Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University

Original Objectives Years 2-4

1. Generate a BAC library from Capsicum frutescens, a major source of disease resistance in pepper

2. 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

1. Comparative structural analysis of orthologous R gene clusters

Jahn Lab Dept. of Plant Breeding & Genetics, Cornell University

Current Objectives Years 2-4

1. Generate a BAC library from Capsicum frutescens, a major source of disease resistance in pepper

2. Fine map the L locus in Capsicum and assess homology to R3/R7/potato and I2/tomato

3. Provide 10 Capsicum BACs to TIGR for draft sequencing, identify clones for full sequencing

4. Comparative structural analysis of homologous NBS-LRR containing regions

1. Provide Capsicum-derived transgenesfor resistance to PVY for use inpotato transformation and breeding

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

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

Some cross-generic R gene groups in Solanaceae

Potato 11 Tomato 11 Pepper 11

TG105, I2

TG36

TG46

TG105, I2

TG36

TG46

CT107

T142, CDI 1366H15LTG36T1151T386

TG46

TG105, I2?CT107

R3R3 BAC contig

AFLP markers < 1.0

> 30 cM

6.0 cM

Comparative mapping R3/R7TG105 and I2 orthologs in pepper map 20 cM

north of Capsicum L locuspotato 11 tomato 11 pepper 11

TG105, I2

TG36

TG46

TG105, I2

TG36

TG46

CT107

T142, CDI 1366H15LTG36T1151T386

TG46

TG105, I2?CT107

R3BAC contig

AFLP markers < 1.0

Overview of our strategy to characterize I2-homologous Capsicum regions

BAC library screening

I2, GP250

Positive BAC clone analysis

BAC fingerprinting and contig mappingPCR screening using primers developed in potato

Candidate BAC clone draft sequencing

Identify BAC clones orthologous to R3/R7

Map BAC sequences on a pepper linkage map

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

Implies significant differences in genome organization between

potato and pepper in this region

There are 17 copies of GP250 and I2 homologs within

the R3/R7 cluster in potato.

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 68Clone 41

Clone 56

Clone 61 Clone 1

Clone 40Clone 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, 90Clone 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 27Clone 25

Clone 4

Clone 28

Clone 26

Clone 72

Clone 23

Clone 8, 9

Clone 10Clone 14, 17

Clone 14, 15

Clone 2

Clone 13, 18

Clone 6

Clone 5, 11, 13, 20

Analysis of I2 positive pepper clones

■ PCR screening I2 positive BAC clones

Primers: R7-1, R7-2, LRR, BAC end sequences

■ Cloning PCR fragments and sequencing

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

The BAC clones obtained by LRR primers.

Sequence alignment of LRR fragments

Nucleotide Substitutions (x100)0

61.0

102030405060

LRR7LRR8LRR6LRR2LRR16LRR9LRR11LRR5LRR1LRR13LRR20LRR17LRR19LRR4LRR10LRR12LRR22LRR18

1

234

5

678910

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

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

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

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

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)

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 R R

pvr11 R S S

pvr12 R R S

Pepper infectivity for potato PVY strains

PVY N PVY NO PVY NTN PVY O

Pvr1+ S R S R

pvr1 R R R R

pvr11 R R R R

pvr12 R R R R

- 5 Plants per genotype were screened. - Pvr1+ and 3 different lines containing the pvr1 gene were screened.

Potato infectivity

PVY N PVY NO PVY NTN PVY O

Superior S S S S

Russet Burbank

S S S S

Dark Red

NorlandS S S ½ R

Umatilla S S S ½ R

Katahdin S S S ½ R

■ 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.

pvr1 Transgenic Potatoes

■ 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

Collaboration with Dr. Sandra Austin-Phillips - UW Madison

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

Burbank5 8 8 3

Katahdin 10 0 0 0

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.