Transcript Slide 1

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
4.
Comparative structural analysis of
orthologous R gene clusters
Jahn Lab
Dept. of Plant Breeding & Genetics, Cornell University
Current Objectives Years 2-4
1.
2.
3.
4.
5.
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
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
Tomato 11
Potato 11
Pepper 11
CT107
CT107
TG46
TG46
TG36
TG36
TG105, I2?
TG46
> 30 cM
6.0 cM
R3 BAC contig
TG105, I2
R3
TG105, I2
T142, CDI 136
6H15
L AFLP markers <
TG36
T1151
T386
1.0
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
TG46
CT107
TG46
TG36
BAC contig
TG105, I2
R3
TG105, I2?
TG46
TG36
TG105, I2
T142, CDI 136
6H15
L AFLP markers < 1.0
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
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.
Implies significant differences in genome organization between
potato and pepper in this region
BAC fingerprinting and contig mapping
Clone 75
Clone 70
Clone 68
Clone 41
Clone 36
Clone 39
Clone 59, 92
Clone 56
Clone 61
Clone 1
Clone 81
Clone 33
Clone 40
Clone 82
Clone 47, 29
Clone 50
Clone 49
Clone 83
Clone 67
Clone 63
Clone 19
Clone 91
Clone 42, 85
Clone 38, 51, 69
Clone 37
Clone 77
Clone 31, 79
Clone 30, 35, 44, 71
Clone 62
Clone 87
Clone 7
Clone 35, 43, 54, 73, 74, 78
Clone 12
Clone 22
Clone 21
Clone 45, 46, 58,
65
Clone 27
Clone 25
Clone 4
Clone 28
Clone 48, 60
Clone 32, 34
Clone 76
Clone 26
Clone 72
Clone 66
Clone 52, 84
Clone 57
Clone 86, 90
Clone 88, 89
Clone 80
Clone 23
Clone 2
Clone 13, 18
Clone 8, 9
Clone 10
Clone 14, 17
Clone 14, 15
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
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.
Sequence alignment of LRR fragments
LRR7
LRR8
1
LRR6
LRR2
LRR16
LRR9
LRR112
LRR5 3
LRR1 4
LRR13
LRR205
LRR17
LRR196
LRR4 7
LRR10
LRR128
LRR229
LRR18
10
61.0
60
50
40
30
20
Nucleotide Substitutions ( x100)
Ten BAC clones were chosen for draft sequencing
Sent to TIGR mid-January, 2006
10
0
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
■ 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
S
S
S
Russet
Burbank
S
S
S
S
Dark Red
Norland
S
S
S
½R
Umatilla
S
S
S
½R
Katahdin
S
S
S
½R
-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
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.