Dept. of Plant Breeding & Genetics, Cornell University 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 Thank you for the kind introduction. It is my great honor to have an opportunity to give a job talk today. And I would like to express my thanks to search committee members and chairman of this department. The title that I’m going to talk today is “Developing a platform for the future vegetable breeding: Capsicum as an example”.

Dept. of Plant Breeding & Genetics, Cornell University 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 Thank you for the kind introduction. It is my great honor to have an opportunity to give a job talk today. And I would like to express my thanks to search committee members and chairman of this department. The title that I’m going to talk today is “Developing a platform for the future vegetable breeding: Capsicum as an example”.

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

Some cross-generic R gene groups in Solanaceae

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

Comparative mapping R3/R7 TG105 and I2 orthologs in pepper map 20 cM north of Capsicum L locus potato 11 tomato 11 pepper 11 CT107 CT107 TG105, I2? TG46 TG46 TG46 TG36 TG36 TG105, I2 TG105, I2 T142, CDI 136 BAC contig R3 6H15 AFLP markers < 1.0 L TG36 T1151 T386

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean. Identify BAC clones orthologous to R3/R7 Complete sequencing orthologous clones

Cloning homologs of BAC end sequences

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. Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean. Implies significant differences in genome organization between potato and pepper in this region

BAC fingerprinting and contig mapping Clone 70 Clone 36 Clone 59, 92 Clone 81 Clone 82 Clone 47, 29 Clone 49 Clone 83 Clone 67 Clone 63 Clone 19 Clone 39 Clone 91 Clone 42, 85 Clone 75 Clone 68 Clone 41 Clone 56 Clone 61 Clone 1 Clone 40 Clone 48, 60 Clone 50 Clone 31, 79 Clone 30, 35, 44, 71 Clone 62 Clone 87 Clone 7 Clone 35, 43, 54, 73, 74, 78 Clone 32, 34 Clone 76 Clone 66 Clone 52, 84 Clone 57 Clone 86, 90 Clone 88, 89 Clone 80 Clone 33 Clone 38, 51, 69 Clone 37 Clone 77 Clone 45, 46, 58, 65 Clone 12 Clone 22 Clone 21 Clone 27 Clone 25 Clone 4 Clone 28 Clone 26 Clone 72 Clone 23 Clone 8, 9 Clone 10 Clone 14, 17 Clone 14, 15 Clone 2 Clone 13, 18 Clone 6 Clone 5, 11, 13, 20 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean. ■ Cloning PCR fragments and sequencing The BAC clones obtained by LRR primers.

Sequence alignment of LRR fragments 1 2 3 4 5 6 7 8 9 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean. 10 Ten BAC clones were chosen for draft sequencing Sent to TIGR mid-January, 2006

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

Potato Virus Y ■ Potato virus Y ■ Strains - 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 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

PVY symptoms ■ PVY O ■ PVY N and PVY NTN - 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) Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

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 PVY - 0 PVY-1 PVY-1-2 pvr1 R pvr11 S pvr12 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.

Pepper infectivity for potato PVY strains PVY N PVY NO PVY NTN PVY O Pvr1+ S R pvr1 pvr11 pvr12 Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean. - 5 Plants per genotype were screened. - Pvr1+ and 3 different lines containing the pvr1 gene were screened.

Potato infectivity ■ Sandra or Chuck Brown sent 10 plants per cultivar which were screened for resistance to the 4 PVY strains listed. PVY N PVY NO PVY NTN PVY O Superior S Russet Burbank Dark Red Norland ½ R Umatilla Katahdin 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.

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

Transgenics potatoes (as of 12/05) ■ Independent transgenic events received: ■ Received 1-2 plants per event (Confirmed by Sandra via nptII PCR). Pvr1 pvr1 pvr11 pvr12 Russet Burbank 5 8 3 Katahdin 10

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 pvr12 were 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. Just to give you a big picture of my talk. At first, I’m going to talk about a brief history of cop breeding in order to get you oriented. An then I am going to talk about my work to date which is mostly on pepper. After the research talk, I’ll be describing my future plans for education and research in Korean.