Godwin Macharia, University of Minnesota

1. 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
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2. Outline
• Section 1: General overview, definitions
• Section 2: Materials, Methods, Pre-analyses
• Section 3: Association mapping results
• Section 4: Research highlights
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3. Section 1
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4. Cereal Production targets
yield gap
Source: Tester and Langridge, 2007
• 37% increase required to meet 2050 demand
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5. Wheat: projecting ahead
• Wheat needs to contribute 25% by 2025
Global wheat yield growth
4 3.6
3 2.8
Rate (%)
2
1 1.1
0
1966-79 1984-94 1995-05
Period
Data source: CIMMYT
• At 1.1% growth, wheat can only contribute 17%
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6. Confronting the yield gap
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7. Source: CIMMYT, 2011
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8. 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
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9. Association Mapping
• Identification of significant statistical
association between a phenotypic trait
and molecular marker based on LD
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10. Linkage Disequilibrium
• Non-random co-inheritance of alleles at
different Loci
…higher marker density
Source: Hamblin et al, 2011
…greater resolution
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11. Population Structure
• Subpopulations at differing allele frequencies
Mapping panel
P1 P2
P3
• Could arise from adaptation, domestication
• Creates LD between unlinked loci
(false positives)
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12. Section 2
Materials, Methods & pre-analyses
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13. Bread wheat mapping panel
• 160 Kenyan lines
• 70 Ethiopian
Historicals to recent releases
• 37 Founders
• Additionally, 35 Minnesota lines
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14. 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
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15. Disease scoring
• 2 to 3 scores taken for each line
• Last score converted to Coefficient of infection
Modified Cobb Scale
Severity (%) × Infection type = Coefficient of infection
0 - 100 R (0.0)
MR (0.4)
e.g. 60MS MS (0.8) (60 x 0.8) = 48
S (1.0)
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16. Stem rust
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17. Leaf rust
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18. Genotyping
• 9000 SNP Illumina’s infinium assay
• GenomeStudio® for ‘Allele calling’
Polymorphic Ambiguous
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19. Genotyping
• Low quality SNPs, MAF < 0.05 eliminated
• 5987 polymorphic SNPs used
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20. 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
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21. Subdivisions inferred
• Four, plus a ‘Mixed group’
‘CIMMYT_1’ ‘North_ Am.’ ‘CIMMYT_2’ ‘Landraces’ ‘Mixed’
Mean Fst = 0.1329
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22. Principal Component Analysis
• 3 sets (each 500 random SNPs) used
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23. Kinship (K)
Based on 5965 SNPs
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24. Linkage Disequilibrium (LD)
• Software: TASSEL v3.0 (maizegenetics.net)
• 2547 unlinked SNPs
• r2 used to estimate LD
• r2 plotted against genetic distance
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25. LD decay
A- 13cM B- 18cM D- 11cM
Estimated LD extent
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26. Section 3
Association mapping results
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27. Mixed-effects Linear Model
y=Qv + Mα + Kµ + e
y: vector of phenotype
Q: structure matrix
v: random effects due to structure
M: vector of marker genotype
α: marker fixed effect
K: Kinship matrix
µ: fixed effects due to ancestry
e: residual effects
Implemented in TASSEL v.3 (maizegenetics.net)
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28. Njoro stem rust_2011
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown
Chromosome
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29. Njoro stem rust_2012
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown
Chromosome
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30. St Paul stem rust_2010
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D 5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown
Chromosome
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31. St Paul stem rust_2011
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D 5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown
Chromosome
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32. St Paul stem rust_2012
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D 7A 7B 7D Unknown
Chromosome
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33. St Paul leaf rust_2010
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown
Chromosome
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34. St Paul leaf rust_2011
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown
Chromosome
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35. St Paul leaf rust_2012
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown
Chromosome
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36. El Batan leaf rust_2011
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown
Chromosome
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37. Crookston leaf rust_2012
FDR=0.01
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A4B4D5A 5B 5D 6A 6B 6D7A 7B 7D Unknown
Chromosome
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38. Summary:
Map positions of Significant markers
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39. Stem rust (FDR=0.01)
5.72
16.7
75.9
124.4
143.4
163.7
211.7
2B 3B 3D 4A 4B
17.1
45.9
Unknown
Unknown
66.7 Unknown
Njoro_2011
St Paul_2010
St Paul_2011
156.9 St Paul_2012
193.3
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5A 6B R2 = 6% to 15%

40. Leaf rust (FDR=0.01)
13.1
28.1
57.7
94.1
123.8 136.3
184.3
387.2
1A 1B 1D 2B 3A 3B 5A 5B
Lr9,Lr14
28.5
115.6 32.2
68.1
Lr61 Unknown
137.0
El Bata_2011
St Paul_2010
St Paul_2011
Crksto._2012
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6A 7D R2 = 6% to 13%
6B 7B

41. Section 4
Research highlights
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42. • Utility of AM for APR QTLs demonstrated
• The 9K chip assay invaluable (90K better!)
• > 20 significant associations (needs validation)
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43. Acknowledgements
Germplasm Field Experiments
• Peter Njau (K.A.R.I.) • Ravi Singh (CIMMYT)
• Solomon Gelalcha (E.A.R.I.) • Yue Jin (USDA ARS CDL)
• Harold Bockelman (USDA ARS NSGC) • Jim Kolmer (USDA ARS CDL)
• Matt Rouse (USDA ARS CDL)
Integrated SNP Map Adviser
• Eduard Akhunov et al (Kansas St Univ.) • Jim Anderson (Univ. of Min.)
Study Leave Funding
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44. Thank you
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45. BGRI Workshop_2012

46. Summary: Detected Stem Rust ‘APR’ QTLs
False Discovery Rate =0.001 Total= 18 R2 = 6% to 15%
SNP marker Chrom. Pos Environment logpvalue markerR2
wsnp_Ex_c23509_32746909 5A 66.7 Njoro_2011 3.7746503 0.1002736
wsnp_Ku_rep_c102220_89250165 5A 17.11 Njoro_2011 3.7220425 0.0979537
wsnp_Ex_c23509_32746806 5A 67.23 Njoro_2011 3.5750289 0.0915756
wsnp_Ex_c22018_31193171 2B 163.67 Njoro_2011 3.1322173 0.084855
wsnp_Ex_c2718_5038502 5A 45.9 Njoro_2011 3.0466039 0.0772597
wsnp_Ex_c1383_2652398 6B 193.34 Njoro_2011 3.0078695 0.0741373
wsnp_Ex_c3044_5620102 2B 163.67 Njoro_2011 3.0067282 0.0827768
wsnp_Ex_c2718_5038582 5A 65.62 Njoro_2011 2.9157554 0.0766549
wsnp_BE490763A_Ta_1_3 unknown unknown St Paul_2010 3.2363714 0.1497071
wsnp_Ex_rep_c70265_69211592 4B 75.95 St Paul_2010 3.2237278 0.1489716
wsnp_BE405849A_Ta_1_1 5A 156.98 St Paul_2010 3.1839175 0.1466602
wsnp_Ex_c7489_12809914 4A 5.72 St Paul_2011 3.2203025 0.06896
wsnp_Ex_c5457_9632050 3B 143.44 St Paul_2011 3.0865325 0.0656068
wsnp_Ra_rep_c70864_68811286 unknown unknown St Paul_2012 4.3207025 0.0967225
wsnp_Ex_c9758_16120551 unknown unknown St Paul_2012 4.0542393 0.0916218
wsnp_Ex_c13505_21253168 3B 211.65 St Paul_2012 3.6055684 0.0793914
wsnp_JD_rep_c53979_36270209 3B 124.4 St Paul_2012 3.1090116 0.0650714
wsnp_BE444579D_Ta_2_3 3D 16.66 St Paul_2012 3.0200302 0.0643022
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47. Summary: Detected Leaf Rust ‘APR’ QTLs
False Discovery Rate =0.001 Total= 19 R2 = 6% to 13%
SNP marker Chrom. Pos Environment LogPvalue markerR2
wsnp_Ex_c8588_14419007 1A 123.8 St Paul_2010 3.9900017 0.1163485
wsnp_Ku_c13229_21142792 1B 13.07 ElBatan_2011 3.287268 0.0807828
wsnp_Ex_c1130_2166731 1D 57.66 ElBatan_2011 3.6755068 0.0935952
wsnp_Ex_c16720_25268525 1D 57.66 ElBatan_2011 3.6755068 0.0935952
wsnp_Ex_rep_c108057_91436561 1D 57.66 ElBatan_2011 3.6755068 0.0935952
wsnp_Ra_c58860_60407020 2B 387.23 St Paul_2011 3.2171866 0.0889992
wsnp_Ex_rep_c104884_89459472 3A 136.29 ElBatan_2011 3.6811409 0.094023
wsnp_Ex_rep_c67033_65490126 3B 31.01 Crookst._2012 3.4344695 0.0766409
wsnp_Ex_rep_c67625_66279491 3B 28.39 Crookst._2012 2.9931129 0.0588606
wsnp_Ex_c31799_40545376 5A 184.34 St Paul_2011 3.1406512 0.0656255
wsnp_Ku_c6464_11320381 5B 94.06 St Paul_2011 3.5574842 0.0770563
wsnp_BF202329A_Ta_2_2 6A 115.65 ElBatan_2011 3.0862858 0.0746064
wsnp_BM134512A_Ta_2_2 6A 115.65 ElBatan_2011 3.0862858 0.0746064
wsnp_Ex_c9502_15748469 6A 137.04 ElBatan_2011 3.1096994 0.0765279
wsnp_Ex_rep_c115803_95396724 6B 28.49 ElBatan_2011 4.8781808 0.1316431
wsnp_Ex_c11106_18002976 7B 32.15 Crookst._2012 3.0027851 0.0588665
wsnp_Ku_c27221_37168065 7D 68.1 Crookst._2012 3.0509121 0.059267
wsnp_Ex_c23850_33089300 unknown unknown ElBatan_2011 4.5646064 0.121684
wsnp_be490219A_Ta_1_1 unknown unknown ElBatan_2011 3.5737042 0.0904964
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