGRC, University of California, Davis: Roger Chetelat, Sheh May Tam

16. Richard Grazzini GardenGenetics LLC Email: rick@gardengenetics.com Web: http://gardengenetics.com Richard Vierling Indiana Crop Improvement Association (Purdue University) Email: vierling@indiacrop.org Web: http://www.indianacrop.org Ainong Shi Indiana Crop Improvement Association (Purdue University) EmaiL: shia@purdue.edu Web: http://web.ics.purdue.edu/~shia

17. Thank you!