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

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

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

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

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

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

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

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

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

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

FLA456

Senegal Indonesia

FLA456 CLN1466J

Taiwan

FLA456 H24 (Ty-2)

El Salvador

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

Severity Scale

Severe (3): stunting, curling Moderate (2): symptoms obvious but not as severe

Slight (1): evident only after examination

TYLCTHV Reactions of Parents and F1

FLA456 F1 CLN1621L

Severity Score Distribution of RILs

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

Severity score means of F6-RIL groups with the same combination of TYLCD resistance QTLs.

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)

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

Bacterial Wilt of Tomato

• Caused by Ralstonia solanacearum • Soilborne pathogen attacking 44 plant families • Major tomato disease in the tropics

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)

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

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

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

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?

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

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

BW Resistance Sources and Worldwide Exchange

Daunay et al., 2011

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

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

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

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

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.

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)

“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

Thank You