jahn lab dept. of plant breeding & genetics, cornell university original objectives years 2-4
Post on 01-Jan-2016
30 Views
Preview:
DESCRIPTION
TRANSCRIPT
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.
top related