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