Genome-Wide Association and Fine Mapping
of Genetic Loci Predisposing to Water Use
Efficiency in Brassica oleracea (L.)
Sajjad Awan1, Miriam Gifford1, John Hammond2 and Andrew Thompson3
1. University of Warwick, University of Warwick; 2. University of Western Australia; 3. Cranfield University
Introduction
 Rapid increase in human population has put extreme pressure on agricultural resources especially irrigation water and threatens the food security
Brassicas are important vegetable crops grown in the UK for edible oil/vegetables and face periodic water shortages in the UK especially during winter
The Current project focuses on locating genes responsible for water use efficiency (WUE) in these plants without the use of transgenic technology. It was
achieved as follow;
The double haploid A12 (Chinese kale) and GD33 (broccoli) cross pollinated to obtain a mapping population in the ‘C’ genome (Brassica oleracea)
The QTL for WUE was found on Chr-7; evidence from substitution line 118 (BC1F1) shown in Figure.1
The BC1F3 (selfing BC1F2)population showed reduction in WUE (Figure.3) due to introgression of GD33 on A12 on Chr-1 & 7. Fine mapping was carried out by
using SNP markers (KASPar).
 The RFLP and SSR markers were used to genotype the
introgressed regions (Fig.1)
sora93
BRAS023
A48350
FITO302
Na12F03
BRAS019
Ca72
LG C7
Marker Position
0.6
1.1
1.6
2.8
2.8
2.8
3.3
3.5
2.2
Marker
M1
M2
M3
M4
M5
M6
M7
M8
M9
11
13
14
14
14
14
17
17
17
17
18
19
24
27
29
30
Marker score
BAT070-02
BC1F3
T:T
C:C
T:T
G:G
T:T
A:A
A:A
C:C
G:G
A:A
C:C
A:A
A:A
T:T
G:G
A:A
G:G
C:C
A:A
T:T
T:T
C:C
C:C
T:T
G:G
C:C
T:T
BAT090-04
BC1F4
T:T
C:C
T:T
G:G
T:T
-
A:A
C:C
-
A:A
C:C
A:A
A:A
-
G:G
A:A
A:A
A:A
G:G
C:C
A:A
T:T
T:T
C:C
G:G
C:C
T:T
BAT075-06
BC1F3
T:T
C:C
T:T
G:G
T:T
A:A
A:A
C:C
C:C
G:G
T:T
G:G
G:G
C:C
A:A
G:G
-
A:A
G:G
C:C
A:A
T:T
T:T
C:C
G:G
C:C
T:T
G
G
A
A
A
A
A
A
A
BAT011
SL118-11_H9 A
A
A/H
G
H
H
G/H
H
H
H
BAT047
BAT074-05
BC1F3
A:A
C:T
C:C
T:T
C:C
G:G
A:A
C:C
G:G
A:A
C:C
A:A
A:A
T:T
G:G
A:A
G:G
C:C
-
T:T
T:T
C:C
C:C
T:T
G:G
C:C
T:T
SL118-11_C7 A
A
A/H
H
G
G
G/H
H
A
A
BAT048
BAT089-06
BC1F4
A:A
C:T
C:C
T:T
C:C
G:G
C:C
C:T
C:C
A:A
C:C
A:A
A:A
T:T
G:G
A:A
G:G
C:C
A:A
T:T
T:T
C:C
C:C
T:T
G:G
C:C
T:T
SL118-11_G3 A
A
A/H
A
G
G
G/H
G
G
G
BAT056
BAT074-09
BC1F3
A:A
C:T
C:C
T:T
C:C
G:G
A:C
C:T
-
A:G
C:T
A:G
A:G
T:C
G:G
A:A
G:G
C:C
A:A
T:T
T:T
C:C
C:C
T:T
G:G
C:C
T:T
SL118-8_B1 A
A
A/H
A
H
H
G/H
H
H
G
BAT057
BAT075-03
BC1F3
A:A
C:T
C:C
T:T
C:C
G:G
C:C
C:T
C:C
G:G
T:T
G:G
G:G
C:C
A:A
G:G
A:A
A:A
G:G
C:C
A:A
C:C
C:C
T:T
G:G
C:C
T:T
SL118-8_C11 A
A/H
H
T:T
G:G
T:T
A:A
A:A
C:C
G:G
A:A
C:C
A:A
A:A
T:T
G:G
A:A
G:G
C:C
A:A
T:T
T:T
C:C
C:C
T:T
G:G
C:C
T:T
G
G
BAT058
BC1F1
C:C
G/H
H
T:T
A
H
AGSL118
A
A12
Recurrent
Parent
A:A
C:T
C:C
T:T
C:C
G:G
C:C
C:T
C:C
G:G
T:T
-
G:G
C:C
A:A
G:G
A:A
A:A
G:G
C:C
-
T:T
T:T
C:C
G:G
C:C
T:T
1
1
1
1
1
1
2
2
3
4
4
4
4
4
5
5
6
6
6
6
6
7
7
7
SL118-10_C1 A
A
A/H
A
H
G
G/H
G
G
G
BAT059
A
A
A/H
A
G
G
G/H
G
G
H
BAT060
SL118-8_C1 A
A
A/H
A
G
G
G/H
G
H
H
BAT061
SL118-9_D6 A
A
A/H
A
H
H
G/H
H
H
A
BAT068
SL118-11_E8 A
A
A/H
A
H
H
G/H
H
H
A
BAT069
SL118-8_B3 G
G
G
A
G
G
G/H
G
G
G
BAT70
SL118-10_G6 G
SL118-11_C6 G
G
G
A/H
G
A
A
H
H
H
H
G/H
G/H
H
H
H
H
H
H
BAT071
BAT072
BINS
Fig 1. Genotyping results matrix for BC1F2 lines (left Panel) and BC1F3/BC1F4 lines (upper panel) derived from
AGSL118 x A12DHd. Dark green cell colour denotes a GD33 allele; Yellow cell colour denotes an A12 allele; H
denotes both parent alleles detected i.e. heterozygous result. A/H or G/H indicates that the marker result is
partially ambiguous; - denotes a missing value. The 6 SSR markers were used on Linkage group-7 to select
BC1F3 lines. The BC1F3/BC1F4 lines originated from selfing of BC1F2 lines preselected by using SSR/SNP markers.
Upper panel shows the genotyping results from KASPar markers in BC1F3/BC1F4 lines.
 Introgression of GD33 on Chr-7 significantly increased the gs
(Figure-2) and reduced WUE (Figure-3)
 Selected lines were self pollinated to achieve homozygous
BC1F3/BC1F4 population
 The Selected lines were genotyped using KASPar markers on
LG-07 only (Fig.1, lower panel)
 Statistical analysis of BINS in Fig.1 revealed significant
association of BIN-3 and BIN-7 with WUEp
 Genes present in the selected locus will be identified and
knockout mutant will be produced to confirm the ‘selected’
genes function.
Fig ure.2. Glasshouse characterisation for plant
stomatal conductance in selected BC1F3 lines. BAT70
reveals a significantly higher stomatal conductance
compared to recurrent parent (A12).
Fig ure.3. Glasshouse characterisation for water use
efficiency in selected BC1F3 lines. BAT70 reveals a
significantly lower WUE compared to recurrent parent.
Genome Wide Association Mapping
 96 ecotypes from Arabidopsis were used in the analysis
 SNPs with relatively high ‘diff’ value and OR low ‘P-value’ (<0.0005) were plotted to
find the clustering hits (Figure.4)
 Using ‘R’ and ‘Virtual Plant’ genes were selected associated with the SNPs (Circled in
Fig.4).
Figure.4. Selection of putative SNPs involved
in controlling water use efficiency in
Arabidopsis.
Figure.5. Polymorphism in Beta
Glucosidase Gene. Green Nucleotides
represent confirmed SNPs, red
‘rejected SNPs’
32
M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27
Genotype Pedigree
Backcross
Progeny
14
SL118-8_A6 G
SL118-8_E6
 Three genotypes were selected to analyse the effect of
introgression of GD33 on plant stomatal conductance (gs) and
WUE
At3g01180
Sample
Name
Ni4B10
Fine mapping the introgressed regions in
A12DHd x SL118 progeny
At3g03380
LG
LG C1 C6
 The PCR products from the selected genes were sequenced for polymorphism (Fig.5)
Conclusion
 Use of recent technologies such as SNP based assays (KASPar) has made it possible for efficient genotyping on large scale. This technology can be used to find
QTL responsible for nitrogen and phosphorus use efficiency in relatively short period of time.
 The orthologues controlling the WUE in arabidopsis can be identified with the availability of well annotated whole genome sequence of Brassica oleracea .
 The approaches implemented in the current research project can also be used in other important crops such as oil seed rapes, wheat , soybean and maize.
Warwick Crop Centre
www.warwick.ac.uk/go/wcc