A genome-wide SNP scan for QTL associated with adult plant rust resistance

in East African bread wheat

G. Macharia1 , S. Chao2 and J. Anderson1

1University of Minnesota, Department of Agronomy and Plant Genetics, ST PAUL MN

2USDA-ARS Biosciences Research Laboratory, FARGO ND

BGRI Workshop_2012

Outline

• Section 1: General overview, definitions

• Section 2: Materials, Methods, Pre-analyses

• Section 3: Association mapping results

• Section 4: Research highlights

BGRI Workshop_2012

Section 1

BGRI Workshop_2012

Source: Tester and Langridge, 2007

Cereal Production targets

• 37% increase required to meet 2050 demand

BGRI Workshop_2012

yield gap

Wheat: projecting ahead

• Wheat needs to contribute 25% by 2025

1966-79 1984-94 1995-050

1

2

3

4 3.6

2.8

1.1

Global wheat yield growth

Period

Rate

(%)

• At 1.1% growth, wheat can only contribute 17%Data source: CIMMYT

BGRI Workshop_2012

Confronting the yield gap

BGRI Workshop_2012

Source: CIMMYT, 2011

BGRI Workshop_2012

Breeding for Durable resistance

• Key to circumvent rust-associated losses

• An opportunity to tap cutting edge techniques Marker assisted recurrent selection Genomic selection Association mapping

• Gene/QTL discovery a pre-requisite

BGRI Workshop_2012

Association Mapping

• Identification of significant statistical association between a phenotypic trait and molecular marker based on LD

BGRI Workshop_2012

Linkage Disequilibrium

Source: Hamblin et al, 2011

• Non-random co-inheritance of alleles at different Loci

…higher marker density

…greater resolutionBGRI Workshop_2012

Population Structure

P3

P1 P2

• Subpopulations at differing allele frequencies

Mapping panel

• Could arise from adaptation, domestication

• Creates LD between unlinked loci (false positives)

BGRI Workshop_2012

Section 2

Materials, Methods & pre-analyses

BGRI Workshop_2012

Bread wheat mapping panel

• 160 Kenyan lines• 70 Ethiopian

Historicals to recent releases

• 37 Founders

• Additionally, 35 Minnesota lines

BGRI Workshop_2012

Phenotyping Trait Location Year

Stem rust APR St Paul, MN 2010 St Paul, MN 2011 St Paul, MN 2012 Njoro, Ken 2011 Njoro, Ken 2012

Leaf rust APR St Paul,MN 2010 St Paul, MN 2011 St Paul, MN 2012 El Batan, MX 2011 Crookston, MN 2012

BGRI Workshop_2012

BGRI Workshop_2012

Modified Cobb Scale Severity (%) × Infection type = Coefficient of infection0 - 100 R (0.0) MR (0.4) e.g. 60MS MS (0.8) (60 x 0.8) = 48 S (1.0)

Disease scoring• 2 to 3 scores taken for each line

• Last score converted to Coefficient of infection

Stem rust

BGRI Workshop_2012

Leaf rust

BGRI Workshop_2012

Genotyping

• 9000 SNP Illumina’s infinium assay

• GenomeStudio® for ‘Allele calling’

BGRI Workshop_2012

Polymorphic Ambiguous

BGRI Workshop_2012

• Low quality SNPs, MAF < 0.05 eliminated

• 5987 polymorphic SNPs used

Genotyping

Population Structure (Q)

• Estimated using 235 SNPs >4 cM apart

• Software: ‘STRUCTURE v.2.3.4’ (Pritchard et al., 2000)

• K=1…10 subpopulations assumed

Each K= 4 replications

BGRI Workshop_2012

‘CIMMYT_1’ ‘North_ Am.’ ‘CIMMYT_2’ ‘Landraces’ ‘Mixed’

Subdivisions inferred

• Four, plus a ‘Mixed group’

Mean Fst = 0.1329 BGRI Workshop_2012

Principal Component Analysis

• 3 sets (each 500 random SNPs) used

BGRI Workshop_2012

Kinship (K)Based on 5965 SNPs

BGRI Workshop_2012

Linkage Disequilibrium (LD)

• Software: TASSEL v3.0(maizegenetics.net)

• 2547 unlinked SNPs

• r2 used to estimate LD

• r2 plotted against genetic distance

BGRI Workshop_2012

LD decay

Estimated LD extent

A- 13cM B- 18cM D- 11cM

BGRI Workshop_2012

Section 3

Association mapping results

BGRI Workshop_2012

Mixed-effects Linear Model

y=Qv + Mα + Kµ + e

y: vector of phenotypeQ: structure matrixv: random effects due to structureM: vector of marker genotypeα: marker fixed effect

K: Kinship matrixµ: fixed effects due to ancestrye: residual effectsImplemented in TASSEL v.3 (maizegenetics.net)

BGRI Workshop_2012

Njoro stem rust_2011

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown

Chromosome

FDR=0.01

BGRI Workshop_2012

Njoro stem rust_2012

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown

Chromosome

FDR=0.01

BGRI Workshop_2012

St Paul stem rust_2010

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D 5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

St Paul stem rust_2011

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D 5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

St Paul stem rust_2012

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

St Paul leaf rust_2010

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

St Paul leaf rust_2011

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

St Paul leaf rust_2012

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

El Batan leaf rust_2011

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

Crookston leaf rust_2012

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown

FDR=0.01

Chromosome

BGRI Workshop_2012

BGRI Workshop_2012

Summary: Map positions of Significant markers

BGRI Workshop_2012

163.7

2B

Stem rust (FDR=0.01)

124.4

143.4

211.7

3B 4A

5.72

75.9

4B

5A

17.145.9

66.7

156.9

6B193.3

UnknownUnknownUnknown

Njoro_2011 St Paul_2010 St Paul_2011 St Paul_2012

16.7

3D

R2 = 6% to 15%

BGRI Workshop_2012

123.8

1A

Leaf rust (FDR=0.01)

3B 5A

28.1

136.3

387.2

7B

184.3

57.7

El Bata_2011 St Paul_2010 St Paul_2011 Crksto._2012

3A

R2 = 6% to 13%

1B

13.1

1D 2B 5B

94.1

6A

115.6

137.0

6B

28.5 32.2

7D

68.1 UnknownLr61

Lr9,Lr14

Section 4

Research highlights

BGRI Workshop_2012

BGRI Workshop_2012

• Utility of AM for APR QTLs demonstrated

• The 9K chip assay invaluable (90K better!)

• > 20 significant associations (needs validation)

AcknowledgementsGermplasm

• Peter Njau (K.A.R.I.)• Solomon Gelalcha (E.A.R.I.)• Harold Bockelman (USDA ARS NSGC)

Field Experiments

• Ravi Singh (CIMMYT)• Yue Jin (USDA ARS CDL)• Jim Kolmer (USDA ARS CDL)• Matt Rouse (USDA ARS CDL)

Integrated SNP Map• Eduard Akhunov et al (Kansas St Univ.)

FundingStudy Leave

BGRI Workshop_2012

Adviser• Jim Anderson (Univ. of Min.)

Thank you

BGRI Workshop_2012

BGRI Workshop_2012

Summary: Detected Stem Rust ‘APR’ QTLs

SNP marker Chrom. Pos Environment logpvalue markerR2wsnp_Ex_c23509_32746909 5A 66.7 Njoro_2011 3.7746503 0.1002736wsnp_Ku_rep_c102220_89250165 5A 17.11 Njoro_2011 3.7220425 0.0979537wsnp_Ex_c23509_32746806 5A 67.23 Njoro_2011 3.5750289 0.0915756wsnp_Ex_c22018_31193171 2B 163.67 Njoro_2011 3.1322173 0.084855wsnp_Ex_c2718_5038502 5A 45.9 Njoro_2011 3.0466039 0.0772597wsnp_Ex_c1383_2652398 6B 193.34 Njoro_2011 3.0078695 0.0741373wsnp_Ex_c3044_5620102 2B 163.67 Njoro_2011 3.0067282 0.0827768wsnp_Ex_c2718_5038582 5A 65.62 Njoro_2011 2.9157554 0.0766549wsnp_BE490763A_Ta_1_3 unknown unknown St Paul_2010 3.2363714 0.1497071wsnp_Ex_rep_c70265_69211592 4B 75.95 St Paul_2010 3.2237278 0.1489716wsnp_BE405849A_Ta_1_1 5A 156.98 St Paul_2010 3.1839175 0.1466602wsnp_Ex_c7489_12809914 4A 5.72 St Paul_2011 3.2203025 0.06896wsnp_Ex_c5457_9632050 3B 143.44 St Paul_2011 3.0865325 0.0656068wsnp_Ra_rep_c70864_68811286 unknown unknown St Paul_2012 4.3207025 0.0967225wsnp_Ex_c9758_16120551 unknown unknown St Paul_2012 4.0542393 0.0916218wsnp_Ex_c13505_21253168 3B 211.65 St Paul_2012 3.6055684 0.0793914wsnp_JD_rep_c53979_36270209 3B 124.4 St Paul_2012 3.1090116 0.0650714wsnp_BE444579D_Ta_2_3 3D 16.66 St Paul_2012 3.0200302 0.0643022

False Discovery Rate =0.001 Total= 18 R2 = 6% to 15%

BGRI Workshop_2012

SNP marker Chrom. Pos Environment LogPvalue markerR2wsnp_Ex_c8588_14419007 1A 123.8 St Paul_2010 3.9900017 0.1163485wsnp_Ku_c13229_21142792 1B 13.07 ElBatan_2011 3.287268 0.0807828wsnp_Ex_c1130_2166731 1D 57.66 ElBatan_2011 3.6755068 0.0935952wsnp_Ex_c16720_25268525 1D 57.66 ElBatan_2011 3.6755068 0.0935952wsnp_Ex_rep_c108057_91436561 1D 57.66 ElBatan_2011 3.6755068 0.0935952wsnp_Ra_c58860_60407020 2B 387.23 St Paul_2011 3.2171866 0.0889992wsnp_Ex_rep_c104884_89459472 3A 136.29 ElBatan_2011 3.6811409 0.094023wsnp_Ex_rep_c67033_65490126 3B 31.01 Crookst._2012 3.4344695 0.0766409wsnp_Ex_rep_c67625_66279491 3B 28.39 Crookst._2012 2.9931129 0.0588606wsnp_Ex_c31799_40545376 5A 184.34 St Paul_2011 3.1406512 0.0656255wsnp_Ku_c6464_11320381 5B 94.06 St Paul_2011 3.5574842 0.0770563wsnp_BF202329A_Ta_2_2 6A 115.65 ElBatan_2011 3.0862858 0.0746064wsnp_BM134512A_Ta_2_2 6A 115.65 ElBatan_2011 3.0862858 0.0746064wsnp_Ex_c9502_15748469 6A 137.04 ElBatan_2011 3.1096994 0.0765279wsnp_Ex_rep_c115803_95396724 6B 28.49 ElBatan_2011 4.8781808 0.1316431wsnp_Ex_c11106_18002976 7B 32.15 Crookst._2012 3.0027851 0.0588665wsnp_Ku_c27221_37168065 7D 68.1 Crookst._2012 3.0509121 0.059267wsnp_Ex_c23850_33089300 unknown unknown ElBatan_2011 4.5646064 0.121684wsnp_be490219A_Ta_1_1 unknown unknown ElBatan_2011 3.5737042 0.0904964

Summary: Detected Leaf Rust ‘APR’ QTLs

False Discovery Rate =0.001 Total= 19 R2 = 6% to 13%

BGRI Workshop_2012