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Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University Original Objectives Years 2-4

Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University Original Objectives Years 2-4 Generate a BAC library from Capsicum frutescens , a major source of disease resistance in pepper Fine map the L locus in Capsicum and assess homology to R3/R7 /potato

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Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University Original Objectives Years 2-4

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  1. Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University Original Objectives Years 2-4 Generate a BAC library from Capsicum frutescens, a major source of disease resistance in pepper Fine map the L locus in Capsicum and assess homology to R3/R7/potato and I2/tomato 3. Provide 3 BACs to TIGR for sequencing Comparative structural analysis of orthologous R gene clusters

  2. Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University Current Objectives Years 2-4 Generate a BAC library from Capsicum frutescens, a major source of disease resistance in pepper Fine map the L locus in Capsicum and assess homology to R3/R7/potato and I2/tomato Provide 10 Capsicum BACs to TIGR for draft sequencing, identify clones for full sequencing Comparative structural analysis of homologous NBS-LRR containing regions Provide Capsicum-derived transgenes for resistance to PVY for use in potato transformation and breeding

  3. Current Project Objectives ■Generate a BAC library from Capsicum frutescens ■Improve map of the L locus in Capsicum with clones from potato and tomato maps ■ Assess homology to R3/R7/potato and I2/tomato ■ Build Capsicum contig through selected region (I2) ■ Obtain draft sequence to identify BAC clones for full sequence; obtain full sequence ■Comparative assessment of sequence from tomato, potato and pepper chromosome 11 ■Developing potyvirus resistant potato using Capsicum eIF4E sequences

  4. Comparative study of R3/R7 loci ■BAC library from Capsicum frutescens Leaf tissues of C. frutescens - # of clones: 221,184 clones - average insert size: 125-130 kb BAC library has already been widely used in Jahn lab: for studies of the Pun1 locus for studies of the L locus in Paran lab: for studies of a number of loci involved with fruit color and fruit quality available at cost from AGI

  5. Some cross-generic R gene groups in Solanaceae

  6. Tomato 11 Pepper 11 Potato 11 CT107 CT107 TG105, I2? TG46 TG46 TG46 > 30 cM TG36 TG36 6.0 cM T142, CDI 136 TG105, I2 TG105, I2 R3 BAC contig 6H15 R3 AFLP markers < 1.0 L TG36 T1151 T386

  7. Comparative mapping R3/R7TG105 and I2 orthologs in pepper map 20 cM north of CapsicumL locus tomato 11 pepper 11 potato 11 CT107 CT107 TG105, I2? TG46 TG46 TG46 TG36 TG36 T142, CDI 136 TG105, I2 TG105, I2 BAC contig 6H15 R3 AFLP markers < 1.0 L TG36 T1151 T386

  8. Overview of our strategy to characterize I2-homologous Capsicum regions BAC library screening I2, GP250 Positive BAC clone analysis BAC fingerprinting and contig mapping PCR screening using primers developed in potato Candidate BAC clone draft sequencing Map BAC sequences on a pepper linkage map Identify BAC clones orthologous to R3/R7 Complete sequencing orthologous clones

  9. Cloning homologs of BAC end sequences

  10. Identification of BAC clones ■ Probes -I2C-1 gene: 89 positive pepper clones - GP250: 7 positive pepper clones These sets did not overlap There are 17 copies of GP250 and I2 homologs within the R3/R7 cluster in potato. Implies significant differences in genome organization between potato and pepper in this region

  11. Clone 75 Clone 38, 51, 69 Clone 12 Clone 70 Clone 22 Clone 37 Clone 68 Clone 36 Clone 21 Clone 41 Clone 77 Clone 39 Clone 45, 46, 58, 65 Clone 27 Clone 59, 92 Clone 56 Clone 25 Clone 61 Clone 1 Clone 81 Clone 4 Clone 33 Clone 82 Clone 48, 60 Clone 28 Clone 40 Clone 47, 29 Clone 32, 34 Clone 50 Clone 76 Clone 26 Clone 49 Clone 72 Clone 31, 79 Clone 66 Clone 83 Clone 30, 35, 44, 71 Clone 23 Clone 52, 84 Clone 67 Clone 62 Clone 2 Clone 8, 9 Clone 57 Clone 63 Clone 13, 18 Clone 87 Clone 86, 90 Clone 10 Clone 19 Clone 6 Clone 88, 89 Clone 14, 17 Clone 7 Clone 91 Clone 5, 11, 13, 20 Clone 80 Clone 14, 15 Clone 35, 43, 54, 73, 74, 78 Clone 42, 85 BAC fingerprinting and contig mapping

  12. Analysis of I2 positive pepper clones ■ PCR screening I2 positive BAC clones Primers: R7-1, R7-2, LRR, BAC end sequences R7-1: 37 BAC clones, 1.5 kb R7-2: 52 BAC clones, 0.8 kb to 1.3 kb LRR: 22 BAC clones, 1.2 kb BAC end sequences: none ■ Cloning PCR fragments and sequencing The BAC clones obtained by LRR primers.

  13. Sequence alignment of LRR fragments 1 2 3 4 5 6 7 8 9 10 Ten BAC clones were chosen for draft sequencing Sent to TIGR mid-January, 2006

  14. Summary and Future Plans ■ A total of 89 positive BAC clones were identified by I2. Seven BAC clones positive to GP250 were also identified. None of GP250 clones cross hybridized with I2 positive clones.■ Ten BAC clones were chosen for draft sequencing by BAC fingerprinting and PCR analysis.■ Draft BAC sequences will be mapped on a pepper linkage map to find pepper sequences orthologous to R3/R7. ■ Complete sequences of the orthologous pepper BAC clones will be determined and compared with potato sequences

  15. Summary and Future Plans ■ A total of 89 positive BAC clones were identified by I2. Seven BAC clones positive to GP250 were also identified. None of GP250 clones were cross hybridized with I2 positive clones.■ Ten BAC clones were chosen for draft sequencing by BAC fingerprinting and PCR analysis.■ Draft BAC sequences will be mapped on a pepper linkage map to find orthologous sequences to R3/R7. ■ Complete sequences of the orthologous BAC clones will be determined and compared with potato sequences

  16. Project Goals ■Comparative study of R3/R7 loci in potato and their orthologous in pepper ■Developing potyvirus resistant potato using potyvirus resistance genes from pepper

  17. Potato Virus Y • ■Potato virus Y • - Infects the Solanaceae crops: potato, tobacco, tomato and pepper • - One the most economically important plant pathogens • - Transmitted by aphids non-persistently • - Tuber-borne virus in potato • ■Strains • - PVY N: causes veinal necrosis and mild mottling on N. tabacum cv. • Xanthi, and occasional necrotic leaves on potato • - PVY O: mottling and mosaic symptom on tobacco and mild to • severe mosaic and leaf drop on potato • - PVY C: induces triple streak symptoms on some potato cultivars

  18. PVY symptoms ■PVY O - causes a green and yellow mosaic pattern in leaves, and/or as roughness in the leaves in most hosts - yield reduction, but no tuber quality loss ■PVY N and PVY NTN - necrotic strains which both cause veinal necrosis on tobacco - causes superficial necrotic ringspots on infected tubers in certain potato varieties - necrosis is referred to as potato tuber necrotic ringspot disesase (PTNRD)

  19. PVY infectivity in pepper (previous work) ■ Pepper, tobacco and potato isolates have their own classification based on biological criteria ■PVY infectivity in pepper - Strains were classified into 3 pathotypes based on their ability to overcome certain resistance genes

  20. Pepper infectivity for potato PVY strains - 5 Plants per genotype were screened. - Pvr1+ and 3 different lines containing the pvr1 gene were screened.

  21. Potato infectivity ■Sandra or Chuck Brown sent 10 plants per cultivar which were screened for resistance to the 4 PVY strains listed. • Tubers from these infected plants were harvested and are currently in cold storage to allow for symptom development. They will be screened for tuber necrosis in a few weeks.

  22. pvr1 Transgenic Potatoes Collaboration with Dr. Sandra Austin-Phillips - UW Madison ■Constructs - Sent 1/05: Pvr1+ - susceptible allele derived from C. annuum pvr1 – resistant allele derived from C. chinense pvr11 – resistant allele derived from C. annuum pvr12- resistant allele derived from C. annuum ■Potato cultivars being transformed: Katahdin Russet Burbank Superior Umatilla (supplied by Chuck Brown) Dark Red Norland

  23. Transgenics potatoes (as of 12/05) ■Independent transgenic events received: ■Received 1-2 plants per event (Confirmed by Sandra via nptII PCR).

  24. Summary and Future Plans ■ All potato varieties for transformation were found to be susceptible to all 3 PVY N strains screened – PVY N, NO and NTN. Tubers of these infected plants are currently in cold storage to allow for PTNRD symptom development – they will be screened for tuber necrosis in a few weeks. ■ All pepper lines containing one of the 3 pvr1 resistance alleles:pvr1, pvr11 and pvr12were found to be resistance to all 3 PVY N strains, while susceptible allele Pvr1+ was susceptible to both PVY N and PVY NTN. ■ All successful transformation events have been received up to this point. They will be confirmed for transgene copy number as well as for transgene expression. ■ Cuttings will be made of all single copy transformants which will be subsequently screened for resistance to PVY O, N, NO and NTN.

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