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Tomato Breeders’ Roundtable Chiang Mai Thailand
6-8 February 2013
Recent Progress in Mapping Begomovirus Resistance and Marker-Assisted Selection for Bacterial Wilt Resistance in
Tomato at AVRDC
P. Hanson, P. Kadirvel, R. Schafleitner, R. de la Peña, S. Geethanjali, L. Kenyon, W-S Tsai, J-F Wang, F-I Ho, S-M Huang, C-W Tan
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Presentation Outline
1. Mapping of Tomato yellow leaf curl disease (TYLCD) resistance in tomato line FLA456
2. Validation of SSR markers associated with the bacterial wilt resistance gene Bwr-12 on chromosome 12
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Major AVRDC Tropical Tomato Line Profiles
Highland and Mid-altitudes Semi-determinate, indeterminate plant
habits Fresh market Late blight, bacterial wilt , Tomato yellow
leaf curl virus disease (TYLCD) and early blight resistances
Lowland Tropics Heat tolerance 32-34/ 23-25 °C TYLCD and bacterial wilt resistances Early blight, fusarium wilt, TMV Determinate, dual purpose fresh
market/processing • High yield, fruit quality, and nutrient contents
important for all market types
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Tomato Yellow Leaf Curl Disease
• TYLCD devastates tomato in the tropics, subtropics • 100% yield loss from early infection • Prompts farmer over-use of pesticides • Resistant cultivars the foundation of sustainable control
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Begomoviruses
• Begomoviruses cause TYLCD • >70 distinct tomato-infecting
begomoviruses • Transmitted by whiteflies
(Bemisia tabaci)
Bg/II2288
DNA-A 2,647 bp
AC1
AC4
AC2
AC3
SpeI107
AV1
CR CR
BC1 BV1
DNA-B 2,569 bp
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Major Tomato-infecting Begomoviruses in Taiwan
Name Abbreviation Type Comment Tomato leaf curl Taiwan virus
ToLCTWV Mono-partite
• Ty-2, Ty-3 genes each provide effective resistance
Tomato yellow leaf curl Thailand virus
TYLCTHV Bipartite • Introduced into Taiwan mid-2000’s and largely displaced ToLCTWV
• Ty-3 offers effective resistance but not Ty-2
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Sources of TYLCD Resistance (‘Ty’ Genes)
Sources Species Gene/Alleles Chrom Reference
LA1969 Solanum chilense Ty-1 6 Zamir et al., 1994
B6013 S. habrochaites Ty-2 11 Hanson et al., 2000
LA1932 LA2779
S. chilense Ty-3, Ty-3a, Ty-3b
6 Ji et al., 2007
LA1932 S. chilense Ty-4 3 Ji et al., 2009
PI126926 PI126930 PI390681 ‘TyKing’
S. peruvianum? S. lycopersicum
Ty-5 ty-5
4 4
Anbinder et al., 2009 Hutton et al., 2012
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Pursuit of Broad and Durable TYLCD Resistance
• Pyramiding Ty genes • Vector (acyl sugar-based) resistance in collaboration with
Prof. Martha Mutschler (Cornell University) • Encourage farmers to adopt additional management
practices: – Seedling nets – Imidacloprid application through flowering – Prompt destruction of infected crop debris
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9
FLA456
• Early TYLCD-resistant line from Jay Scott’s program
• TYLCD resistance derived from LA2779 (S. chilense) and ‘Tyking’
• High and stable resistance in Africa, Asia, Central America
• Resistance is recessive and not due to Ty-1/Ty-3
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Entry Taiwan Mali Senegal Indonesia (Marco Polo)
Indonesia (East West)
India
CLN1466J 3.0 3.6 3.0 3.0 3.0 3.0 CLN2123A (Ty-2)
1.6 2.3 2.8 3.0 3.0 1.2
FLA456 0.0 0.5 1.2 1.4 1.0 1.1 LSD (0.05) 0.7 - 0.5 0.3 0.2 0.6 Scores based on symptom severity including leaf yellowing, leaf curling, stunting Scores ranged from 1 (no symptoms) to 3 (severe symptoms) at Senegal, Indonesia, and India Score ranges at Taiwan and Mali were 0-3 and 0-4, respectively Trials conducted by Remi Nono-Womdim (Senegal), Thierry Jaunet (Marco Polo-Indonesia), Nurul Hidayati (East West Indonesia), and V. Muniyappa (India)
TYLCD Severity Scores of Selected Tomato Entries Evaluated in Africa and Asia
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FLA456
Senegal Indonesia
FLA456 CLN1466J
Taiwan
FLA456 H24 (Ty-2)
El Salvador
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Methods: FLA456 Resistance Mapping
• FLA456 (resistant) x CLN1621L (susceptible, heat tolerant) • 167 F6 recombinant inbred lines (RIL) • 10 seedlings (15-day old) of parents, F1 and RILs exposed two weeks
to viruliferous (TYLCTHV) whiteflies in the greenhouse and transplanted
• Symptom severity assessed 14, 42, 70 DAT • 123 polymorphic SSR markers for genotyping, linkage map
construction
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Severity Scale
Severe (3): stunting, curling Moderate (2): symptoms obvious but not as severe
Slight (1): evident only after examination
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TYLCTHV Reactions of Parents and F1
FLA456 F1 CLN1621L
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Severity Score Distribution of RILs
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QTL in FLA456 Conferring Resistance to TYLCTHV
Kadirvel et al. 2012
0.0 cM
123.4 cM
0.0 cM
83.5 cM
23.9 cM
87.1 cM
3.0 cM
102.4 cM
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Severity score means of F6-RIL groups with the same combination of TYLCD resistance QTLs.
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Summary
• Four QTLs detected in FLA456 collectively contributed 60.5% of resistance to TYLCVTHV
• qTy10.1 mapped to lower part of chromosome 10 (59 cM) was significant at all scoring dates and contributed the most to resistance. First reported TYLCD resistance gene on chromosome 10
• qTy 4.1 mapped between markers SINAC1 and SLM4-34 on chromosome 4 and is likely ty-5 from ‘Tyking’ (Hutton et al., 2012)
• qTy11.1 mapped to lower part of chromosome 11 and possibly allelic with Ty-2
• qTy6.1 located about 740 bp from Beta on chromosome 6 and probably one of the resistance factors reported by Agrama and Scott (2006)
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Implications
• Possible undetected TYLCD resistance QTLs in FLA456 • qTy10.1interval includes a candidate gene encoding the
eukaryotic translation factor ieIF4 associated with reduced potyvirus cell-to-cell movement
• ty-5 (=tgr-1) associated with impaired viral systemic infection or reduced viral replication of TLCV (Bian et al. 2007)
• Complementary resistance mechanisms offer broad resistance?
• Recessive TYLCD resistance under-appreciated by the breeding community
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Bacterial Wilt of Tomato
• Caused by Ralstonia solanacearum • Soilborne pathogen attacking 44 plant families • Major tomato disease in the tropics
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Specific PCR
ITS Multiplex PCR
Phylogeny/ specific PCR
PFGE/rep-PCR/ AFLP etc.
Clonal Lines
‘Species’ “Ralstonia solanacearum
species complex”
Phylotype Phylotype II “America”
Phylotype I “Asia”
Phylotype IV “Indonesia”
Phylotype III “Africa”
Sequevars
Courtesy of P. Prior
Classification system proposed by Fegan and Prior (2005)
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Nature of BW Resistance in Tomato
• Resistance assessed as percentage of healthy or wilted plants in “sick plots”, greenhouse trials
• Pathogen strain, temperature, soil moisture, nematodes affect expression of resistance
• Hawaii 7996 (H7996) among best sources of resistance
Drench inoculation Hawaii 7996
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Bacterial Wilt Resistance QTL’s Mapped in H7996 (Wang et al. 2012)
• 188 RILs from H7996 (resistant) x WV700 (susceptible) • RILs, parents screened for BW reaction (% wilted plants) by seedling
inoculation / field trials in Taiwan, Thailand, Indonesia, Philippines, Reunion • Chromosome 12 QTL (Bwr-12) is a 2.8 cM region and accounted for most
(17.9-56.1%) of resistance to phylotype I (Asian) strains • Chromosome 6 (Bwr-6) a 15.5-cM region with ≥1 QTLs explained 11.5-
22.2% of variation, and critical for resistance to phylotype 2 strains
Major BW resistance QTLs from H7996 on chromosomes 6 and 12
Courtesy JF Wang
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Percent wilt means of H7996 x WV700 RIL Groups with (++) or without (--) Bwr-12 and Bwr-6
Trial/Isolate Phylotype Bwr-12 Bwr-6 %wilt Taiwan/Pss4 I ++ ++ 27.3 a
++ -- 46.0 b -- ++ 70.6 c -- -- 83.2 d
Thailand-CM I ++ ++ 9.9 a ++ -- 23.9 b -- ++ 44.4 c -- -- 74 d
Wang et al., 2012 Bwr-6 genomic region extends 15.5 cM with 1-4 QTLs
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SSR Markers Associated with Bwr-12
• SSR markers SLM12-2 and SLM12-10 linked to Bwr-12 • Effective Bwr-12 markers allow identification of
homozygous F2 plants, early elimination of susceptibles, and reduction of the number of BW seedling screens during generation advance
• However, BW resistance in AVRDC lines NOT directly derived directly from H7996
• Presence and frequency of Bwr-12 in AVRDC lines?
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Selection Scheme in Development of CLN3125 ‘Multiple TY’ Lines and Frequency of BW Screens
Season Action Traits for Selection 2010 Fall Seed multiplication/ international
distribution International distribution
2010 Spr F6 BW screen Drench inoculation (46 lines x 20 plants/line) 2010 Spr Preliminary Yield Trial Yield, fruit quality and nutrition, horticultural 2010 Spr Fusarium wilt (race 2) screen I2 gene marker, seedling screen 2009 Fall Select among/within F6 lines TYLCD, visual yield, fruit quality 2009 Spr F5 BW greenhouse screen BW: drench inoculation (151 lines x 20 pl/line) 2009 Spr Select among/within F5 rows TYLCD (whitefly + markers), visual yield, fruit
quality 2008 Fall Select among/within F4 rows TYLCD, visual yield, fruit quality 2008 Spr Select among/within F3 rows TYLCD, visual yield, fruit quality 2007 Fall Select single F2 plants TYLCD (whitefly), visual yield, fruit quality 2007 Spr-Sum
F1 field evaluation & F1 BW greenhouse screen
TYLCD, visual yield, fruit quality, BW drench inoculation (80 F1 x 20 plants /F1)
2006 Fall Cross and create F1 Crossing block
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Bwr-12 in AVRDC tomato lines: 1970’s-2000’s
Entry name Source Period w% SLM12-9 SLM12-8 SLM12-10 SLM12-12 SLM12-2
Hawaii7996 resistant check 7 H H H H H
CLN698BC1F2-358- AVRDC 1980's 3 W W W / H
CLN2413D AVRDC 2000's 7 H H H H H
CLN2498E AVRDC 2000's 10 H H H H H
CLN2585D AVRDC 2000's 10 H H H H H
CLN1462A AVRDC 1990's 13 H H H H H
CLN2026D AVRDC 1990's 13 H H H H H
CLN475BC1F2-265- AVRDC 1980's 17 H H H H H
CLN2116B AVRDC 1990's 17 H H H H H
CLN2418A AVRDC 2000's 20 W W W H H
CL143-0-10-3-0-1-10AVRDC 1970's 23 H H H H H
CLN2123A AVRDC 1990's 24 H H H H H
CL9-0-0-1-3 AVRDC 1970's 30 H H H H H
CL1131-0-43-8-1 AVRDC 1970's 30 W W W / H
CL5915-206D4-2-2-0AVRDC 1980's 47 H H H H H
CLN657BC1F2-274- AVRDC 1980's 53 H H H H H
CL5915-93D4-1-0-3 AVRDC 1980's 60 W W W H W
CLN2545B AVRDC 2000's 70 W W W W W
CL11d-0-2-2-0-3-0 AVRDC 1970's 71 W - W / W
L390 susceptible check 90 W W W W W Courtesy JF Wang
SSR markers SLM12-2 and SLM12-10 linked to Bwr-12 H=H7996 allele, W=WVA700 allele, / =neither w%= percent wilted plants after drench inoculation with R. solanacearum isolate Pss4
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BW Resistance Sources and Worldwide Exchange
Daunay et al., 2011
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Validation of Bwr-12 in AVRDC lines: Drench inoculation vs Bwr-12 marker genotypes
Entry Bacterial wilt (% survival)
SLM12-2 35.6 cM
SLM12-10 33.1 cM
CLN3125A-23 27 cd H H CLN3125L 45 bc H H CLN3070J 66 a R R CLN3078G 63 ab R R CLN3125O 78 a R R CLN3125P 73 a R R CLN3078A 16 c-e R S CLN3078C 22 b-d R S CLN3125G 7 e S S CLN3125E 13 de S S CLN3125Q 8 de S S H7996 (R) 82 a R R WVa700 (S) 20 de S S L390 (S) 7 e - -
R,S: homozygous for H7996, WVa700 alleles, respectively H=heterogeneous Percent survival three weeks after drench inoculation with R. solanacearum strain Pss4 Means followed by the same letter not significantly different
Drench inoculation
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Development of Bwr-12 Near Isogenic Lines
• F6 – derived and F7 –derived lines CLN3125A-23 and CLN3125L segregate for Bwr-12
• Marker validation Plan (2011-2012): 1. Apply markers SLM12-2 and SLM12-10, find individual
plants homozygous with or without Bwr-12 within both lines
2. Harvest plants individually to produce near isogenic lines with Bwr-12 (Resistant NIL) or without Bwr-12 (Susceptible NILs)
3. Evaluate NILs, parents, original lines by drench inoculation in greenhouse (2 reps and 20 plants/rep)
4. Estimate effect of Bwr-12
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BW reactions of NIL groups with or without Bwr12 associated with SLM12-2 and SLM12-10, Summer 2011.
Line NIL Group Marker % Survival
SLM12-2 SLM12-10
CLN3125A-23 Resistant (n=22) R R 34 a
Susceptible (n=19) S S 7 b
CLN3125A-23 (n=1) H H 26 ab
CLN3125L Resistant (n=22) R R 34 a
Susceptible (n=19) S S 11 b
CLN312L (n=1) H H 6 b
R,S: homozygous for H7996, WVa700 alleles, respectively. H=segregating % survival = percent healthy plants after drench inoculation in the greenhouse
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NIL Groups to Assess Association of Bwr12 with SLM12-2 and SLM12-10.
Line NIL Group Marker % survival
SLM12-2 SLM12-10 CLN3125A-23 A-1 (n=5) R R 57 a
A-2 (n=10) R S 37 b A-3 (n=13) S R 39 b A-4 (n=6) S S 16 c CLN3125A-23 (n=1) H H 33 b
CLN3125L B-1 (n=5) R R 81 a B-2 (n=12) R S 56 b B-3 (n=4) S R 30 c B-4 (n=4) S S 29 c CLN312L (n=1) H H 41 c
R,S: homozygous for H7996, WVa700 alleles, respectively. H=segregating
• Crossovers detected between SLM12-2 and SLM12-10 in 2011 • MAS applied to create additional NIL groups
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Selected Entries Contrasting for BW Resistance within NIL groups.
Line PCR Code Marker % survival
SLM12-2 SLM12-10 CLN3125A-23 5S5 R R 65
5S12 R S 67 5S41 R S 5 5U21 S R 82 5T23 S R 6
CLN3125L 5W49 R R 87 5W80 R S 83 5X20 R S 12 5W50 S R 40 5W58 S R 9
R,S: homozygous for H7996, WVa700 alleles, respectively.
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AVRDC Marker-assisted selection for BW
• Bwr-12 lies between markers SLM12-2 and SLM12-10 • Difference between Group NIL % survival means with (RR) or
without (SS) Bwr-12 were 41% and 52% (2012) and 27 and 23 (2011)
• Collaboration with National Taiwan University to clone Bwr-12 • Ongoing dissecting and fine-mapping Bwr-6 region
CLN3125L (Bwr-12)
CLN3125L (bwr-12)
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“Working Together Works”-Rob Gilbert
• Thanks to East-West Seed for the invitation to present this work and hosting the TBRT and to Prof. Scott for sharing seed of FLA 456
• We are grateful to our collaborators worldwide who conducted BW and TYLCD evaluations
• Financial support from the Council of Agriculture-Taiwan, the National Science Council of Taiwan and the Federal Ministry for Economic Cooperation and Development Germany , and the Asia and Pacific Seed Association are gratefully acknowledged
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Thank You