7 GEM Cooperators Me..

Report
Genetic Architecture of
Kernel Composition in the
Nested Association
Mapping (NAM) Population
Sherry Flint-Garcia
USDA-ARS Columbia, MO
Outline
Development of NAM Population
Kernel Composition
Joint Linkage Mapping
Genome-Wide Association Mapping
Linkage-Based QTL Mapping
“Genome Scan”
Identify genomic regions that contribute to
variation and estimate QTL effects
140
130
120
110
100
90
80
70
60
50
40
30
20
Parent 2
0
Parent 1
10
Position (cM)
9
6
5
4
3
2
1
b1520
b2077
b1225
b2248
m231
b2277
0
u1552
F2 population
Composite
Interval Mapping
7
u1622
F1
8
LOD Score
Genotype
Phenotype
Linkage (QTL) Mapping
Genome scan
Structured population
High power
Low resolution
Analysis of 2 alleles
Association Mapping
Candidate gene testing
Unstructured population
Low power
High resolution
Analysis of many alleles
Nested Association Mapping
Structured families nested within an unstructured population
High Power
High Resolution
Analysis of many alleles
CM37
R4
K148
Ky228
NC344
DE-3
Oh7B
K4
NC360
A682
NAM
Founders
NC362
CI91B
NC262
NC342
NC364
NC258
CMV3
Mt42
Mo17
CI187-2
Yu796-NS
Mo45
B97
A556
CI3A
Hi27
Mo47
CO106
W401
NC222
Mo46
B77
B103
W117HT
Tzi16
Tzi25
DE811
B105
MS153
DE1
SD40
NC290A
A641
A214N
B164
NC236
DE-2
CM7
B57
N7A
N28HT
NC250
H100
STIFF STALK
H84
I205
C123
CO109
ND246
B64
H105W
A635
A632
C103
CO125
B79
H91
A634
B68
B84
B14A
Hy
Ky21
A661
CI21E
B76
CM174
WD
CM105
A554
B104
B75
38-11
B37
Os420
NC260
MS71
Mo1W
NC328
Mo44
A679
NC294
NC368
B109
N192
CH9
NC292
WF9
SD44
OH43
A239
A188
Pa762
VA26
Ky226
C49A
Va102
Oh43E
Va35
Va14
Oh40B
NC372
NC310
B10
B46
Pa880
T8
NC324
NC330 NC322
NC308
NC312 NC306
NC268
H49
B73
NC326
NC314
W64A
Pa875
A619
R229
A680
B73Htrhm
R168
Va59
C49
A654
W153R
A659
Va17
CI-7
R177
Va22
W22
H95
W182B
Va99
PA91
H99
M14
CI90C
NON STIFF STALK
33-16
Va85
CH701-30
NC33
VaW6
4226
NC232
L317
B115
R109B
MoG
I137TN
K55
CI66
CI44
NC230
81-1
CI31A
MEF 156-55-2
CI64
IL677A
M162W
K64
E2558W
Ia5125
N6
IA2132
T234
CML52
CML69
CML103
L578
CML14
IL14H P39
SC357
IL101
CML38
SWEET
B52
CML287
EP1
Tzi11
NC366
CML108
F2
F7
SC213R
CO255
CML9
GT112
CML61
NC238
CML254
CML5
T232
CML314
GA209
CML264
CML258
CML10
B2
CML11
CML45
CML341
CML261
CML331
CML332
Sg1533 SG18
Mo24W
IDS28
CML238
CML321
A6
Ki14
F2834T
M37W
CML157Q
SA24
F44
Ki11
Ki2021
F6
Ki44
4722
I-29
Oh603
Ki43
POPCORN
CML328
Ki21
D940Y
HP301
CML277
CML247
CML322
U267Y
MS1334
IDS69
IDS91
TROPICALSUBTROPICAL
Mp339
CI28A
Q6199
NC340
Ki2007
CML323
CML92
CML228
Ki3
NC338
TZI18
NC300
NC358
A272
NC356
NC302
NC354
CML218
CML77
NC332
Tx303
NC318
SC55
A441-5
NC334
CML154Q
NC370
TZI10
NC264
Ab28A
CML220
Tzi9
NC350
NC320
TX601
CML349
CML158Q
NC304
CML91
CML333
Tzi8
CML311
Mo18W
MIXED
0.1
Based on 89 SSR loci
CML281
NC296A NC346
parvi-03
NC336
NC296
NC352
NC348
NC298
parvi-30
ssp. parviglumis
parvi-14
parvi-49
parvi-36
Flint-Garcia, et al. (2005) Plant J.
NAM Development
B97



Linkage
Current genetic
map consists of:
CML103
CML228
CML247
CML277
CML322
4699 RILs
1106 SNP loci
CML333
CML52
CML69
Association
Hp301
Ki11
Ki3



Il14H
Ky21
Average marker
density - one
marker every 1.3
cM
M162W
M37W
Mo18W
MS71
NC350
NC358
Oh43
Oh7B
P39



Tzi8



Tx303
25 DL
SSD
Yu, et al. (2008) Genetics;
McMullen, et al. (2009) Science
Kernel Composition in NAM
Fiber
Starch
Amylose
Zeins
Amylopectin
Protein
Oil
Amino Acid
Profiles
Fatty Acid
Profiles
The Phenotypic Data
7 locations of NAM –
2006: MO, NY, NC, PR, FL
2007: MO, NY
Self pollinated seed samples
NIR analysis for starch, protein, and
oil content (% kernel - dry matter basis)
Two sweet corn families excluded
>6000 rows per location
Phenotypic Data Statistics
Heritability
(23 Families)
Trait Correlations
r
H2
Starch 0.85
Protein 0.83
Oil
0.86
Starch
Protein
Protein
Oil
-0.65 -0.40
0.32
NAM Analysis in SAS
Permutations for selection thresholds ~10-5
Joint stepwise regression; Proc GLMSelect
Family main effect & markers within families
Final model; Proc GLM
Estimate effects (P = 0.05)
Genome Scan; Proc Mixed
Maximum likelihood with background cofactors
Epistasis; all (611,065) pair-wise combinations
NAM Kernel Quality Architecture
80
70
60
LOD
50
Starch
Protein
Oil
40
30
20
10
0
Trait
N
R2(family)
R2(QTL)
Starch
Protein
Oil
21
26
22
28.7
25.8
44.5
58.1
59.9
69.0
R2(QTL+family)
59.1
61.0
69.7
No Epistasis
Observed at
the NAM Level
Sig. Alleles
N
(P = 0.05)
Starch
Protein
Oil
180
206
174
Min
Max
(%)
(%)
%
-0.5
-0.3
-0.1
0.0
0.1
0.3
0.5
-0.62 0.65
-0.38 0.34
-0.12 0.21
62.3
63.3
63.4
63.8
63.9
64.8
65.2
65.3
65.5
66.2
66.3
66.8
67.3
67.3
67.5
67.5
67.6
67.6
67.6
67.8
68.2
68.7
68.9
HP301
CML247
Ki11
CML322
Ki3
CML333
MS71
CML228
Tzi8
CML69
Oh7B
NC350
Ky21
CML52
CML277
Tx303
M37W
Mo18W
M162W
NC358
B97
Oh43
CML103
1_20.1
1_70.3
1_146
2_210.9
2_277.2
2_306.1
3_420.5
3_478.8
3_503.4
4_650.2
5_724.7
5_753.5
6_819.4
6_836.8
7_993.1
7_1013
8_1102.7
8_1141
9_1224.4
9_1245.5
10_1338.2
Starch
Additive
Allelic Effects
-0.52 -0.32 -0.30 -0.34
-0.31
-0.55
-0.45
0.56
-0.35
-0.33 0.51
-0.25 -0.30
B73
%
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
12.3
12.5
12.6
12.7
12.7
12.9
13.1
13.2
13.3
13.3
13.6
13.8
13.8
14.0
14.0
14.2
14.2
14.4
14.6
14.7
14.8
15.2
15.3
B97
NC358
CML277
Mo18W
M162W
Tx303
Ki3
Oh43
HP301
CML103
CML69
CML333
Ky21
MS71
CML228
Ki11
CML52
M37W
CML247
CML322
Oh7B
NC350
Tzi8
-0.10
-0.10
-0.18
0.10
-0.12
0.15
-0.27 -0.26 -0.46 0.25
0.25
-0.24 -0.40 -0.41 0.29 -0.62
-0.42
-0.28
-0.25
-0.37 -0.20
0.20
-0.44
-0.33
0.19 -0.33
-0.23
-0.25
-0.22 -0.26
0.28
0.23
-0.40 -0.47
-0.21 0.24 0.27
0.26 -0.26
-0.20
0.16
-0.25
0.11
0.15 -0.16
0.15
0.15
0.10
-0.12
-0.13
-0.34
0.32
0.18
0.21 -0.20
-0.19
0.33
0.16
-0.38
0.17
0.15 -0.15
0.12
0.34
0.13 -0.15
0.13
0.14
-0.19
-0.11
0.12
-0.15
-0.13
-0.15
-0.20
0.20
-0.10
0.15
-0.19 -0.36
0.12
-0.15
0.10
0.17
0.16
-0.13
-0.18
0.15
0.13
0.10
0.17
-0.11
0.13
0.11
-0.11
0.23
0.20 -0.13
0.14
-0.17
0.20
-0.16
0.14
-0.13
0.17
0.25
0.18
0.23
0.32
-0.12
0.20 -0.22
-0.11
-0.17
0.19
0.13
0.15
-0.13
0.22
-0.26
0.13
-0.12 -0.13
0.19 -0.17
0.18
0.13 -0.14 -0.20 -0.17
0.18
0.22
0.24
0.15
0.11
0.16
0.21
-0.15
-0.23
-0.24 -0.12
0.16
-0.12
-0.18
0.19
-0.45 -0.34
-0.27 0.23
0.22 0.24
-0.38
0.20
%
-0.15
-0.10
-0.05
0.00
0.05
0.10
0.15
0.18
-0.21 0.19
-0.25
-0.46
0.40
-0.20 -0.47
0.27 -0.21 -0.32
0.31 0.28
-0.52
-0.25 0.17
-0.27
-0.30
0.34
-0.22
-0.25
-0.25 0.22
0.40 -0.47
0.23
-0.27
-0.27
0.31 0.25
-0.23 -0.29 0.33
0.30
-0.34
-0.28 0.28
0.20 0.21
0.25
0.20
0.25
-0.29 0.22
-0.29
-0.26
-0.24
-0.35
-0.23 -0.23 0.36
0.23 -0.43
-0.55 0.34 -0.31 0.38 0.26 0.19 -0.55 -0.21 0.35
0.22
-0.27
0.30
-0.34
-0.37
0.43 0.27 -0.23
-0.20
-0.25 -0.32
0.27 -0.22 0.43
3.5
3.6
3.6
3.6
3.9
3.9
4.0
4.0
4.1
4.2
4.2
4.3
4.4
4.4
4.6
4.6
4.7
5.1
5.2
5.3
5.3
5.4
5.5
0.36
^
1_33.1
1_116.5
1_141.9
1_199.4
2_266.6
2_282.2
2_308.5
3_414.1
3_462.1
4_571
4_592
4_642.9
5_668
5_692.6
5_715.3
5_741.9
6_841.5
6_869.5
7_969
8_1135.1
9_1240.3
10_1355.8
10_1399.3
-0.27
0.29
-0.18
-0.11
-0.16
10_1353.5
10_1324.2
0.18
0.12 -0.19 -0.18 -0.14 -0.11
0.12
-0.14 -0.27
0.11 -0.23 -0.31
-0.23
0.20 -0.14 -0.11
0.24
0.14
-0.13
-0.11 -0.12
0.15
-0.16
0.14
-0.12
0.11
-0.22
0.13
-0.18 -0.13
0.13
-0.11
0.11
-0.25
0.20
-0.19
0.12
0.10
0.19
-0.22
-0.12
0.10
0.12
-0.11
0.15
0.23 -0.16 -0.22
0.15
-0.20
B73
0.10
0.21
0.15
-0.12
-0.13
0.15
-0.13
0.10
-0.13
-0.22
-0.18
0.13
-0.18 -0.22
0.17
0.12
0.15
0.39
0.38
0.19 -0.19 0.29 0.26
Oil
-0.18 -0.15
-0.17 -0.15
-0.11
-0.12 -0.34
9_1256.6
9_1222.6
8_1149.3
8_1128.9
-0.13
0.11
-0.26
-0.19
-0.25 0.50 0.20 -0.32 -0.50
-0.22 -0.28
-0.39 0.39
0.36
0.29 -0.25 -0.24 -0.26 -0.23
0.43
-0.21 -0.25
-0.28 0.42
-0.61 -0.33 0.28 0.65
-0.23 -0.41
-0.21
-0.32
-0.22 -0.20
^
0.15 -0.20 -0.11
-0.18
0.16
-0.19
8_1106.7
-0.15
-0.17
-0.18
0.15
7_1013
7_987.6
6_914.7
0.18
0.22
-0.10
6_862.1
6_836.8
6_819.2
5_756.6
5_723.4
4_595.3
4_575.4
3_503.4
3_420.5
2_355.8
2_297.3
2_255.7
1_162.2
1_137.6
1_56.5
-0.27
-0.22 -0.34
-0.38 0.50
B73
Protein
-0.38 -0.26 -0.20 -0.26
-0.28 0.47
Oh43
M37W
Ky21
CML247
NC358
Tx303
CML277
B97
Mo18W
MS71
HP301
CML103
M162W
CML52
Ki3
CML69
Ki11
CML228
Tzi8
Oh7B
NC350
CML322
CML333
0.11
0.05
-0.10
-0.06
-0.09
0.06 -0.05
0.04
-0.07 -0.08
-0.04
0.05
0.06
0.06
-0.11
-0.05
0.10 0.18
-0.06 -0.11
-0.06 0.07
-0.05
0.10 0.05
0.05
-0.05 0.05 -0.11 0.08
0.11
-0.09
-0.08
0.06
0.06
-0.07 0.06 -0.12 0.09
0.11
-0.07
0.07 0.07
0.21 0.07
0.05
-0.05
-0.09 -0.07
0.05
-0.07
0.06
-0.06
0.04
0.05
-0.06
-0.05 0.05
0.06 -0.10
-0.06
0.05 0.07 -0.10
0.05
0.06
-0.11
0.10 0.07 -0.05
0.08 0.06
0.06
-0.10
-0.05 0.07
-0.07
-0.05
-0.05
0.05
-0.07 0.07 0.07
0.10
-0.11
0.12
-0.06
0.08
0.04 0.08
0.07 0.09 -0.07 0.08
-0.05
-0.08
-0.04 -0.06
0.07
-0.09
0.07
0.12
0.04
-0.07 0.06 0.07 0.06 0.05 0.10 0.10
0.13
-0.07
0.05
0.12
0.09 0.07 0.12 0.12
0.08 0.06 -0.06
-0.05
0.12
0.12
-0.10
-0.06
0.12
-0.10
0.06
0.06 -0.05
0.05 0.11 0.11
0.07
0.08
-0.06
0.06
0.10 0.06
0.08
-0.09
-0.06
0.12
0.05 0.11
0.05
0.06
0.12
-0.05
-0.06
0.06 0.06 -0.11
0.08 -0.10 -0.10
0.16 0.06
0.14
-0.10 0.10
-0.04
0.10
0.09
0.10 -0.06 -0.06
0.05
0.10
0.08 0.05
0.11 -0.04
Validation Efforts
Near Isogenic Lines (NILs)
Genome Scan Association Analysis
Candidate Genes Association Analysis
Candidate
Floury1
Opaque2 Modifier/Mucronate
Brittle Endosperm1
DGAT1-2
Waxy1
Marker
m221
m261
m619
m708
m968
Chr.
2
2
5
6
9
Dist.
266.6
297.3
724.7
841.5
1224.4
Fine Mapping
Trait
Oil
Oil
Oil
Oil
Protein
Starch
Allele
Tx303
CML322
CML228
Tx303
CML103
Tzi8
Marker
m707
m401
m94
m565
m657
m353
Chr.
6
3
1
5
5
3
Dist. (cM)
841.5
462.1
116.5
692.6
756.6
420.5
Jason Cook
Trait
Oil
Protein
Starch
Oil
Starch
Estimated
Effect (%)
0.21
0.11
0.11
- 0.12
0.21
0.43
Genetic vs. Physical Distance
80
70
Joint Linkage Mapping - Oil
60
40
30
20
10
0
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
Genetic Distance (cM)
90
80
Joint Linkage Mapping - Oil
70
60
LOD
LOD
50
50
40
30
20
10
0
-
500,000,000
1,000,000,000
Physical Distance (bp)
1,500,000,000
2,000,000,000
Genome Wide Association (GWAS)
90
80
Joint Linkage Mapping - Oil
70
LOD
60
50
40
30
20
10
0
100.0 90.0
500,000,000
1,000,000,000
1,500,000,000
2,000,000,000 80
70
GWAS - Oil
80.0
60
BPP
BPP
70.0
50
60.0
50.0
40
40.0
30
30.0
20
20.0
10
10.0
0.0
0
-
500,000,000
1,000,000,000
1,500,000,000
Physical Distance (bp)
1.6 Million HapMap v1 SNPs projected onto NAM
Bootstrap (80%) sampling to test robustness
2,000,000,000
Chr. 6 Oil Candidate: DGAT1-2
Oil Effect (%)
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.10
M37W
Oh43
CML103
CML322
HP301
MS71
B97
NC358
CML69
Ki11
CML277
CML247
Mo18W
CML333
CML52
NC350
Ki3
CML228
Tzi8
M162W
Oh7B
Ky21
Tx303
Encodes acyl-CoA:diacylglycerol
acyltransferase
Fine mapped by Pioneer-Dupont
Zheng, et al. (2008) Nature Genetics
High parent = 19% oil
High allele = 0.29% additive effect
DGAT is the largest effect
kernel quality QTL in NAM
4.4%
5.3%
3.6%
3.9%
Phenylalanine insertion in
the C-terminus of the protein
DGAT 1-2 (Chr6: 105,013,351-105,020,258)
M1
M2: Phe Insertion
M3
Marker Trait
M1
Oil
M2
Oil
M3
Oil
M4
Oil
M4
Starch
M5
Oil
M5
Starch
Population
282 Assn.
282 Assn.
NAM
282 Assn.
NAM
NAM
NAM
M4
Analysis Method
MLM (Q+K)
MLM (Q+K)
GWAS - Bootstrap
MLM (Q+K)
GWAS - Bootstrap
GWAS - Bootstrap
GWAS - Bootstrap
BPP
31
51
67
11
M5
P-Value
1.2E-04
9.9E-04
4.3E-05
-
Effect
0.18
0.16
0.18
0.19
-0.38
0.13
-0.31
NAM Population: 24 Total HapMap.v1 SNPs in DGAT
Association Panel: 2 Total 55K SNPs in DGAT
DGAT 1-2 (Chr6: 105,013,351-105,020,258)
M1
M2: Phe Insertion
Oil Effect (%)
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.10
M37W
Oh43
CML103
CML322
HP301
MS71
B97
NC358
CML69
Ki11
CML277
CML247
Mo18W
CML333
CML52
NC350
Ki3
CML228
Tzi8
M162W
Oh7B
Ky21
Tx303
M3
M4
M5
= B73 Allele
= Non-B73 Allele
?
What’s Next for NAM?
NextGen sequencing of the 5000 NAM RILs
Potentially 30-50 Million SNPs
Identify very precisely where recombination
events are in the mapping population.
This will VASTLY improve the mapping
resolution of NAM and GWAS.
Conclusions
Genetic Architecture of Kernel Quality Traits
Governed by many QTL (N = 21-26)
Many QTL in common with prior studies
Effect sizes are small to moderate
Allele series are common
Genome Wide Association Studies (GWAS)
Results confirm many QTL and candidate genes
Resolution will improve with more markers on
NAM RILs (define recombination events)
What Does This Mean To You?
Identifying Functional Markers for MAS
(Distantly) Linked markers not accurate
Parent Selection = Allele Mining
Valuable alleles are often masked.
Selection for specific alleles is more accurate
than selecting based on parental phenotype.
Acknowledgements
NSF Maize
Diversity Project
www.panzea.org
Syngenta
Joe Byrum & Kirk Noel
GEM Allelic Diversity Project
250
Races
B47 (SS)
PHZ51 (NSS)
Allele Library
2500 lines
Genome Wide Association Analysis
“mini-NAM”
Allele Mining

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