application of molecular tools for wheat quality ...aciar.gov.au/files/node/3875/session...

Application of Molecular Tools for Wheat Quality Improvement: Indian Scenario

H. S. BalyanCh. Charan Singh University,

Meerut

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

Wheat Grain Quality1. GPC & micronutrients (Fe, Zn)

- Nutritional quality

2. Pre-harvest Sprouting (PHS)- Activates hydrolyzing enzymes- Endosperm constituents are broken down- Decreased volume & poor crumb of bread- Lowers quality of grain for bread/feed and seed

3. GW- Aesthetic, market & industrial quality (flour recovery)

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

Wheat Grain Quality4. GPC, GW, sedimentation value, kernel

hardness, amylose content- Bread, biscuit, chapati and noodle quality

5. HMW-GS and LMW-GS- Flour processing properties/dough quality such as viscoelasticity & extensibility

6. Phytic acid- Reduces absorption of Fe and Zn

Applications of Molecular Markers

- Preparation of genetic maps- QTL analysis- High density mapping- Validation of linked markers- Molecular marker assisted selection

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

RIL Populations Used

- RIL pop for GPC (HD2329 × PH132)- RIL pop for PHST (WL711× SPR8198)- RIL pop for GW (CS × Rye Selection111)- RIL population for GH (K65 × Lerma Rojo)

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

PAU & IARI

Framework Molecular Maps GPC Map: 214 loci (CCSU &NCL)GW Map:298 loci (CCSU)PHST Map:214 loci (CCSU)GH Map: Under preparation (IARI)

Prasad et al. (2003). TAG 106:659-667

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15PH13

2

WL7

11

- 10 QTLs for GPC detected (LOD 2.5/above threshold LOD)

- 9 chromosomes: 2A, 2B, 2D, 3D, 4A, 6B & 7A

- Range of PVE: 2.95% to 32.44%Prasad et al. (2003). TAG 106: 659-667

Composite Interval Mapping (CIM) (QTL Cartographer)

QTL Analysis for GPC

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 CSSU

Xgwm830

Xgwm1063Xgwm164Xgwm357

2A

Xgwm359Xgwm1198

Xgwm1256

Xgwm1052Xgwm515Xgwm372

Xgwm895

Xgwm1115

C

1D

C

Xgwm957

Xgwm608

2B

Xgwm319Xgwm1249

Xgwm374

Xwmc272

Xgwm1027

Xgwm429Xgwm410

C

2D

CXwmc41

Xgwm1264Xgwm1204

C

3A

Xgwm1171

C

7A6B

Xgwm680

Xgwm133Xgwm771Xgwm889C

Xgwm456

3D

Xgwm892C

Xgwm894

Xgwm601

Xgwm397

C

4A

Xgwm940b

4B

C

Xgwm513Xgwm805

C

5D5B

Xgwm831

C

7D

C

NIL2233

Xgdm86

C

Xgwm666

2B

CXgwm429Xgwm410

Xgwm1027

3B

C

Xgwm493

5A

C

NIL2210

Xgwm456

Xgwm493

2B

CXgwm429Xgwm410Xgwm319

3D

C

Xgwm1249

Xgwm374

Xwmc272

3B 4B

C

Xgwm513

C

2A

C

Xgwm830

NIL2215

Xgwm940b

2A

Xgwm940b

NIL2218

Xgwm1027

2B

Xgwm429C

Xgwm1249

3B

C

Xgwm493Xgwm726

C

4B

C

NIL2232

1A

Validation of SSR markers Linked With QTLs for GPC in NILs (CCSU)

ICIM mapping of QTLs for GW using IciMapping v1.4

• 34 QTLs on 18 chromosomes

• 3 major QTLs detected

• QTL on1B explained 26.0 to 82.0% and another QTL explained 10.6- 45.7 % PV

• QTL on 2B explained 11.37-32.77 % PV

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

Kumar et al. (2006). Euphytica 151:135-144CSSU

QTL Analysis for PHSTCIM

• 7 QTLs on 1A, 2A, 2D, 3A, 3B

• One major QTL each on 2A and 3A

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 CSSU

HD2329 SPR8198 1 2 3 4

5 6 7 8 9

Kulwal et al. (2005). TAG 111: 1052-1059

Two-Locus QTL AnalysisQTLMapper

PVE due to M-QTLs:GPC=7.22 to 7.22%; GW=28.11%; PHST=37 to 48%

PVE due to Q×Q:GPC= 2.68 to 6.04%; GW=42.12%; PHST=27 to 28.73%

PVE due to Q×E:GPC= 25.00 to 47.99% ; GW= nil; PHST= 3.24%

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 CSSU

1A1B1D2A2B2D3A3B

3D4A4B4D5A5B5D6A6B6D7A7B7D

: a : a + ae: ae : aa : aae : aa + aae : centromere

Q × Q and Q × E Interactions Resolved Through Two-Locus QTL Analysis for GPC in Bread Wheat

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 CSSU

GPC: gwm1264, gwm608, gwm1171, gwm508, gwm193, gwm361, gwm644, PCR-CAPS marker, ASA marker, uhw89

PHST: cdo795, psr115, bcd1431.1, bcd1431.2, bcd1380, fbb370, bcd907, fbb283, ATPase.2,wmc104, Mst101,wmc153,gwm155,GW: wmc333, gwm419,E35M4714, E35M4718Leaf rust: Lr9 (SCS5550); Lr24(SCS73719); Lr28(SCS421570); Lr19 (SCS265512); (SCS253736)

Linked Markers and Their Validation

CSSU

Strategy for MAB• Three step selection during backcrossing

- Foreground selection for alleles for donor-parent markers flanking the QTL

- Background selection for the recipient-parent-alleles on loci of individual chromosome carrying the QTL

- Background selection for the recipient-parent-alleles for the whole-genome

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007CSSU

MAB of GPC-B1,a high GPC QTL, into 10 Indian Wheat Genotypes

Yecora Rojo

F1 s ×

Foreground & background selections

BC3F1s (85-100% genomic similarity)

BC3F2s Selection of individuals homozygous for GPC-B1a

Indian cultivars

Respective RP

BC1F1S

10 Recipient Parents (RP) High GPC Donor Parent (DP)×

×

Xuhw89

DP RP BC3F2 progenies

* ** * * * *ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

Asterisk indicate individuals homozygous for the GPC-B1 allele

CSSU

10

11

12

13Pr

otei

n co

nten

t (%

)

HD

2687

HD

2329

PBW

343

PBW

343(

Lr19

)

PBW

343(

Lr9)

PBW

343(

Lr24

)

K91

07

PBW

373

Raj

3765

HI9

77

Performance of BC3F1 Containing GPC-B1 QTL Allele(Change in GPC by -0.93 to 1.72%)

CSSU

Summary of MAS for Gpc-B1

Keylong--2004F1

2007

2006-07

2006-07

2005-06

2004-05

Year

PAU, Ludhiana25~50F4

PAU, Ludhiana74167BC1F1

PAU, Ludhiana168300BC2F2

KeylongIn progress

DNA isolation

BC2F3/F5

Keylong110330BC2F1

Location+ve for ASA

Total plants analysed

Generation

Recipient Parents- BW3652, C273, C518, DBW16, DBW17, BW4406, BW4426, PBW509, PBW554, PBW563, PBW564, PBW568, PBW571

PAU

20

15

10

5

0

Grain Protein content of F4s progenies of the crosses ofGluPro with wheat

%GP

C

PBW

343

Glup

ro

13.0 to 14.3%

11.3%

15.6%

Marker assisted selection for HGPC gene GpcB1 using allele specific amplification marker (ASA)

PAU

PHS

scal

e

Parents and Progeny0

3

6

9

HD23

29 (R

P)

BC3F

1

SPR8

198

(DP)

PHS scaleSusceptible =9 Resistance =1

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 CSSU

Graphical genotypes of PBW343/SPR5 introgression lines

Xwmc153 and Xgwm155 suitable for marker assisted mobilization of PHST to elite wheat backgrounds

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 PAU

Transfer of new GluAx and GluAy subunits to bread wheat

X

BC2F3(2006-07)

Half seed analysis

X

X

X

Half seed analysis

Half seed analysisHalf seed analysis

Half seed analysis

T. durum-T. dicoccoidesPBW343

F1 PBW343

BC1F1 PBW343

BC2F1PBW343

BC3F1BC2F2

BC3F2(2006-07)

~300 half seeds analysed and 8 homozygous and 11 segregating progenies identified PAU

0102030405060708090

100

Ae. ko

tschy

i 357

3Ae.

kotsc

hyi 3

774

Ae. pe

regrin

a 379

1

Ae. pe

regrin

a 137

72

Ae. lon

gissim

a 3770

Ae. lon

gissim

a 3507

T. dico

ccoide

s 464

0

T. dico

ccoide

s 477

2

T. aes

tivum PBW 34

3

T. duru

m PDW 274

Accessions

Iron

and

Zinc

(mg/

kg)

Iron (mg/kg) Zinc (mg/kg)

Iron and zinc of wild relatives as compared with cultivars

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 IARI, ARI, PAU, IITR

Chinese Spring (PhI) F1 CS (PhI) XAe. kotschyi

Ae.kotschyi

Spikes of Parents and F1 of wide crosses

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 IARI, ARI, PAU, IITR

a b

dc

fe

Meiotic chromosome pairing in F1 of wide crosses

CS (PhI) X Aegilops kotschyi 3573

(a) 2II (b) 4II

CS (PhI) X Aegilops kotschyi 396

(c) 4II (d) 6II

CS (PhI) X Aegilops kotschyi 3790),

(e) 3II;,1III (f)2II

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 IARI, ARI, PAU, IITR

Sterile

Colchicine treatment

Synthetic amphiploid

AABBDDUUSS

Chinese Spring (PhI) Ae.kotschyiF1 CS (PhI) XAe. kotschyi

UUSSABDUSAABBDDSynthetic amphiploids

IARI, ARI, PAU, IITR

Grain Iron and Zinc of Amphiploids

41.6774.55Ae. kotschyi 3790

17.2330.87WL 71119.6130.84CS (Phi)

32.7577.27CS(Phi) x Kot 379033.1370.00WL711 x Kot 39133.6870.73CS(Phi) x Kot 39625.2071.96WL711 x Kot 377451.0777.00Ae. kotschyi 396

41.2282.67Ae. kotschyi 3774

Parents/ Amphiploid Fe (ppm) Zn (ppm)

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 IARI, ARI, PAU, IITR

0

20

40

60

80

100

120

140

160

180

a b c d e f g h i j k l m n o

Recipients, F1 hybrids, Donors

Iron

cont

ent (

mg/

kg)

Recepient F1 Hybrid Donor

0

10

20

30

40

50

60

70

a b c d e f g h i j k l m n o

Recipients, F1 Hybrids, Donors

Zinc

con

tent

(mg/

kg)

Recipient F1 Donor

Leaf iron and zinc content in sterile F1 hybrids between wheat and Aegilops donors

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007 IARI, ARI, PAU, IITR

Future Research Programmes to Identify Novel QTLs & Linked Markers

• AB-QTL analysis• LD and association analysis• Identification of novel positive alleles• Transciptome analysis • Expression QTL (e-QTL) analysis• Genetic mapping of newly identified gene

sequences including regulatory sequences

ICAR-ACIAR Workshop on Molecular Marker Assisted Breeding In Wheat, October 12, 2007

Participating Institutions

• CCSU, Meerut• IARI, New Delhi• PAU, Ludhiana• DWR, Karnal• ARI, Pune• NCL Pune• IIT, Roorkee

ACKNOWLEDGEMENTS

Funds provided by DBT and NATP are gratefully acknowledged

Thank You