PAG XXII 2014 – Genomic resources applied to marker-assisted breeding in cowpeas – BL Huynh

Genomic resources applied to marker-assisted breeding in cowpea

GCP Tropical Legumes 1: “Improve cowpea productivity for marginal environments in sub-Saharan Africa”

Philip Roberts, Timothy Close, Bao Lam Huynh

Mitchell Lucas, Arsenio Ndeve, Steve Wanamaker – UC Riverside

Ousmane Boukar, Christian Fatokun, Sam Ofodile – IITA, Nigeria

Ndiaga Cisse, Penda Sarr – ISRA, Senegal

Issa Drabo, Jean-Baptiste Tignegre – INERA, Burkina Faso

Rogerio Chiulele – E. Mondlane U, Mozambique

Batieno T. Benoît Joseph – WACCI, Ghana

Ndeye N. Diop, Xavier Delannay et al. – IBP, CIMMYT, Mexico

PAG XXII – GCP Workshop, Jan 2014

Cowpea – Vigna unguiculata

• Tropical legume.

• Nutritious, high-protein food.

• Major food crop in sub-Saharan Africa.

• Nitrogen fixation to enrich soil fertility.

• Tolerance to drought, heat, poor soils.

Yard-long bean/asparagus bean

Blackeye bean

Diverse seed types

Cowpea production zones in Africa

200mm

800mm ISRA INERA

IITA

EMU

200mm

800mm

Yield constraints

Seedling Flowering Pod filling Post-harvest

Aphids Flower thrips

Drought

Pod-sucking bugs

Striga

Root-knot nematodes

Weevils

Heat

African cowpea varieties yield ~20% of potential

Pod borer

Macrophomina

Bacterial blight

Viruses

Genetic variation – basis for QTL discovery and marker-assisted breeding

Heat

Striga

Root-knot

nematodes

Aphid

T S

R

S R S

R S

Jean-Baptiste, Burkina Faso

V1: 2009 PNAS 106:18159-18164

V4: 2011 Plant Genome 4:218-225

V6: 2013 http://harvest.ucr.edu/

QTLs for biotic and abiotic traits

Cowpea consensus genetic map

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11

11 RIL populations, 1091 SNPs, 815 bins, 680 cM

http://harvest.ucr.edu/

http://harvest.ucr.edu/

1536 SNPs

> 400 accessions from Africa and the world

SNP TVu-9522 TVu-9556 TVu-9557 TVu-9620 TVu-9651 TVu-969 TVu-972 TVu-9749 TVu-9761 TVu-9801 TVu-9820

1_1431 GG GG GG GG GG AA GG GG GG AA AA

1_0721 CC CC CC CC CC CC CC CC CC CC CC

1_1392 GG GG GG GG GG GG GG GG GG GG GG

1_1157 AA AA AA AA AA AA AA AA AA GG GG

1_0595 GG GG GG GG CC CC CC CC CC CC CC

1_0741 AA AA AA AA AA AA GG AA AA AA AA

1_0482 GG CC CC GG CC CC GG CC CC GG GG

1_0791 AA AA AA AA AA AA GG AA AA GG GG

1_1490 GG GG GG GG GG GG CC GG GG GG GG

1_1033 GG GG GG GG GG GG AA GG GG GG AA

1_0144 GG GG GG GG GG GG AA GG GG AA AA

1_0328 GG GG GG GG GG GG GG GG GG GG AA

1_0240 CC CC CC CC CC CC CC CC CC CC CC

1_0985 AA TT TT TT TT AA AA TT AT AA AA

1_0041 GG GG AA GG GG AA GG GG GG GG GG

1_1470 AA AA AA AA AA AA AA AA AA GG GG

1_1535 GG GG GG GG GG GG GG -- GG AA AA

1_1230 AA AA AA AA AA AA AA AA AA AA AA

1_1108 CC CC CC CC CC CC AA CC CC CC AA

1_0670 AA GG GG AA AA AA GG GG AG AA AA

SNP database for germplasm collection

2013 The Plant Genome 6(3)

BreedIt (breedit.org), GDMS (IBP tool, ICRISAT)

Select informative SNPs for a MAS project Consensus map SNP genotypes of parents SNPs selected for MAS QTL

http://breedit.org/

Selection Crossing Planting

New genotyping

1 2 3

4

5 6 7

8

SNP genotyping (KASP platform-LGC Genomics)

Employ marker-assisted backcrossing (MABC) to improve local cowpea varieties

Country 11 MABC populations (Recurrent/Donor) Main donor Traits

Nigeria IT93K-452-1/IT97K-499-35 IT89KD-288/IT97K-499-35 SuVita 2/IT97K-499-35

Striga Striga Drought, Striga

Burkina Faso Moussa/IT93K-693-2 KVX745-11P/KVX414-22

Striga Striga, Large Seed

Senegal Melakh/IT97K-499-39 Striga

Mozambique IT85F-3139/CB27 CB27/INIA-41

Large seed, Heat Nematode, Drought

USA/ Mozambique

CB27/IT97K-556-6 CB46/IT97K-556-6 CB50/IT97K-556-6

Aphid

MABC example – Aphid resistance

IT97K-556-6 (resistant)

Blackeyes (susceptible)

Aphid resistance screening

Big Buff

Recombinant inbred lines CB27 (S) x IT97K-556-6 (R)

R S

Big Buff

Kearney, CA

Aphid-resistance QTLs

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11

Major QTL 66% phenotypic variance

Minor QTL 9% phenotypic variance

QTL IciMapping: http://www.isbreeding.net/

http://www.isbreeding.net/

04 05 11 12 24 26 35 38 40 46 48 53 57 62 63 66 67 69 71 79 80 CB46A

AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA

AB AB AA AB AA AB AB AB AB AB AB AB AB AA AB AB AB AB AB AB AB AA

AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB AA




AB AB AB AB AA AB AA AA AA AA AA AB AB AA AA AB AB AB AA AA AA AA

AA AB AB AB AA AA AB AA AB AA AA AA AB AA AA AB AB AB AA AB AA AA







AA AA AB AB AB AB AA AA AA AA AA AA AA AA AB AA AA AB AB AB AA AA




AA AA AB AB AB AA AB AB AB AB AA AA AA AA AB AB AA AB AB AB AA AA

AB AA AB AA AB AA AB AB AB AB AA AA AA AA AB AB AB AB AB AB AA AA






AB AB AA AB AB AA AB AB AA AA AB AA AA AA AA AB AB AA AB AA AA AA

AB AB AA AA AB AB AB AB AA AA AB AA AA AA AA AB AB AA AB AB AA AA







AA AA AA AA AA AA AA AA -- AA AA AA AA AA AA AA AA AA AA AA AA AA


QTL introgression

2 3

4 5 6

1

BCnF1 plants Leaf sampling SNP genotyping

Backcrossing Background selection Foreground selection

Resistant genotypes

Resistant RIL x Recurrent P

Employ marker-assisted recurrent selection (MARS) to develop improved breeding lines P

op

ula

tio

n

de

velo

pm

en

t Q

TL

Re

com

bin

atio

n

Lin

e d

evel

op

me

nt

Cycle 1

Multilocation phenotyping

Parent 1 × Parent 2 F1

F2

F3 Genotyping

Multilocation phenotyping

A B C D E F G H

F1 F1 F1 F1

F1 F1

F1

F2

F3

F3:4

QTL detection –estimate marker effects

Cycle 2

Cycle 3

4 populations Traits

Burkina Faso (Issa Drabo et al.)

Yield, Drought, Striga, Macrophomina

Nigeria (Ousmane Boukar et al.)

Earliness, Striga, Heat

Senegal (Ndiaga Cisse et al.)

Drought, Striga, Nematode, Macrophomina

Mozambique (Rogerio Chiulele et al.)

Heat, Large seed, Grain quality

Ideotype

Single seed descent

• Developed F2 from elite parents (Suvita 2, IT97K-499-35).

• Genotyped 300 F2s with 164 poly SNPs every 2 cM interval.

• Phenotyped F2:3 families

₋ Pobe (low-yielding site)

₋ Saria (high-yielding site)

Pope

Saria

Burkina Faso – MARS example

300 mm

1000 mm

QTL detection

VuLG1 VuLG2 VuLG3 VuLG4 VuLG5 VuLG6 VuLG7 VuLG8 VuLG9 VuLG10 VuLG11

Yield Yield

Yield Grain size

Grain size

Favorable alleles from IT97K-499-35

Favorable alleles from Suvita 2

QTL IciMapping: http://www.isbreeding.net/

http://www.isbreeding.net/

Striga

YLD-4 YLD-6 YLD-8 GDW-3 GDW-11 Rsg 3

243 0.83 1.00 0.50 0.50 1.00 1.00 1.00 Yes

71 0.83 1.00 1.00 0.50 1.00 0.50 1.00 Yes

281 0.79 1.00 0.50 0.98 0.50 1.00 0.73 Yes

51 0.75 1.00 0.50 1.00 1.00 0.50 0.50 Yes

190 0.75 0.50 1.00 0.98 0.50 1.00 0.50 Yes

154 0.71 0.50 1.00 1.00 0.50 0.50 0.73 Yes

112 0.42 1.00 0.50 0.50 0.50 0.00 0.00 No

235 0.42 0.50 0.00 0.50 0.50 0.50 0.50 No

251 0.42 0.50 0.00 0.50 0.50 0.50 0.50 No

199 0.19 0.62 0.00 0.00 0.00 0.50 0.00 No

285 0.17 0.00 0.00 0.50 0.00 0.00 0.50 No

99 0.17 0.00 0.00 0.00 0.00 0.00 1.00 No

SelectedF2:3

family

Molecular

score

Yield QTLs Kernel-size QTLs

Select best families with OptiMAS (IBP tool, INRA)

The striga QTL is incorporated from prior publication 2002 Genome 45:787-793 (Ouédraogo et al.)

(Example list)

Striga

YLD-4 YLD-6 YLD-8 GDW-3 GDW-11 Rsg 3

71 F2 0.83 1.00 1.00 0.50 1.00 0.50 1.00

71-03 F4 0.92 1.00 1.00 0.50 1.00 1.00 1.00 Yes

71-02 F4 0.92 1.00 1.00 1.00 1.00 0.50 1.00 Yes

71-10 F4 0.92 1.00 1.00 1.00 1.00 0.50 1.00 Yes

71-01 F4 0.75 1.00 1.00 0.52 1.00 0.00 1.00 No

71-04 F4 0.75 1.00 1.00 0.00 1.00 0.50 1.00 No

71-05 F4 0.75 1.00 1.00 0.50 1.00 0.00 1.00 No

71-07 F4 0.67 1.00 1.00 0.00 1.00 0.00 1.00 No

SelectedPlant ID Cycle MSYield QTLs Kernel-size QTLs Plant Cycle 1_0853 1_0447 1_0146 1_0937 1_0031

2010-057-190 F2 GG GG AA GG GG

2010-057-190-01 F4 GG GG AA GG GG

2010-057-190-02 F4 GG -- AA -- GG

2010-057-190-03 F4 GG GG AA GG GG

2010-057-190-04 F4 GG GG AA GG GG

2010-057-190-05 F4 GA GC GA GC GA

2010-057-190-06 F4 GG GG AA GG GG

2010-057-190-07 F4 GG GG AA GG GG

2010-057-190-08 F4 GG GG AA GG GG

2010-057-190-09 F4 GG GG AA GG GG

2010-057-190-10 F4 GA GC GA GC GA

QTL combination

2 3

4 5 6

1

10 members per family/cross Leaf sampling KASP genotyping

Intercrossing OptiMAS to select best plants Outcrosses eliminated

OptiMAS summary: Frequency of favorable alleles at different selection steps in Burkina Faso MARS (on average and for each QTL)

P F1

F2

F4 F5

C1

C2

“Ideotype” Local check

Intercross to

recombine QTLs

Intercross progeny

MARS activities in Burkina Faso (Issa Drabo et al.)

Leaf sample

for genotyping

Acknowledgements University of California, Riverside

Roberts, Philip A.

Close, Timothy J.

Huynh, Bao Lam

Wanamaker, Steve

Lucas, Mitchell

Ndeve, Arsenio

Jansen Santos

Xu, Shizhong

Ehlers, Jeff D.

Diop, Ndeye N.

Muchero, Wellington

Pottorff, Marti

Hu, Zhiqiu

22

National Agricultural Research System (NARS)

Cisse, Ndiaga, ISRA-Senegal

Drabo, Issa, INERA-Burkina Faso

Tignegre, Jean-Baptiste, INERA-Burkina Faso

Joseph, Batieno T. Benoît – WACCI

Chiulele , Rogerio, EMU-Mozambique

International Inst. of Tropical Agriculture (IITA)

Boukar, Ousmane

Fatokun, Christian

Ofodile, Sam

LGC Genomics

Vyas, Darshna et al.

IBP-GCP

Delannay, Xavier et al.

PAG XXII 2014 – Genomic resources applied to marker-assisted breeding in cowpeas – BL Huynh

Technology

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