Recent Progress in Mapping Begomovirus Resistance and Marker-Assisted Selection for Bacterial Wilt Resistance in Tomato at AVRDC

Tomato Breeders’ Roundtable Chiang Mai Thailand 6-8 February 2013 Recent Progress in Mapping Begomovirus Resistance and Marker-Assisted Selection for...
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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

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) SpeI107

AC4 AC1

Bg/II2288

CR

DNA-A 2,647 bp AC2

AC3

CR

DNA-B 2,569 bp

AV1 BC1

BV1

Major Tomato-infecting Begomoviruses in Taiwan Name Abbreviation Type Tomato leaf ToLCTWV Monocurl Taiwan partite virus Tomato yellow TYLCTHV Bipartite leaf curl Thailand virus

Comment • Ty-2, Ty-3 genes each provide effective resistance • 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

S. peruvianum?

Ty-5

4

Anbinder et al., 2009

S. lycopersicum

ty-5

4

Hutton et al., 2012

PI126926 PI126930 PI390681 ‘TyKing’

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

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

9

TYLCD Severity Scores of Selected Tomato Entries Evaluated in Africa and Asia Entry

Taiwan

Mali

Senegal

Indonesia (Marco Polo)

Indonesia (East West)

India

CLN1466J CLN2123A (Ty-2) FLA456 LSD (0.05)

3.0 1.6

3.6 2.3

3.0 2.8

3.0 3.0

3.0 3.0

3.0 1.2

0.0 0.7

0.5 -

1.2 0.5

1.4 0.3

1.0 0.2

1.1 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)

FLA456 Senegal

Indonesia

Taiwan

FLA456

CLN1466J

El Salvador

H24 (Ty-2)

FLA456

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 0.0 cM

123.4 cM

87.1 cM

0.0 cM

3.0 cM

83.5 cM

Kadirvel et al. 2012

23.9 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

Classification system proposed by Fegan and Prior (2005)

‘Species’

Phylotype

“Ralstonia solanacearum species complex” Phylotype I

Phylotype II

“Asia”

“America”

Phylotype III Phylotype IV “Indonesia” “Africa”

Specific PCR

ITS Multiplex PCR

Sequevars

Phylogeny/ specific PCR

Clonal Lines

PFGE/rep-PCR/ AFLP etc. Courtesy of P. Prior

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 11.522.2% 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 Taiwan/Pss4 I ++ ++ --Thailand-CM I ++ ++ ---

Bwr-6 ++ -++ -++ -++ --

%wilt 27.3 a 46.0 b 70.6 c 83.2 d 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 SprSum

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

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

susceptible check

90

W

W

W

W

W

L390

w% SLM12-9

SLM12-8

SLM12-10 SLM12-12 SLM12-2

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

Courtesy JF Wang

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)

7e

-

-

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 CLN3125A-23

CLN3125L

NIL Group

Marker

% Survival

SLM12-2

SLM12-10

Resistant (n=22)

R

R

34 a

Susceptible (n=19)

S

S

7b

CLN3125A-23 (n=1)

H

H

26 ab

Resistant (n=22)

R

R

34 a

Susceptible (n=19)

S

S

11 b

CLN312L (n=1)

H

H

6b

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. • Crossovers detected between SLM12-2 and SLM12-10 in 2011 • MAS applied to create additional NIL groups Line CLN3125A-23

CLN3125L

NIL Group

Marker

% survival

SLM12-2

SLM12-10

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

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

Selected Entries Contrasting for BW Resistance within NIL groups. Line CLN3125A-23

CLN3125L

PCR Code

Marker

% survival

SLM12-2

SLM12-10

5S5

R

R

65

5S12

R

S

67

5S41

R

S

5

5U21

S

R

82

5T23

S

R

6

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

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