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