DOFCO(NDDB) University of Delhi

Biotech Project

Breeding for Yield

Stabilization in Mustard

(Brassica juncea)

Rapeseed and mustard growing area in India

Breeding objectives in B. juncea

Increase in yield

Increase in yield potential

Hybrids / pure line breeding

Stabilization breeding

Disease and pest resistance

Quality

Oil

Meal

17 10 13 26 30 4

Blight White rust Powdery mildew

Low erucic, low glucosinolate varieties Aphid Others

Status of research in B. juncea

Productivity increase

2 Hybrids

DMH-1, DMH-11

Molecular Breeding

Traits Tagged

Erucic acid

Glucosinolates

Seed coat color

White rust resistance

Ongoing

Alternaria resistance

Stem rot

MAS in progress

Mustard Hybrid DMH-1

• A CMS based hybrid

• Yield advantage >20%

• Resistant to white rust

Performance of hybrid DMH-1 in coordinated trials

during rabi 2004-05, 2005-06 and 2007-08

S.

No.

Strain /

Hybrid

Seed Yield Oil Yield 1000

seed

weight

(g)

Oil

Content

(%) kg/ha %

increase

over

kg/ha %

increase

over

1 DMH-1 2053 - 812 - 3.6 38.38

2 Varuna (NC) 1592 29 616 32 5.4 37.29

3 Kranti (NC) 1701 21 677 20 4.1 39.19

4 RL-1359 (ZC) 1811 13 719 13 4.3 38.95

NC - National Check; ZC - Zonal Check

Reference: AICRPRM reports (2005, 2006, 2008)

Released by ICAR in 2009

(Hissar, Sriganganagar, Navgaon, Delhi, Bawal, Ludhiana, Bathinda)

Performance of DMH-1 in farmers field

Location No. of

Farmers

surveyed

Average

yield of

DMH-1 (q/ac)

Average

yield of

Check

(q/ac)

%

increase

over

check

Alwar & Dousa Distt 25 8.60 6.39 34.59

Sriganganagar 13 9.4 8.11 15.91

Rewari 11 8.60 7.85 9.55

Bharatpur & Agra 14 8.10 7.95 1.89

Yield trial of DMH-11 hybrid Male sterile barnase line

• DMH-11 is barnase-barstar based

• Yield advantage >20%

• Resistant to white rust

• Biosafety tests in progress

bar-spacer-TA29(870)bn

RB LB 35SPr bar barnase 35SpA TA29(870) ocspA spacer AMV

Constructs used for developing transgenic male

sterile and restorer lines

RB

Hind III Pst I

LB 35SdePr bar Bsmod 35SpA TA29(279)

ocspA

bar-TA29(279)-bsmod

Performance of DMH-11 under bio-safety trial

S No Entry

ICAR Centre

Total Mean % Increase

over Kumher Alwar Sgnagar

1 Varuna (barnase) 1986 1789 2513 6287 2096

2 EH-2 (Barstar) 1730 1842 2455 6026 2009

3 Varuna 1866 1741 2670 6278 2093 24%

4 EH-2 1793 1716 2182 5691 1897

5 DMH-11 2285 2515 3000 7801 2600

6 Maya/RL-1359 (ZC) 2057 1767 2287 6112 2037 28%

BRL-I Trial, Kumher

BRL-I Trial, Navgaon

Crossability Study at Bawana

BRL-I Trial, Sri Ganganagar

Application of molecular markers in mustard breeding

By 2012

1. Total no. of markers 2160

2. Types of markers

a) AFLP

b) RFLP

c) SSR

d) Gene markers

e) Intron spanning markers

1282

70

279

13

778

3. Total length (cM) 1850.2

4. Assigning LGs to A and

B genomes

10 A genome LGs

8 B genome LGs

5. Traits tagged

a) Erucic acid

b) Linoleic acid

c) Seed coat colour

d) Glucosinolates

e) White rust resistance

Candidate genes

Candidate genes

SSR markers

Candidate genes

IP markers

Molecular mapping in B. juncea (Varuna x Heera)

wg2a4a 0.0

e39m55h163 7.1

p65t76v198 20.2

p79t78v127 29.2

p65m62h265 33.3

e54t75h129 40.9

e47t64v352 45.9

e39m50h446 54.8

p62t78h185 63.1

p53t76h368 68.4

FAE1.2 72.6 e79t78v205 76.1

e50t67v375 87.1

p32t77v268 110.1

p65m62h181 118.4

e31m50v273 122.9 ea-2

(39

%)

B7

e31m60v98 0.0 e48t78v400 3.6

e34m50v82 12.6

wg2h1 19.4

p62m39v124 27.8

e39m50v500 36.0

e54t71h380 46.7 e32t66h142 49.2

e32t66v191 FAE1.1 50.1

e31m60v195 52.6

p65t90v258 59.9

ea-1

(60

.8%

)

A8

Tagging of Two Loci of Erucic

Acid (ea1 and ea2) by

Candidate Gene Polymorphism

At5g05920 2.4

At5g13190a 12.8

At5g53920 31.9

At1g74640a 48.5

At5g27740b 77.6

ELONG R1 90.1

At5g61970 95.4

Msl

Sqm

s

At4g35530 6.9

At4g21105 18.3

At1g74640b 27.8 ELONG R4/Myb28a 29.9

At3g54670b 37.1

At3g10572b 50.6

At4g01897a 62.5 ALK R1 64.2

At2g30200a 77.5 Tsw

Sq

l P

br

ALK R2 0.6 8.6

At5g63905 21.5

At2g14170a 34.2

At2g19260 44.9

At3g46560 57.9

At2g03060 68.3

At1g22860 78.5

At3g55430a 90.6

At3g62620 104.4

At2g20490b 118.0

At2g22370 129.7

At3g06483 8.8

At3g10572a 19.1

At3g20920 30.1 At5g47890 44.6 At5g27830

46.2

At5g66290 53.2

At1g58220a 55.3

At3g23980 61.6

At3g14850 74.3

At3g12260b 84.9

At3g13062 100.4

A2 A3 A9 B1

138

75

25 113

12

134

4-5

63520

ELONG R1

61970

?

27740b

74640b

ELONGR4 Myb28a

14045

05755 05590 54670b

Myb28b

Tagging of loci involved in glucosinolate biosynthesis in B. juncea

?

Negative yield QTL from low GSL parent

Ramchiary et al 2007, TAG Bisht et al 2009, TAG

Gsl1

Gsl2

Gsl3 Gsl5

At5g27560

Myb28c 17.4

Status of mapping in different B. juncea populations

Cross Type of

Population

Total markers

available

Traits of

interest Value

Varuna x Heera

(VH)

1200 DH lines,

465 RILs

AFLP- 1290

RFLP- 70

IP- 797

SSR- 287

Gene markers- 32

Seed size,

Oil content,

Erucic acid,

Glucosinolate,

White rust resistance

5.2 (TSW) 2.6

41% 36%

52 % 0%

114 uM 14

S R

TM4 x Donskaja

(TD)

134 DH lines AFLP- 585

IP- 318

SSR- 8

Oil content,

White rust resistance 40% 45%

S R

Donskaja x EH2

(DEH)

296 DH lines IP- 323

SSR- 241

Gene markers- 8

Oil content ,

Erucic acid 45% 36%

25% 0%

EH2 x J8

(EHJ8)

1000 DH lines IP – 348

SSR- 144

SNP - 239

Oil content,

Oil and meal quality 36% 50%

EH2 x Pusa Jai Kisan

(EHPJ)

300 DH lines IP- 797

SSR- 287

Gene markers- 32

Seed size,

Oil content,

Erucic acid,

glucosinolate

2.6 (TSW) 6.7

36% 42%

52% 0%

115 uM 14

Limitations in mapping

Markers available for ‘A’ genome but not enough for saturating QTL regions of interest Not enough markers available for ‘B’ genome Need to generate more makers – microsatellite and SNPs

Transcriptome sequencing of Brassica species

• Illumina GA II Technology • Samples used: leaf, stem, inflorescence and developing siliqua • Species used

B. rapa B. juncea

Indian East European

Chiifu ssp Chinensis Candle ssp Oleifera YSPB-24 ssp Trilocularis Tetra ssp Trilocularis

Varuna Pusa Jai Kisan

Heera EH-2 Donskaja

J8

SNP data generated and SNP based map being developed in B. juncea

SNP data generated and SNP based map developed in B. rapa

White rust

Resistance available in east European B. juncea lines

Cause

Losses in yield

Albugo candida 17- 34%

• Two mapping populations used Varuna x Heera (VH) and TM4 x Donskaja (TD)

• Phenotyping of VH and TD populations identified two different loci for white rust resistance

• The two loci are being transferred to different B. juncea lines using tightly linked markers

Mapping for white rust resistance in B. juncea

A4

At3g63420b*(3g150) 0.0 At3g54650(3g66) 2.4 At3g58500a*(3g165) At3g55440b(3C-18) 4.7 p62m59h197 5.5 At5g37480(5gAJ30k) At1g77550(174)

At4g13940*b(4C3) WR3 At5g37580(288)

6.4

At5g37630(5g59) 7.3 At5g37850(5gAJ31) 8.2 WR17 9.2 WR22 9.9 At5g41560(5gAJ35) 10.6 At1g66410(254d) At2g21150(2g135) WR26 11.8

At5g36880*(5g57v) At2g22000a(2g136) 12.5 At2g22640(2g137) 13.3 At2g24765a*(2C-9) 15.8 At2g25737(2C-10) p53m47h285 17.4 At2g28880b*(2C-15) 22.8 At1g69980(1gLP78) 25.2 e47m43h330 At2g30130*(AJ-2g11) 26.2 At2g31490a(2g59a) 29.8 At2g33040(417) At2g07440b*(2g105) At2g32520(2g61) 32.5

e31m58v101 36.4 GE-R3 At2g35790(2g66) 39.8 At2g36530c*(2C-22) 42.1 At2g37040*(ec4c11) 43.1 At2g37150*(wg4a4) 43.7 At2g40690*(wg5b2) At2g40765b(2g74b) 53.3 At1g76540(183b) 55.3 At2g42040(2g76) 56.6 p63t78v400 62.2 At2g44520(2g167) 74.9

Wh

ite

rust

Varuna-Heera

WR locus:7-17cM

TM4-Donskaja

WR locus:18-24cM

A5

Wh

ite

rust

At2g46390(2g82) 0.0

2g472.2 15.1 2g428 20.9 e47t70v258 e51t78t290 2g337 p32t77d380 27.3

2g415(b) e55t73t240 29.1 2g415(a) e50t66t380 29.6 e31m42v144 e35t63v351 e48t78v225 msa21 e34m62v89 e31m58v430 e31m61v136 e34m62v157 e31m49v336 e34m62t160 2g55(NCB)

32.6

e31m49v295 34.4 e31m50t98 43.9

p32t77v301 p32t77d310 56.9 3g14 57.5

2g385 64.5 e34m62d143 66.5 5g218 68.6 e35t63v240 70.4

Marker-assisted introgression of two white rust

resistance loci into popular Indian varieties of mustard

Donskaja IV AcB1-A5.1

X EH-2 AcB1-A4.1

F1

DH (296 lines)

Foreground selection

AcB1-A5.1 – (At2g36360)

AcB1-A4.1 - (At5g41560 and At5g41940)

Line no. 67, 98, 144 and 207

• contain two resistance loci

• Early maturity

• Good plant type

Pusa bold

Varuna

Pusa Jai Kisan

Rohini

EH-2

X

F1 BC1

Foreground and background

selection

BC3 Selfing / DH

Alternaria blight

Cause

Losses in yield

Alternaria brassiceae

35- 46% in mustard

up to 70% in toria and yellow

sarson (B. campestris)

No resistance source available in cultivated Brassica species

• One major resistance locus identified on chromosome 2 of Arabidopsis using RIL between a resistance and susceptible parent

• Using phenotype genotype correlation resistance locus mapped down to 1 MB

• The region has now been fine mapped and further

narrowed down to 32Kb by phenotyping and genotyping of recombinants

Mapping of Alternaria resistance in A. thaliana

Alternaria resistance locus in Arabidopsis

587

608

613

626

OXF

644

PVU II 120 kb 32 kb

7 genes

Cv x Gre

26

Cloning of the candidate genes for

complementation studies

Candidate gene

Candidate gene

Gre 3.2 MP 132

• At2g25409

• At2g25410

• At2g25420

• At2g25430

• At2g25440

• At2g25450

Stem rot

• No source of resistance in cultivated Brassica species

• Transgenics being developed using Oxalate oxidase gene

from Barley and PGIP gene from Arabidopsis in B. juncea var.

Varuna

Cause

Losses in yield

Sclerotinia

sclerotiorum

Depends on % incidence

RB

Kan 35S AtPGIP 1 HA

LB

RB

Kan BaR 35S AtPGIP 2 HA

LB

35S BOXO

LB RB

BaR

Spec

CD3-687-AtPGIP1

CD3-687-AtPGIP2

pZP200-baR-35SBOXO

i

ii

iii

Constructs used for developing transgenics in B. juncea for Stem rot

BaR

In vitro testing of transgenics for stem rot resistance

48hrs

72hrs

Collaborations

Prof Deepak Pental

Dr. Arundhati Mukhopadhyay Prof. Akshay Pradhan

Dr. Vibha Gupta Dr. Pradeep Burma

Dr. Y. S. Sodhi Dr. Naveen Bisht

Dr. N. Arumugam Dr. Indira Sivaraman

Dr. Arun Jagannath Dr. N. Ramchiary

Mr. Jagdish Verma Dr. Priya Panjabi

Mr. B. S. Yadav Dr. Amrendra

Dr. Sarita Sharma Dr. Lakshmi K. Padmaja

Dr. Satish Yadav Mr. Diwakar Nandan

Dr. Jagreet Kaur