Molecular Breeding in Vegetable Crops- Challenges and ... - icrisat

Molecular Breeding in Vegetable Crops-
Challenges and Opportunities

India is the second largest producer of
vegetables in the world….
Per capita consumption of vegetables is about 174 g/day against
300 g/day
Vitamin A and iron intake is inadequate in most states of the
country
Productivity of vegetables is also lower (12.7 t/ha) than average
productivity of the world (16.9 t/ha)
India accounts for one-third of all pesticide poisoning cases in the
world and 50-60% of vegetables are contaminated with insecticide
residues
Poor availability of good varieties and lack of improved production
technologies
AVRDC-Regional Center for South Asia

Research and Development: Four global themes
Germplasm
Germplasm conservation,
evaluation and gene discovery
Breeding
Production
Consumption
Genetic enhancement, varietal
development, selection of
indigenous lines, seed production
Safe and sustainable vegetable
production systems
Postharvest management and
market opportunities; nutritional
security, diet diversification and
human health
Slide 1 (06/2010)
www.avrdc.org

Crops on Focus
Bulb
Allium
Tomato
Pepper
Cucurbits
Vegetable
soybean
Mungbean
Crucifer

MAS for Tomato Yellow Leaf Curl
Virus Resistance in tomato

TYLCVD - a major challenge for tomato
production
• Tomato Yellow Leaf Curl Virus Disease (TYLCVD)
devastates tomato in the tropics, subtropics
• 100% yield loss from early infection
Peter Hanson

Diversity of Tomato yellow leaf curl virus
100% 90% 80% 70% 60% 50% 40%
30%
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TbLCJV JR
ToLCJV ID
AYVV CN
ToLCDaV PH p162
ToLCLV LA
ToLCMYV MY
PaLCuCNV CN
ToLCKHV KH
ToLCV LA LA2
TbLCYuV CN Y161
ToLCVV VN
ToLCCNV CN G32
ToLCGuV CN G2
TYLCGuV CN G3
ToLCTWV TW Tainan
ToLCPV PH
ToLCHsV TW Hsinchu
ToLCBDV BD
TbCSV CN Y41
ToLCPK PK
ToLCGV IN Vadodara
ToLCGV IN Varanasi
ToLCGV NP
ToLCIRV IR
ToLCKV IN Karnataka
TYLCTHV MM
TYLCTHV CN Y72
TYLCTHV TW
TYLCCNV CN
ToLCSLV LK fsl1
ToLCBV IN
ToLCV AU
TYLCV JP
TYLCV TR Tu6
TYLCV IL
TYLCV IR
TYLCMLV ML
TYLCMLV ET ET4
TYLCMalV ES Malaga
ToLCSDV SD Shambat
ToLCYTV YT
ToLCUGV UG ug11uc8
ToLCTZV TZ TT11
TYLCSV IT
ToCSV ZA
ToLCMLV ML
ToLCNDV PK
ToLCNDV IN ND mild
ToLCIDV ID Lembang
TYLCKaV TH Kanchanaburi
TYLCKaV VN
PepYLCIDV ID
ToMoTV CU
ToMoV USA Florida
CdTV IC
CdTV MX H6
ToLCSinV NI Santa Lucia
ToMHV CU Qui
ToRMV BR Ube
ToSRV BR
ToCMoV BR
TGMV YV
ToMLCV VE
TYMLCV VE
ToChLPV MX
ToGMoV GT
WDV
AVRDC- Virology
based on complete
DNA-A sequences
● = bipartite
Asia +Australia
Africa
Europe
Americas

Sources of resistance to TYLCD found only in
the wild species
S. habrochaites
S. chilense
S. peruvianum

Mapped Ty Resistance Genes in Tomato
Chromosome 6 Chromosome 11 Chromosome 3 Chromosome 4
Ty-1
Ty-3
Ty-5
Ty-4
Ty-2
Peter Hanson

MAS helps for early selection of TYLCV
resistant lines
Peter Hanson

MAS in tomato breeding lines
Ty-2 check
variety
Ty-3 check
variety
Ty-1 check
variety

MAS in Pedigree Breeding Programs
Parent 1 x Parent 2
MAS
F1
Selection unit Selection criteria Evaluation
MAS
F2
F3
F6
Single plant
Progeny row
Disease
resistance
Fruit-set (yield)
Fruit size,
Fruit shape,
Fruit firmness
Disease resistance:
Begomovirus
Bacterial wilt, others
MAS
F7/F8
Yield trials
Homozygous
“pure lines”
Nutrition:
lycopene, β-carotene,
vitamin C, flavonoids
Quality:
solids, color
Peter Hanson

2009 Spring Preliminary Yield Trial in
• To determine which combinations of
Ty-1, Ty-2, Ty-3 offer high and stable
begomovirus resistance
Taiwan
• 24 F 6 lines that together represent
most combinations of Ty-1, Ty-2, Ty-3
• Seedlings exposed to Taiwan
begomoviruses (ToLCTWV) and
(TYLCTHV)
• Transplanted to field after exposure, 2
reps in RCBD, 30 plants per plot
• Evaluation of symptom severity 40
and 75 DAT
Peter Hanson
• PCR testing of 4 plants per plot for
each begomovirus 40 DAT

Ty genes-pyramided breeding lines
Ty-1+ Ty-2
Ty-1+ Ty-2 + Ty-3
Ty-1 + Ty-3
Peter Hanson
No Ty genes

Group Mean Comparisons of TY lines, Taiwan
TY Genotype
No.
entries
Mean TY
severity score
Mean MY
(t/ha)
Ty-1+Ty-2+Ty-3 9 0.8 a 38.7 a
Ty-1+Ty-3 4 0.9 a 41.8 a
Ty-1+Ty-2 2 1.6 b 37.0 a
Ty-2 7 2.1 c 25.4 b
Susceptible 2 3.0 d 3.5 c
Severe (=3) Moderate (=2) Slight (=1)
Peter Hanson

AVRDC Multiple TY F 6 Lines
CLN3125G
Ty-1+Ty-2+Ty-3
CLN3078C
Ty-2+Ty-3
CLN3079C
Ty-2+Ty-3
Peter Hanson

Exploring new sources of TYLCV resistance
genes
Resistance to
TYLCV in FLA456
appears to be
recessive

Mapping in RILs
Phenotyping
A set of 167 F6 Recombinant Inbred Lines
(RILs) were evaluated for TYLCV resistance
Two weeks exposure in virus
screenhouse
Follow up of symptom
development after transplanting

A significant QTL mapped on chromosome 4
Composite Interval Mapping
Ty5
tyQTL4.1
LOD – 7.6 (2)
R 2 – 20%
Qgene 4.3.7
Joehanes & Nelson (2008)

A minor QTL for TYLCV detected on chromosome 6
Mi-1
Ty-1
Ty-3
tyQTL6.1
LOD – 2.2 (2)
R 2 – 6%
Qgene 4.3.7
Joehanes & Nelson (2008)

MAS for tyQTL4.1(t) in the tomato breeding lines
derived from FLA456
300 bp
250 bp
200 bp
SSR43 marker helps to predict resistant or susceptible progenies
AVRDC-Tomato Breeding

AVRDC Tomato Disease Marker
Protocols (current)
Disease Genes (symbol) Protocol type
Begomoviruses (TYLCVD) Ty-1, Ty-2, Ty-3, Ty-5 PCR
Root-knot nematode Mi PCR
Fusarium wilt I2 PCR
Late blight Ph-3 PCR
Gray leaf spot Sm CAPS
Tomato mosaic virus Tm2a hypocotyl color (ah
linked to Tm2a)
Peter Hanson

Introgressions from wild species –
0.0 cM T1928
6.0 cM
8.0 cM
10.0 cM
Mi-1
Ty-1
C2_At4g01900, TG97
cLET-5-A4
Linkage Drag
Tomato DNA
• Ty-1/Ty-3 resistance in AVRDC
lines originated from Univ. Florida
(Jay Scott)
16.0 cM
T1563
• Ty-1 and Ty-3 part of a large 32 cM
region from S. chilense
20.0 cM
cLEG-31-P16
Wild DNA
• AVRDC lines carrying Ty-1+Ty-3
likely carry entire introgression
27.0 cM
Ty-3
C2_At4g27700
• Lines homozygous for this region
may show some reduced fruit and
tendency for puffy fruit
32.0 cM
T0834
Tomato DNA
Peter Hanson

Problem: linkage of Ty-2 and i-2 +
(FW susceptibility)
77.0 cM
82.0 cM
82.5 cM
84.0 cM
87.0 cM
89.0 cM
TG546
cLET-5-E4
C2_At1g07960
TG36
cLEN-11-F24
T0302
Ty-2
Wild DNA
• Ty-2 linked to i-2 + form of the gene
(allele) which conditions
susceptibility to FW (repulsion
linkage)
• Use of Ty-2 brings along i-2 + for
FW susceptibility, a win-lose
situation
91.5 cM
i-2 +
• Crossovers between Ty-2 and I-2
are rare but occur.
• Objective: identify rare Ty-2/I-2
recombinant plants and make a
win-win situation
103.0 cM
TG393
Peter Hanson

Linkage of Ty-3 and mi-1+ (susceptibility)
0.0 cM T1928
6.0 cM
8.0 cM
10.0 cM
Tomato DNA
Mi-1 +
C2_At4g01900, TG97
cLET-5-A4
Ty-1
16.0 cM
S. chilense DNA
T1563
Mi-1 mi-1 +
20.0 cM
27.0 cM
32.0 cM
cLEG-31-P16
C2_At4g27700
T0834
Ty-3
• Mi-1 gene conditions resistance to the
root-knot nematode (Meloidogyne
incognita)
• Ty-1/Ty-3 resistance linked to the allele
for susceptibility (mi-1 + )
• Identify lines with Ty-3 and Mi-1
• Loci are over 20 cM apart
Peter Hanson

Traits and Genes of Interest for Future MAS
Trait
Bacterial wilt resistance
Late blight
Gene/QTL mapping
progress
Important genomic
regions identified
Ph-2, Ph-4 genes
mapped
Action required
/challenge
Lack of polymorphic
markers
Identify linked
polymorphic markers
Fusarium wilt (race 3), I-3 Marker probably
available in literature
High lycopene
High T°fruit-set (heat
tolerance)
Drought
hp-1, og c genes mapped
in literature
Ongoing evaluation of
mapping population
Important genomic
regions identified
Salinity ? ?
Peter Hanson
Identify linked
polymorphic markers
Lack of polymorphic
markers
?

Mapping of Bacterial Wilt Resistance QTLs in
Tomato
Rhizoctonia solanaceraum

Jaw-fen Wang
BW QTL on Chromosome 6

Jaw-fen Wang
BW QTL on Chromosome 12

Mapping of Heat Tolerance
CLN1621L x CA4 – Recombinant Inbred population (F5)
(96 lines)
AVRDC-Tomato Breeding

Screening for heat tolerance in RILs – Yield
components and pollen fertility under heat stress
Plant height
Days to flowering
Days to maturity
Flower number
Fruit number
Fruit set (%)
Fruit size
Fruit weight
Yield
Seed number
Seed weight
Dorthe Mussman and Joyce Yen

QTLs associated with heat tolerance in tomato
Fruit set (%)
Fruit load
Pollen viability (%)
Composite Interval Mapping
Chr QTL LOD Variance (%) Additive effect Donor parent
6 TES0111-SLM6-5 2.3 10.6 9.12 CLN1621L
12 SLM12-31-SLM12-50 2.6 13.0 5.81 CLN1621L

Mapping Drought Tolerance
Mapping population
Backcross inbred
lines (BC1F3:4) of
CLN2498E x LA1579
fixed with Ty-2 locus
(96 lines)
S. pimpinellifolium
Phenotyping
• Plant height
• Days to flowering
• Days to maturity
• Fruit set
• Fruit yield
• Fruit weight
• Chlorophyll content
• Shoot dry weight
Genotyping

Phenotyping: Backcross Inbred Lines
Screening in rain-out shelters
Drought stress
Control
Measuring physiological and yield traits under stress and non-stress
conditions
Rachael Symonds and Joyce Yen

QTLs associated with yield-related traits under drought
stress and control experiments (greenhouse)
Jean Lin and Joyce Yen
Dreb1a mapped to Chr. 6
and associate with yield
under drought stress

Jean Lin and Joyce Yen
Putative QTLs co-localize
with reported wild
introgressions contributing
drought tolerance traits

Association mapping for salinity
tolerance traits in tomato
Dr E. Sreenivasa Rao, BOYSCAST Fellow, IIHR,
Bangalore

Salt stress affects yield components in
tomato
Arka Meghali
• Progressive
salt stress
until
maturity
• Effect on
physiologic
al and yield
traits
Control 50 mM 100 mM 200 mM NaCl
Vincent Ezin

Salt tolerance exists in tomato germplasm
LA1606 -
S. pimpinellifolium
Vincent Ezin
Control 50 mM 100 mM 200 mM

Discovering desirable alleles in germplasm:
Allele mining through Association Mapping
SNP 1
SNP 2
SNP 3
INDEL1
INDEL2
0 1300 bp
SNP 4
SNP 5
SNP 6
SNP 7
Acc. 354 361 506 752 831 1017 1018 1068 1183 1189 Na
1 A G 0 0 T T G A T C 4867
2 A T 0 0 T T G A T C 3300
3 A G 0 0 T T G A T C 3450
4 A T 0 0 T T G A T C 4267
5 A T 0 0 T T G A T C 9200
7 T G 3 0 C C T T C A 4967
10 A T 0 0 C C G T C A 6200
12 A T 0 0 C C G T C C 3100
15 T G 3 0 C C T T C A 4067
17 T G 3 0 C C T T C A 3900
20 T G ? 0 C C G T C A 4050
21 T G ? 0 C C G T C A 3767
25 A T 0 3 T T G A T C 3150
64 A T 0 3 T T G A T C 3500
69 A T 0 3 T T G A T C 2867
73 A T 0 3 T T G A T C 3050
75 A T 0 3 T T G A T C 3800
76 A G 0 3 T T G A T C 1767
115 T G ? 3 T C T T ? C 1900
An effective allele of NHX1 gene,
contributing to reduced sodium
accumulation (about 430 ppm)
identified in S. pimpinellifolium
collection
176 A T 0 3 T C T A C C 4650 P=0.0185 R 2 = 6.75%
Sreenivasa Rao
SNP 8
Tolerant
Sensitive

A Dreb1a allele associate with various salt tolerance
traits in S. pimpinellifolium collection
SNP 1
0 1020 bp
SNP 2
INDEL1
SNP 3
INDEL2
Accession 346 504 540 906 969 1003
1 G G 1 T 0 1
101 A T 1 C 0 1
105 G G 1 T 1 1
106 G G 0 T 0 1
107 G G 1 T 0 0
108 G G 1 T 0 0
109 G G 1 T 0 1
11 A T 1 T 0 0
110 G G 1 C 0 1
113 A T 1 C 0 0
115 A G 0 T 1 0
119 A T 1 T 0 0
122 G G 1 T 0 0
124 G G 1 T 0 1
126 G G 1 ? ? ?
131 A T 1 T 0 0
132 A T 1 C 0 0
INDEL3
Traits
Trait R 2 Actual
Leaf chlorophyll content
(P=0.038) 5.91% 5.30%
Leaf sodium concentration
(P=0.0057)
Leaf potassium content
(P=0.019)
10.39% -769.5ppm
7.04% +790ppm
Survival score
(P=0.061) 4.34% +0.55
Shoot dry weight
reduction
(P=0.01) 7.05% +10.88%
Fruit number
(P=0.07) 3.46% +16.46
Fruit yield
(P=0.03) 6.15% +7.94g
Sreenivasa Rao

Next step:
NHX1
Accession 1 Dreb1a Accession 2
0 1300
0 1020
752bp
1003bp
Pyramiding of desirable alleles on different genes
NHX1
0 1300
752bp
+
Dreb1
0 1020
1003bp
Would take 6-7 years for 2 loci

Are tailor-made combinations already available in the
germplasm ?
Accession 3
0
NHX1
1300
+
Dreb1a
0 1020
752bp
1003bp
Genotype NHX1-752bp Dreb -1003bp
2 0 0
4 0 0
7 0 0
11 0 0
15 0 0
17 0 0
20 0 0
21 0 0
22 3 ?
25 3 1
76 3 0 Better allele

Mapping Anthracnose resistance locus from
Capsicum chinense
Molecular markers for anthracnose resistance
Resistant
Susceptible
Hot pepper
susceptible to
anthracnose
CAPS marker was developed
from AFLP marker and genetic
mapping is in progress
Anthracnoseresistant
hot peppers
Vivian Wang, Chien-an Liu, Paul Gniffkke, Hayde Galvez

Identification of bruchid resistance locus from Vigna radiata
ssp sublobata for mungbean breeding
LG 5
13.4
5.6
3.1
17.5
m4pcc585
mg5att46
mg5pat46
m3pca400
m4pcc417
m4pcc579
No. of lines
R
Huei-mei Chen
% of seed damage
C. chinensis (L.)
S
1.4
2.4
1.2
1.2
11.1
3.4
1
1.4
1.6
17.8
1.2
2.6
7.5
CAPS markers
w02s12
w02s6
mg3pag43
mg3ag431
m1pgg256
w02a4
w02s9
mg3pag42
w02s10
w02s11
w02s2
w02s4
w02s3
m5pca598
M13PAG33
mg4pga29
m3pca283
m1pgg258
mg7pcg22
m3pca314
u168a6
u223a7
v02a4
v02a3
m5pca382
m4pcg370
m9pca371

Developing molecular markers
for vegetable breeding -
Public-Private Partnership

Consortia to develop DNA markers
• Marker resources are not
available to many
companies
• Marker development cost is
high
• Sharing of cost to develop
marker resources
• Initially focus on SSRs
SSR
SNP

Genetic linkage map of tomato based on SSR markers – CLN2498E x
LA1940 (S. pennellii)
CH1
CH2
CH3
CH4
CH5
CH6
18.5
19.7
28.0
28.2
29.0
31.2
31.7
31.8
32.0
32.4
32.8
33.2
33.4
33.7
34.0
34.3
34.4
34.5
34.9
35.5
36.5
37.8
38.6
42.4
45.5
46.0
46.7
51.6
55.6
88.0
93.1
104.0
113.8
115.7
123.1
124.8
129.3
132.2
139.4
144.0
149.5
150.0
165.0
TM1034
TM76
TM1040
TM172
TM1042 TM1043
TM1045 TM1046
TM1047
TM1049 TM1050
TM1051 TM1052
TM1053
TM1055 TM1057
TM12
TM9
TM5
TM339
TM415 TM449
TM340
TM597
TM77 TM330
TM336 TM311
TM56 TM377
TM365
TM373
TM245
TM152
TM82
TM483
TM100
TM131
TM290
TM1058 TM1059
TM89
TM198
TM272
TM224 TM1073
TM1074
TM99
TM1061 TM1062
TM144
TM1075 TM1076
TM491
TM164
TM1068 TM1077
TM74
TM106
TM1079
TM319
TM1080
TM1070 TM1071
TM1072
13.0
15.2
18.0
18.7
20.2
27.0
29.3
32.3
36.0
36.5
36.6
37.0
39.0
42.0
49.7
52.6
59.8
67.9
70.0
72.5
73.0
75.3
92.0
92.1
111.0
120.0
120.7
126.5
139.5
141.0
TM1082 TM1084
TM133 TM165
TM1112
TM212
TM352
TM1113
TM206
TM3
TM1087
TM1088 TM1090
TM1092
TM1093 TM1094
TM1095
TM112
TM1096 TM1097
TM188
TM405
TM533
TM284
TM1099
TM1100 TM1101
TM1116
TM210
TM1117
TM214
TM1119 TM1120
TM1102 TM1103
TM1104
TM221
TM1106 TM1107
TM1121 TM1122
TM1108 TM1109
TM1110
0.0
4.6
30.0
54.5
58.0
59.0
60.3
62.7
63.2
68.7
69.5
69.8
70.0
70.4
70.5
70.6
72.0
72.8
73.7
75.6
77.0
78.0
81.6
91.9
92.0
93.0
96.0
105.5
110.0
126.9
128.4
131.8
133.0
141.0
160.0
TM1123 TM1124
TM1150
TM1127 TM1128
TM1130 TM1131
TM1132
TM1133 TM1134
TM1135 TM1136
TM1138
TM92
TM494
TM539
TM14
TM521
TM480
TM127
TM387
TM492
TM425 TM314
TM337
TM1139 TM1151
TM80
TM20
TM414
TM1152
TM1141 TM1153
TM475
TM553
TM1142
TM543
TM1154 TM1155
TM216
TM1143
TM286
TM79
TM274
TM1156 TM1157
TM1146 TM1147
TM1148 TM1149
Shu-mei Huang, Vivian Wang, Lucy Lin, Julie Chu
6.5
22.6
22.7
33.0
33.9
41.4
42.7
42.8
43.2
43.4
45.3
45.4
47.9
52.6
55.0
62.5
66.0
81.0
88.3
93.7
119.5
125.0
127.0
137.0
TM1158 TM1159
TM273
TM1186
TM1163 TM1164
TM291
TM318
TM217
TM499
TM81
TM102
TM345 TM338
TM380
TM473
TM341
TM1165 TM1166
TM1167
TM1169 TM1170
TM1171 TM1172
TM75
TM1175 TM1176
TM1177
TM1179 TM1180
TM1187
TM1183
TM1188
TM1189 TM1190
7.0
9.7
16.0
21.0
30.0
37.0
37.2
46.0
51.0
53.9
54.5
55.2
55.9
56.1
56.5
57.1
57.3
59.2
60.0
60.7
84.0
104.0
115.0
119.0
TM1191 TM1192
TM1193 TM1194
TM95
TM1195 TM1196
TM1197 TM1198
TM1199 TM1202
TM1223
TM1203 TM1204
TM1206 TM1208
TM1225 TM1226
TM522
TM1227
TM1228 TM1229
TM1230
TM322
TM2
TM103
TM8
TM530 TM16
TM18
TM582
TM239
TM166
TM525
TM1209
TM454
TM1233
TM1234 TM1235
TM1210 TM1211
TM1236
TM1213 TM1214
TM1216
9.1
18.5
19.8
22.0
22.1
22.3
23.3
30.7
45.0
47.0
47.2
63.0
73.0
74.1
75.2
97.0
TM136
TM1244 TM1245
TM506
TM428
TM261 TM135
TM117 TM138
TM123 TM233
TM58
TM528
TM68
TM1246 TM1247
TM578
TM1248 TM1249
TM1242 TM1243
TM61
TM179
TM1251
AVRDC-APSA
Tomato SSR Marker
Consortium (18
companies)

Conclusion
• Genomes of vegetable crops are being sequenced. Cucumber
Genome (Nature Genetics 41: 1275-1281)
• A large number of DNA markers are discovered…SSRs and SNPs,
high throughput genotyping and phenotyping platforms are
established
• Tools – Linkage mapping, Association Mapping, Family based QTL
mapping, Functional Genomics etc.,
• Diverse crops and diverse problems in the tropics. New challenges
continue to emerge (Tospoviruses in tomato in India)
• Strengthen wide-hybridization programs to introgress genes from
wild species
• Integration of genomics in plant breeding is the way forward
49

Acknowledgements
Robert de la Peña
Peter Hanson
Rachael Symonds
Jaw-fen Wang
Huei-mei Chen
Paul Gniffke
Andreas Ebert
S. Geethanjali
Lawrence Kenyon
Roland Schafleitner
Mr. Chen
Thank you!