Manipulation of Bolting Time in Lettuce
A. Massiah, J. Taylor, A. Abbott, M. Kerr and S. Jackson
Life Sciences, University of Warwick, Wellesbourne, Warwickshire, CV35 9EF UK
1. Introduction
5. Late bolting phenotypes in Larissa lines are robust
•
• Late bolting phenotypes are robust over a range of temperatures (Fig.
4A) and when plants are grown under different field conditions (Fig. 4B)
A
Average number of days
to bolt
•
Lettuce is an important crop species in the UK
with ~ 6000 hectares grown per year and a home
production value of £84.7 million in 2009
Bolting causes changes in the production of
secondary metabolites, which can be detrimental to
the crop. The timing of bolting, or flowering, is an
important factor since it affects crop quality and
yield (Fig. 1)
160
140
120
100
80
60
LAR WT
40
20
LINE 307
Figure 4 – Bolting times of
late bolting Larissa lines 2,
164 and 307 compared to
wild type
LINE 2
LINE 164
18⁰C
21⁰C
23⁰C
(A) Plants grown under
controlled environment
conditions at specific
temperatures
25⁰C
Temperature
•
Figure 1 – Lettuce
bolting in the field
Bolting is promoted by high temperatures
B
Average number of
days to bolt
•
Several genes involved in regulation of flowering
time in Arabidopsis thaliana are conserved in other
plant species
Wellesbourne I
Wellesbourne II
Spalding
Spring 2010
Summer 2010
Summer 2010
80
70
50
70
(B) Plants grown under
different field conditions at
Wellesbourne, Warwickshire
and Spalding, Lincolnshire.
60
50
60
40
50
30
40
LAR
WT
164
2
307
30
LAR
WT
Late-bolting line
164
2
307
LAR
WT
Late-bolting line
164
2
307
Late-bolting line
2. Research Aim
To increase the ‘holding ability’ of lettuce in the field by identifying alleles
that delay bolting/flowering
3. Research strategy
•
•
•
•
6. Target lettuce flowering time genes isolated
•
Screen chemically (EMS) mutagenised lettuce populations to identify
lines with delayed bolting
Identify lettuce flowering time genes (based on Arabidopsis) and look for
mutations in these genes in late bolting lines
Utilise high-throughput sequencing technologies to identify mutations in
unknown genes in late bolting lines
Back-cross to clean up useful alleles in near-commercial lettuce
varieties to enable breeding for plants with greater ‘holding ability’ in the
field
Key genes involved in the regulation of flowering time in Arabidopsis
thaliana have been identified and homologues isolated from lettuce
(Fig. 5)
Photoreceptors
Ambient
temperature
Circadian
clock
Photoperiodic
pathway
Light
quality
FKF1
FRI
Vernalisation
CDF
FLC
CO
FT
SOC1
FLK, FVE, LD, FY
FLD, FCA, FPA
Autonomous
induction
Gibberellins
Meristem identity genes
4. Chemical mutagenesis generates late bolting lines in
L. sativa cultivars Larissa and Saladin
Late bolting phenotypes are observed in a Larissa EMS-mutagenised
population (Fig. 2). Four late bolting lines are being characterised from
a total population of 62 lines carrying EMS-induced mutations
Bolted
after 8
weeks
Bolted
after 11
weeks
Figure 5 – Key pathways regulating flowering in Arabidopsis thaliana. Red circles indicate
target genes isolated from lettuce. Four FLC-like genes have been isolated, called LsFLClike GENES 1 - 4
7. Lettuce target genes regulate flowering time in
Arabidopsis thaliana
Bolted
after 16
weeks
Distribution of bolting times - Larissa Population
No. of plants bolting
•
FLOWERING
• Lettuce FLOWERING LOCUS T (LsFT) promotes flowering (Fig. 6)
160
140
120
100
80
60
40
20
0
Figure 6 – Flowering times of
plants grown under long days
25
20
8
9
10
11
12
13
14
15
Weeks after sowing
ft-1, Arabidopsis late-flowering mutant
lacking FT
15
16
10
ft-1:AtFT and ft-1:LsFT, early flowering
T1 transgenic ft-1 lines over-expressing
Arabidopsis and lettuce FT respectively
5
wild type bolting time
0
•
GENE 2
Thirty nine late bolting lines have been identified in a Saladin EMSmutagenised population totalling 1,119 lines. A subset is shown in Fig. 3
Bolting times – Saladin lines
200
Days to Bolt
180
flc3 mutant
early
wild
type
160
late
140
flc-3 : LsFLC GENE 2
120
100
100
A
80
80
flc-3, early flowering
Arabidopsis mutant lacking
FLC
60
60
40
40
20
20
0
0
Figure 7 – Flowering
times of plants grown
under long days
flc-3 : LsFLC GENE 2 T1 Lines
Line
flc-3:LsFLC GENE 2, T1
transgenic flc-3 lines overexpressing lettuce FLC GENE
2
100
80
6. Molecular analyses to identify the mutations
M2_88
M2_10
M2_117
M2_138
M2_18
M2_169
M2_126
M2_100
M2_157
M2_183
M2_121
M2_173
M2_44
M2_124
M2_29
M2_159
M2_150
M2_122
M2_168
M2_161
M2_53
M2_148
M2_133
M2_8
M2_171
M2_86
M2_118
M2_70
M2_52
M2_49
60
Line
Figure 3 – Bolting times observed in selected individual M2 plants of Saladin lines carrying
EMS-induced mutations
•
• Lettuce FLOWERING LOCUS C (LsFLC)- like genes repress flowering
(Fig. 7)
Roseteleaf
leafnumber
number at flowering
flowering
Rosette
Figure 2 – Range of bolting times observed in Larissa plants carrying EMS-induced
mutations. Bolting times assessed in 12 M2 plants per individual M1 line
flc3 null
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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41
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46
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48
Larissa plants 14 weeks after sowing
Late bolting lines have been back-crossed twice to wild type, to reduce
background mutations, and homozygous plants have been identified
causing the late bolting phenotypes
The following have been carried out or are under consideration to detect the causative
mutations:
• PCR-based screening for mutations in target gene sequences, including upstream
regulatory regions
• Transcriptome sequencing
• RNASeq expression analysis
• Whole genome sequencing