Germplasm Enhancement of Maize
Racial and Allelic
Diversity
S. Flint-Garcia, USDA-ARS, Columbia, MO
- August 17, 2006 -
Evolutionary Genetics of Maize
Selection
Teosinte Landraces Modern Inbreds
• Domesticated from Zea • Plant architecture
mays ssp. parviglumis e.g. tb1
• Seed coat
• Single domestication event
e.g. tga1
in Mexican highlands
Doebley et al. (1997) Nature 386: 485-488
• 6,000~9,000 years ago Matsuoka et al. (2002) PNAS 99: 6080-6084
Wang et al. (2005) Nature 436: 714-719
Selection Screens
The Bottom Line for Diversity
Unselected Gene Domestication Gene Improvement Gene
Teosintes
Domestication
Maize
Landraces
Plant Breeding
Maize
Inbred Lines
Very low genetic diversity
2 - category do of
In which 4% (~1200)the maize genes
traits have been targets of selection.
Genes that contribute to agronomic have for YOUR trait belong?
genes responsible undergone selection
Population Statistical Analysis
• Diversity Statistics
– π = average number of pair-wise differences
per nucleotide site
0 differences in 66 (12*11/2) comparisons
Inbreds (N=12)
Teosinte (N=13)
42 differences in 78 (13*12/2) comparisons
Average πinbreds = 0.0065
Relative Loss of Diversity (π)
• Average πteosinte = 0.0098 ~ 66% diversity
retained
• Average πinbred = 0.0065
Neutral Gene -adh1 Proline DehydrogenaseAuxin response factor, ARF1
0.08
0.04 Inbreds Inbreds
Teosinte Inbreds Teosinte
0.06 Teosinte
0.02
0.03
π 0.02 π 0.04 π
0.01
0.01 0.02
0 0 0
1 1000 0 2000 (bp)
200 400 1 600 1000
(bp) 2000 3000 (bp)
Yamasaki et al. (2005) Plant Cell 17: 2859-2872
Wright et al. (2005) Science 308: 1310-1314
0.25 35
Teosintes
Landraces 30
0.2
Inbred Lines
25
0.15
% kernel wt
% total AA
20
15
0.1
10
0.05
5
0 0
T vs LR ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **
LR vs Inb ** ** ** ** ** ** ** ** ** ** **
T vs Inb ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **
henylalanine
Serine
Valine
Lysine
Methionine
Proline
Leucine
Glycine
Tyrosine
Cysteine
Tryptophan
Amino Acid
Alanine
Histidine
Arginine
Isoleucine
Threonine
Glutamic Acid
Aspartic Acid
Total
Trans-cinnamic
acid Lignin
PAL
Glucose Phenylalanine Tyrosine
Glycine Serine
O-Acetylserine 3-Phospho-
glycerate Prephenate
Cysteine synthase
Erythrose 4-P Chorismate
Cysteine mutase
2-isopropyl- Phosphoenol
malate pyruvate DAHP Shikimate Chorismate
Leucine synthase
Anthranilate
Pyruvate Pyruvate Alanine Synthase β Anthranilate
Valine
Acetohydroxy Acetyl-CoA Indole-3-glycerol
acid synthase phosphate
Asparagine
Isoleucine Tryptophan
Asparagine Aspartate Synthase β1
synthetase Amino-
transferase
2-Ketobutyrate Aspartate Oxalo- Tryptophan
acetate
Threonine deaminase
Aspartate Glutamate TCA Cycle
kinase
Threonine
Aspartate
4-seminaldehyde α-Keto- NH4
DHDP Arginine
Homoserine synthase glutarate Proline
4-phosphate
Cystathionine γ-synthase Cysteine 2,3-Dihydro- Glutamate Proline
dipicolinate dehydrogenase dehydrogenase
Cystathionine Glutamate
Homocysteine
Lysine
Glutamine
NO3– NO2– NH4
Methionine
Nitrate
Reductase Histidine
SAM synthetase I SAM synthetase II
Hexokinase
S-Adenosyl-
methionine (N:C sensing)
Adapted from Buchanan et al 2000
80
Teosintes
70
Landraces
60 Inbred Lines
50
Percent
40
30
20
10
0
Ash Carbohydrate Crude Fat Crude Fiber Moisture Crude Protein
LR vs Inb ** ** ns * ns **
T vs LR ** ** ** ** ** **
T vs Inb ** ** ** ** ** **
Testing for Phenotypic Effects
Unselected Domestication/ B73
B73
(Neutral) Gene Improvement Gene
Teosinte-B73
NILs B73
Teosintes
B73
Selection
Inbred Lines
Mu transposon B73
insertion knockout
B73
Effect of teo background
& teo allele
Effect of B73 allele
if additive gene action
B73 x teosinte BC1
3’
tall
7-24-06
7-24-06
Teosinte (ssp. parviglumis)
B73 x teosinte BC1
12’ 9’
6’ tall tall
tall
8-7-06 8-7-06
Teosinte BC2
Ames21814 PI384063 PI384065 PI384066
7-24-06 7-24-06 7-24-06 7-24-06
PI384071 Ames21889
7-24-06 7-24-06 8-7-06
8-7-06
Ames21785 Ames21786 Ames21789
7-24-06 8-7-06 8-7-06 8-7-06
Landraces Cateto
Costeno Chapalote Sulino
Bolita
Cristalino Conico Chalqueno
Norteno
Serrano Sabanero Guirua
Tuson Nal-tel Gordo Dzit
Pissccotunto Bacal
B73 x Landraces
7-24-06 8-7-06
CM37
R4
K148
Mo46
Ky228 Hi27
DE-3 NC344 K4 Mo47Yu796-NS
NC360 OH7B Mo45
A682
MO17
CO106
Mt42CMV3 B97
W401
NC362CI91B
NC262 NC222 A556 B103 DE811
NC364 NC258 MS153 B77
NC342CI187-2 CI3A W117HT Tzi25
Tzi16 B105
DE1 NC290A A641
SD40 A214N
NC236 B164 NC250 STIFF STALK
NON STIFF STALK DE-2 B57 CM7 I205 N7AN28HT H100 H84
C123 H105W
A635 B64
ND246 CO109 C103
CO125 B79
A632B68
H91
A634
B14A B84
Hy Ky21 CM174 B76
A661WD B104
CM105
CI21E A554B75
38-11
MS71 B37
Os420 NC260 N R229
Mo1W Mo44
R168
A679
NC294
C
A680
B73Htrhm
N192 B109 NC368 NC326 B73
32
NC324
NC314 NC292
NC322
NC330
W64A 8
Pa875 NC308
NC306
NC312 NC372
NC268
A619 CH9 H49 WF9
SD44 B46 B10
NC310
A239A188
Pa762OH43 T8 Pa880 C49A
Ky226 VA26 Va102 C49
Va35A654 W153R
Oh40B
Va14
Oh43E Va17 Va59 A659
R177
CI-7
Va22 W22
H95 W182B
Va99 PA91
H99
CI90C M14 33-16
Va85
CH701-30 NC33
VaW6 4226
NC232
L317 B115
MoG K55 R109B I137TN
CI66
CI44
NC230
CI31A MEF 156-55-281-1
CI64 IL677A
K64 M162W Ia5125 SWEET
E2558WN6 CORN
IA2132
T234 IL14H P39
L578 SC357
CML52 IL101
CML14 CML69
CML38 B52
Tzi11 CML287 EP1 F2
CML103 CML108 NC366
F7
SC213R CO255
CML9 GT112
CML61 NC238
CML254
CML5
CML314 T232 GA209
CML264
CML258CML10
Q6199 CI28A Mp339
B2
CML341
CML332
CML11CML261
CML45CML331 MS1334 U267Y
Mo24W D940Y
SG18
Sg1533
HP301
IDS69
IDS28F2834T
CML277 SA24
IDS91
M37W
CML322 CML238
CML321
Ki14
CML247 Ki2021 Ki11 A6 F44 4722
F6 I-29
CML157Q Ki44 Oh603
Ki43 CML328
Ki21 CML228
Ki2007 NC340
NC300 CML323
NC302 NC356
A272 CML92
CML218 Tx303
NC320
Ki3 CML77 NC332NC318
NC338
NC358 CML154Q NC334 SC55 A441-5
TROPICAL-SUBTROPICAL TZI18NC354 TZI10 NC370
NC264 POPCORN
Ab28A
Tzi9 CML220 Mo18W
TX601
NC350 CML349
CML158Q
CML333
NC304 CML91 TZI8
CML311
CML281 MIXED
NC346
NC296A
parvi-03
NC336
Linkage Mapping Association Mapping
Structured population Unstructured population
High power Low power
Low resolution High resolution
Analysis of 2 alleles Analysis of many alleles
Moderate marker density High marker density
Genome scan Candidate gene testing
Nested Association Mapping (NAM)
Structured sub-populations nested within an unstructured population
High Power
High resolution
Analysis of many alleles
Moderate marker density
NAM Population Development
The 26 founder inbreds capture 80-85% of the diversity
present in public maize inbreds (i.e. 302 assoc. pop.)
Non-Stiff Stalk Mixed Tropical
B97 M37W CML103
Ky21 Mo18W CML228
M162W Tx303 CML247
MS71 CML277
Oh43 Other CML322
Oh7B HP301 CML333
IL14H CML52
Mo17 (IBM) P39 CML69
Ki11
Ki3
NC350
NC358
Tzi8
NAM
F1s
SSD
25 DL
2
1
200
B97
CML103
CML228
CML247
CML277
CML322
CML333
CML52
CML69
Hp301
×
Il14H
B73
Ki11
Ki3
Ky21
M162W
M37W
Mo18W
MS71
NC350
NC358
Oh43
Oh7B
P39
Tx303
Nested Association Analysis
Tzi8
Yu, Holland, McMullen, and Buckler (in progress)
CM37
R4
K148
Mo46
Ky228 Hi27
DE-3 NC344 K4 Mo47Yu796-NS
NC360 OH7B Mo45
A682
MO17
CO106
Mt42CMV3 B97
W401
NC362CI91B
NC262 NC222 A556 B103 DE811
NC364 NC258 MS153 B77
NC342CI187-2 CI3A W117HT Tzi25
Tzi16 B105
DE1 NC290A A641
SD40 A214N
NC236 B164 NC250 STIFF STALK
NON STIFF STALK DE-2 B57 CM7 I205 N7AN28HT H100 H84
C123 H105W
A635 B64
ND246 CO109 C103
CO125 B79
A632B68
H91
A634
B14A B84
Hy Ky21 CM174 B76
A661WD B104
CM105
CI21E A554B75
38-11
MS71 B37
Os420 NC260 N R229
Mo1W Mo44
R168
A679
NC294
C
A680
B73Htrhm
N192 B109 NC368 NC326 B73
32
NC324
NC314 NC292
NC322
NC330
W64A 8
Pa875 NC308
NC306
NC312 NC372
NC268
A619 CH9 H49 WF9
SD44 B46 B10
NC310
A239A188
Pa762OH43 T8 Pa880 C49A
Ky226 VA26 Va102 C49
Va35A654 W153R
Oh40B
Va14
Oh43E Va17 Va59 A659
R177
CI-7
Va22 W22
H95 W182B
Va99 PA91
H99
CI90C M14 33-16
Va85
CH701-30 NC33
VaW6 4226
NC232
L317 B115
MoG K55 R109B I137TN
CI66
CI44
NC230
CI31A MEF 156-55-281-1
CI64 IL677A
K64 M162W Ia5125 SWEET
E2558WN6 CORN
IA2132
T234 IL14H P39
L578 SC357
CML52 IL101
CML14 CML69
CML38 B52
Tzi11 CML287 EP1 F2
CML103 CML108 NC366
F7
SC213R CO255
CML9 GT112
CML61 NC238
CML254
CML5
CML314 T232 GA209
CML264
CML258CML10
Q6199 CI28A Mp339
B2
CML341
CML332
CML11CML261
CML45CML331 MS1334 U267Y
Mo24W D940Y
SG18
Sg1533
HP301
IDS69
IDS28F2834T
CML277 SA24
IDS91
M37W
CML322 CML238
CML321
Ki14
CML247 Ki2021 Ki11 A6 F44 4722
F6 I-29
CML157Q Ki44 Oh603
Ki43 CML328
Ki21 CML228
Ki2007 NC340
NC300 CML323
NC302 NC356
A272 CML92
CML218 Tx303
NC320
Ki3 CML77 NC332NC318
NC338
NC358 CML154Q NC334 SC55 A441-5
TROPICAL-SUBTROPICAL TZI18NC354 TZI10 NC370
NC264 POPCORN
Ab28A
Tzi9 CML220 Mo18W
TX601
NC350 CML349
CML158Q
CML333
NC304 CML91 TZI8
CML311
CML281 MIXED
NC346
NC296A
parvi-03
NC336
Linkage Mapping
Quantitative Trait Locus (QTL)
Mapping of Stalk Traits
• Identify genomic regions that contribute to
variation and estimate QTL effects
Position (cM)
100
110
120
130
140
50
60
80
90
30
40
70
10
20
Parent 1 Parent 2
0
9
8
Genotype 7
Phenotype
LOD Score
6
5
F1 4
F2 population 3
Composite 2
1
Interval Mapping 0
b1520
b1225
b2077
b2248
b2277
m231
u1622
u1552
Flint-Garcia et al. (2003) Crop Sci. 43: 13-22
Association Mapping
Association Analysis
A C G A G 1.3m
A C G A T 1.4m
A T A A G 1.5m
C T A G T 1.8m
A T G G T 2.0m
A C G G G 2.0m
• Utilize natural populations
– Exploit extensive ancestral recombination
• Evaluate several alleles simultaneously
Linkage Disequilibrium (LD)
dwarf3
Maize
Landraces 100 kb
Arabidopsis 250 kb
• Correlation between polymorphisms
• Extent of LD in the population determines
the resolution of association analysis
0 10 kb 20 kb 30 kb 40 kb 50 kb
Flint-Garcia et al. (2003) Ann. Rev. Plant Biol. 54: 357-374.
Association Analysis
Methodology
Choose
Candidate Gene
Choose Germplasm
PCR Amplify
Choose & Sequence
Obtain Independent
Target Genome-wide
Trait Marker Data Contig and Align
Sequences
Evaluate Trait Estimate Identify
in Replicated Population Polymorphisms
Trials (T) Structure (Q) (C)
Association Analysis
T=Q+C+ε
Population Structure and
Associations
G G G G G T T T G T T T
Andes U.S.
10 200
8 180
Plant Height
Kernel Hue
160
6
140
4
120
2
100
P << 0.001 P = 0.04
0 80
T G T G
True association Non-functional association