Lettuce Crop Improvement Research at
Warwick HRI.
Dr Paul Hand
VeGIN meeting 05 November 2009
General Strategy
Lettuce Crop Improvement
Research
at Warwick HRI
Dr. Paul Hand
• Identify source(s) of variation in the trait
• Develop mapping populations & genetic
maps
• Obtain robust phenotypic data
• Identify QTL & linked markers
• Determine marker assisted selection
strategies
• Fine mapping & identification of genes
‘Warwick HRI’ lettuce diversity set
Genetic Resources
Identifying trait variation
• Lettuce diversity set
• Mapping populations
• Genetic maps
• TILLING population
24 accessions including mapping parents
WHRI lettuce mapping populations
WHRI lettuce mapping populations
• Saladin x Iceberg recombinant inbred population
• >300 F6 recombinant inbred lines (RIL).
• Map construction & phenotypic analysis for QTL discovery
• ~120 most informative lines (MIL) selected for F7 & F8
• F7 map under construction
• Iceberg x L. serriola interspecific F2 population
• Parents from WHRI & U.C. Davis (Michelmore) maps
• Map anchorage: access to EST & SNP markers
• Access wider trait variation
1
Lettuce Crop Improvement Research at
Warwick HRI.
Dr Paul Hand
VeGIN meeting 05 November 2009
Trait examples
Peach-Potato Aphid
Myzus persicae
Downy mildew
Peach-potato
aphid
7000
6000
5000
Bolting
4000
3000
2000
Discolouration
1000
0
2 9 6 22 24 38 1340 39 7 2318 35 37 30 1 31 1110 5 1648 28 47 1541 3234 19 4617 26 8 3 25 33 27 36 1444 2942 45 43 20 21 4 12
Cultivar no
Nitrate content
Mean No. of M. persicae per plant on 240
SSD F6 lettuce lines
80
Mean no. of aphids per plant on the most
resistant & most susceptible lettuce F6 RILs
Most resistant + s.e. Most susceptible + s.e.
70
No. of SSD lines
Nitrate concentration (mg/kg
8000
• Wide host range
• Virus vector
• No resistant lettuce
varieties
• Some partial
resistance
60
50
1.75
0.92 33.5
6.59
40
3.5
1.76 31.5
6.46
30
5.25
1.44 24.25
3.42
5.25
1.89 22.5
5.62
7.0 (Iceberg)
3.19 20.25 (Saladin) 4.96
20
10
0
0-5
6-10 11-15 16-20 21-25 26-30 31-35 36-40
No. of aphids per plant
QTL for M. persicae resistance
SalxIce1
SalxIce2
0
0
20
20
40
ApSur-2
60
ApSur-1
ApSur-4
60
ApRep-5
40
40
60
SalxIce6
ApSur-3
0
20
40
SalxIce5
20
ApRep-1
ApRep-3
0
SalxIce4
ApRep-2
0
20
SalxIce3
0
0
0
20
20
20
40
40
40
60
80
80
100
100
120
Survival =
ApRep-4
80
100
Lettuce nitrate content
SalxIce7 SalxIce8
Background
• Concern over high nitrate levels in the diet
• EU places limits on nitrate in lettuce
– Lower light levels in Northern Europe limit assimilation
– Temporary derogations – until Dec. 2008
• Cultural practices can minimise accumulation
• Can nitrate levels be reduced through breeding?
140
Reproduction =
2
Lettuce Crop Improvement Research at
Warwick HRI.
Dr Paul Hand
VeGIN meeting 05 November 2009
Warwick HRI lettuce diversity set
Nitrate content of 94 F7 mapping
lines – winter experiment
Identifying trait variation
5000
4500
Nitrate (ppm FW)
4000
3500
3000
2500
2000
1500
1000
19
10
51
4
59
37
17
22
12
52
71
76
82
65
43
27
42
38
55
32
30
49
90
74
83
15
14
45
81
88
58
79
62
23
47
25
67
24
95
72
86
28
63
33
26
20
56
6
46
50
11
80
60
64
96
93
35
13
18
92
89
73
21
61
7
48
3
39
75
68
44
8
77
78
2
69
84
57
87
40
53
34
5
1
41
29
36
85
70
0
94
31
91
66
54
9
16
500
Cultivar no
Lettuce Nitrate QTL (2)
Lettuce Nitrate QTL (1)
10
3
c5
27.0
29.2
E51M47_87s
E35M48_347i
E41M47_183s
57.7
E38M54_243s
42.4
E41M62_102s
45.6
E45M48_128i
59.4
E35M47_143i
63.6
E44M48_212s
NitmgWin-3
43.9
E35M47_194i
32.0
33.4
34.5
36.9
E35M47_4>400s
E35M49_2>400i
E54cM48_2>400i
E35M48_377i
NitmgSumm-1
E41M47_66i
E41M50_100i
E41M47_187i
E41M49_183s
NitmgWin-1
34.0
36.5
38.2
40.1
25.4
18.2
E41M59_315i
24.9
E41M62_292i
29.8
32.1
E41M50_166i
E54cM48_126i
36.4
E41M49_90i
c13
0.0
E38M54_270bs
9.5
E60M59_265i
19.3
E35M49_269s
33.7
36.0
37.5
39.0
39.6
40.0
41.3
43.2
46.4
51.1
E35M48_291i
E44M48_335i
E32M47_2a>400s
E55M50_253i
E49M47_308s
E35M47_105i
E41M61_100s
E36M50_166i
E36M50_154s
E41M47_119i
56.5
58.4
E60M59_137i
E44M48_113i
63.2
64.0
E37M59_154i
E37M59_165s
0.0
E38M54_122s
21.9
23.1
25.9
27.8
29.1
31.3
33.5
LsD106_G
E35M49_105i
E32M47_208s
E37M59_250i
E37M59_258s
E41M62_3>400i
E38M54_272ai
43.2
E38M60_272s
NitmgSumm-4
E35M48_1a>400s
E41M50_190s
E35M59_176s
c12
E35M60_2>330i
E44M48_2>400i
E38M60_350s
E45M48_335s
E38M60_218i
E41M61_106s
E41M47_97i
LsA001
E35M49_295s
E41M59_123s
E45M48_112i
E37M59_129i
E41M61_2>400i
E37M59_144s
E35M59_3>330i
E49M47_63i
Dm_5/8
P34M47_140i
E45M48_68i
E33M59_2>330i
E41M62_167s
E41M49_137i
E44M48_297i
E45M48_420s
E41M50_112i
E35M59_240ai
E41M48_190i
E49cM58_1>400s
E41M50_188s
E41M49_213i
E45M48_208s
E35M61_218i
E36M50_318s
E35M59_240bs
E35M49_152i
NitmgSumm-3 NppmSumm-3
21.5
E37M59_88i
5.0
E41M62_300i
0.0
2.3
3.6
4.2
4.8
5.1
5.5
5.6
7.4
9.8
10.5
14.6
15.9
16.5
16.9
17.5
18.9
20.1
21.2
21.5
23.4
24.8
42.3
44.8
45.4
46.4
47.0
47.1
47.2
47.8
48.5
49.4
49.8
51.0
55.2
NppmWin-3
E40M61_168i
E60M59_230s
E49M47_76i
E35M59_22>330i
E41M61_136s
E35M59_2>330i
E41M48_360i
0.0
NppmSumm-2
5.4
6.2
8.5
10.5
11.6
12.8
15.3
E51M47_248i
NppmWin-2
E41M62_1>400s
0.0
NitmgSumm-2
8.1
24.9
E35M49_154i
NppmWin-1
0.0
E41M59_100i
NitmgWin-2
E35M47_150s
3.7
NppmSumm-1
0.0
17.3
c11
c9
Lettuce ESTs related to nitrate metabolism
VeGIN Lettuce Programme
Objectives
• Develop lettuce genetic resources
• Further understand key traits for
sustainable production
3
Lettuce Crop Improvement Research at
Warwick HRI.
Dr Paul Hand
Develop Lettuce Genetic Resources
VeGIN meeting 05 November 2009
Develop Lettuce Genetic Resources
• Map improvement & marker discovery
• Diversity set
– Increase to 96 accessions (domestic & wild)
– Extract & amplify DNA from single plants
• Mapping population
– Produce F8 seed by single seed descent in 2009
Develop Lettuce Genetic Resources
High-throughput genotyping & sequencing
• Current F7 Saladin x Iceberg RIL map
– 140 AFLPs
– 57 SNPs
– 20 ESTs
– 1 cos marker
– 1 gene (seed coat colour: w)
– Total: 219 markers
• 15 linkage groups covering all 9 chromosomes
• Map length of 723.2 cM, covers >80% genome
• All groups link with the integrated lettuce map by Truco et al., 2007.
A lettuce TILLING population
Targeting Induced Local Lesions IN Genomes
• Polymorphic ESTs
– Illumina GoldenGate genotyping of F7 RILs (2 x 384)
• SNP discovery
– Lettuce 6.6 million feature Genechip® (35,000 genes)
M1
M2
• Genome & transcriptome sequencing
– Illumina Genome analyser
• Targets
– 1500 polymorphic SNPs for mapping
– High-density lettuce map anchored to Michelmore
integrated map
– Extensive lettuce genome sequence
From: Henikoff, S. & Comai, L. (2003) Ann. Rev. Plant Biol. 54: 375-401
Effect of EMS on lettuce seed
100
80
80
60
60
40
40
20
20
0
M1 plants yielding
more than 4 seeds (%)
M1 germination (%)
M1 germination and seed yield
100
0
0
0.1
0.2
%EMS v/v
0.3
0.4
Effects of EMS on M1 leaf development & pigmentation
4
Lettuce Crop Improvement Research at
Warwick HRI.
Dr Paul Hand
Lettuce TILLING Populations
VeGIN meeting 05 November 2009
Lettuce TILLING population 2008/09
• Approx 4000 M2 plants sown
• Sampled for DNA extraction & pooling
• Seed collected from 3257 plants
Larissa (65 lines)
Saladin (1,000 lines)
0.1% EMS 18hrs
M0 seed
• Some phenotypes recorded
•Screening for bolting/flowering mutants
M1 seed
M3 seed
M2 seed
M1 plants
M2 plants
Traits for Sustainable Production
Traits for Sustainable Production
Resistance to Nasnovia ribis-nigri
Postharvest Discolouration
• Resistance already
deployed BUT
• New resistancebreaking biotype of N.
ribis-nigri
• Assay diversity set for
resistance
• Fine map main effect QTL
– Linked markers
• Map ESTs of genes in discolouration pathways
– Identify potential target genes through links to QTL
Other linked projects
• Attachment of human pathogens to
salad leaves – BBSRC
• Control of bolting & flowering – BBSRC
& Industry
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