Association mapping inforage crop grasses
Leif Skøt
• Potential for high-resolution mapping
• Evaluation of many alleles
• Complements linkage mapping
• Control for population structure
Association mapping in Lolium perenne
Traits
Water soluble carbohydrates• Ruminant productivity• Abiotic stress tolerance (drought, cold)
Flowering time• Plant development• Forage quality traits• Seed production
60
50
40
30
20
10
GE
NE
TIC
DIS
TA
NC
E (
cM)
0 1 2 3 4 5
LOD SCORE
_
_
__
_
_________________
LG6
RZ87
rv1423
rv1208
M65-1Alkinv 1/4
B6104
rv0641
rv0067
RYE 14
CDO686
rv0196
C37rv0414
rv0449Alkinv 2/3rv0171CDO718rv0307
E39M4908
E41M5712
Turner et al. New Phytol. 2005
Fructose and Glucose
Alkaline Invertase locus map to QTL forGlucose and Fructose
Invertases: 1. Acid (vacuolar and Cell wall) 2. Alkaline
Vacoule
Sucrose
Sucrose
Glucose
Fructose
Glucose
Fructose
Glucose
Fructose
Acid Invertase
FructanFructosyl -transferase
Alkaline Invertase
Sucrose Acid Invertase Chloroplast /Amyloplast
Starch
-substrates for respiration-biosynthesis of primary and secondary compounds- regulation of gene expression by sugars
Cytosol
-sugar storage-osmoregulation-response to cold stress
Cell Wall
Effect of sucrose accumulation on gene expression
Depleted in soluble carbohydrate Plants - low lightshort photoperiod
Excised Leaves
Sucrose solution
Sorbitol solution(osmotic Control)
Dark
Water
24h
Actin
SucW Sor
Alk Inv.
TLC - Water Soluble Carbohydrates
1 2 3 4 5
Ph
oto
peri
od
Sucrose
Alkaline invertase expression along the developing leaf
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
Ct
Ct
Ct
Ct
1 2 3 4 5
Alkaline Invertase6238 bp
SNP492
SNP202
SNP382
SNP805
SNP825 (M to L)
SNP953
SNP1053 (D to N)
SNP1954
SNP1970
SNP2214
SNP2283
SNP2647
SNP4879
SNP5259
SNP5395
SNPs in Alkaline InvertaseLocus of Lolium perenne LG 6
1 kb
M-LD-N
SNP No
Indel 6
Syn 13
Non-syn 8
Non-coding 65
Total 92
Alk. Inv. locus
0
0.2
0.4
0.6
0.8
1
0 1000 2000 3000 4000 5000 6000
Distance (bp)
r2
LD in candidate genes
Alkaline Invertase
123456789
1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586878889909192
686976808183-84
105106126133150173181202234273-274
275286306312320322-324
382383384393395396413424440448464468474478492-560
6246488058258989539679841016
1026
1039
1048
1053
1130
1187
1970
2003
2024
2026
2050
2081
2082
2083
2092
2097
2214
2283
2309
2317
2355
2416
2497
2548
2600
2647
2650
2680
2696
2702
2708
4847
4856
4879
4947
4954
4974
4978
5047
5058
5061
5063
5068
5148
5259
5395
Alkaline Invertase6238 bp
SNP492
SNP202
SNP382
SNP805
SNP825 (M to L)
SNP953
SNP1053 (D to N)
SNP1954
SNP1970
SNP2214
SNP2283
SNP2647
SNP4879
SNP5259
SNP5395
LD in Alkaline invertase locus
Pop Inferred clusters1 2 3 4 5 6 7 8 9
1 0.005 0.000 0.000 0.000 0.000 0.000 0.000 0.989 0.005
2 0.000 0.001 0.000 0.001 0.010 0.001 0.982 0.005 0.000
3 0.001 0.003 0.000 0.971 0.015 0.009 0.000 0.000 0.001
4 0.008 0.946 0.000 0.006 0.013 0.001 0.000 0.000 0.027
5 0.003 0.042 0.000 0.005 0.000 0.000 0.001 0.000 0.950
6 0.004 0.001 0.989 0.000 0.003 0.001 0.000 0.002 0.000
7 0.976 0.014 0.001 0.000 0.001 0.000 0.000 0.007 0.000
8 0.009 0.023 0.007 0.008 0.913 0.017 0.007 0.007 0.011
9 0.002 0.001 0.007 0.001 0.011 0.975 0.002 0.001 0.000
Population structure70 AFLP markers
SNP WSC N DMD
04 05 04 05 04 05
202 1 1 1
382 1 2 1 1
492 1 1 1
805 1 1 1
825 1 1 2
953 1 1 1
1053 1 1 1 2
1954 1
1970 1 1 2 1
2214 1 1
2283 1 2 1
2647 3 1 1 2
4879 1 1
5259 1 1 1
5359 1 1
SNP 04 05
202
382
492
805
825
953
1053
1954
1970
2214
2283
2647 0.05
4879
5259
5359
Association analysisAlkaline Invertase and WSC
Linear mixed model(WSC)
ANOVA
E38M4710
0610121923252627283033343538414243454653555658596985
103
INV1:2R2869
B6102bCDO545
OSWLpF2LpF1LpF3C764LpF4
ACA_CTA_254 ACA_CTA_321E40M5909
RZ144 S2539ACA_CAC_225
E39M4910E37M5601 GSY60.1
CDO385.1PSR690 E41M5710
RZ952C390
LtemCOaLtemCOb
E39M5802E41M5701
ACA_CAC_246ACA_CTA_204
ACP
CAT
ACA_CTA_254ACA_CTA_321
C764
S2539ACA_CAC_225
0 10 20 30 40LOD
AFLP markers associated with flowering timein Lolium perenne
Fructan epistatic QTL
Properties of LpCO gene
• Synteny with rice HD1 locus
• Homologous to CONSTANS
• Complements Arabidopsis co-2 mutant (Martin et al. 2004, PMB 56: 159)
• Upregulated in response to LD
• Map to major QTL for flowering time
Lolium perenne HD1 Locus13243 bp
N-ICAA[C/A]A
V-M
HD1Peroxidase
SNPs in HD1 locus in Lolium perenne LG7
1 kb
SNP No
Indel 0
Syn 2
Non-syn 2
Non-coding 8
Total 12
1
2
3
4
5
6
7
8
320513147521182389444347175443
LD in candidate genes
HD1
Association analysisHD1 locus and flowering time
SNP Flowering time WSC
04 05 04 05
320
513
1475
2118
2389
4443
<0.05 <0.001 <0.001
4717
5443
Linear mixed model(pops random effect)
SNP Ba9955 Ba10113 Ba10158 Ba10278 Ba10284 Ba10732 Ba10870 Ba11304 Ba12945
04 05 04 05 04 05 04 05 04 05 04 05 04 05 04 05 04 05
320
513 ** *
1475 * * **
2118 * *** *
2389 *
4443 ** *** ** * * ***
4717
5443 * * ** * * *
Association analysisHD1 locus and flowering time
ANOVA
* P<0.05; ** P<0.01; *** P<0.001
Lolium perenne HD1 Locus13243 bp
N-ICAA[C/A]A
V-M
HD1Peroxidase
SNP 4443 in HD1 locus
1 kb
CAACA Consensus recognition sequence for RAV1/AP2…
AcknowledgementsIGERJanet HumphreysMervyn HumphreysJoe GallagherDanny ThorogoodIan ArmsteadSue HeywoodMark HirstRuth SandersonLesley Turner
University of BirminghamZewei LuoMike Kearsey