Racial and Allelic Diversity

S. Flint-Garcia, USDA-ARS, Columbia, MO

- August 17, 2006 -

Ger

mp

lasm

En

han

cem

ent

of M

aize

Evolutionary Genetics of Maize

• Domesticated from Zea

mays ssp. parviglumis

• Single domestication event

in Mexican highlands

• 6,000~9,000 years ago

• Plant architecturee.g. tb1

• Seed coat e.g. tga1

Doebley et al. (1997) Nature 386: 485-488Matsuoka et al. (2002) PNAS 99: 6080-6084

Wang et al. (2005) Nature 436: 714-719

Selection

Teosinte Modern InbredsLandraces

The Bottom Line for Diversity

Teosintes

MaizeLandraces

MaizeInbred Lines

Unselected Gene Domestication Gene Improvement Gene

Plant Breeding

Domestication

In which category do the genes responsible for YOUR trait belong?

2 - 4% (~1200) of maize genes have undergone selection

Selection Screens

Very low genetic diversity

Genes that contribute to agronomic traits have been targets of selection.

Te

os

inte

(N

=1

3)

Inb

red

s (

N=

12

)

Population Statistical Analysis

• Diversity Statistics– π = average number of pair-wise differences

per nucleotide site

42 differences in 78 (13*12/2) comparisons

0 differences in 66 (12*11/2) comparisons

Average πinbreds = 0.0065

Relative Loss of Diversity (π)

• Average πteosinte = 0.0098

• Average πinbred = 0.0065

~ 66% diversity retained

Neutral Gene -adh1

1 1000

0

0.01

0.03

0.02

0.04

2000 (bp)

Inbreds

Teosinte

π

Auxin response factor, ARF1

0

0.01

0.02

1000 2000 30001 (bp)

π

Inbreds

Teosinte

Yamasaki et al. (2005) Plant Cell 17: 2859-2872Wright et al. (2005) Science 308: 1310-1314

0

0.02

0.04

0.06

0.08

0 200 400 600 (bp)

InbredsTeosinte

Proline Dehydrogenase

π

0

0.05

0.1

0.15

0.2

0.25%

to

tal A

A

Teosintes

Landraces

Inbred LinesA

lan

ine

Arg

inin

e

Asp

artic

Aci

d

Cys

tein

e

Glu

tam

ic A

cid

Gly

cine

His

tidin

e

Iso

leuc

ine

Leu

cin

e

Lysi

ne

Met

hion

ine

Phe

nyla

lani

ne

Pro

line

Ser

ine

Thr

eoni

ne

Try

pto

pha

n

Tyr

osi

ne

Val

ine

T vs LRLR vs Inb

** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **

T vs Inb** ** ** ** ** ** ** ** ** **** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **

0

5

10

15

20

25

30

35

Tot

al

Am

ino

Aci

d

******

% k

ern

el w

t

TCA Cycle

α-Keto-glutarate

Oxalo-acetate

Glucose

3-Phospho-glycerate

Phosphoenolpyruvate

Pyruvate

Acetyl-CoA

O-Acetylserine

SerineGlycine

Cysteine

Cysteine synthase

Pyruvate

Valine

Leucine

2-isopropyl-malate

synthase

Lysine

2,3-Dihydro-dipicolinate

DHDP synthas

eCystathionine γ-synthase

Cystathionine

Homocysteine

Methionine

S-Adenosyl-methionine

SAM synthetase II

Cysteine

Aspartate Amino-

transferaseAspartate

Aspartate4-seminaldehyde

Asparagine

Asparagine synthetase

Aspartate kinase

Glutamate

Isoleucine

2-Ketobutyrate

Homoserine4-phosphate

Threonine

Threonine deaminase

Acetohydroxyacid synthase

Glutamate dehydrogenase

Glutamate

ArginineProline

Prolinedehydrogenase

Glutamine

Histidine

NitrateReductase

NH4NO3– NO2

–

NH4

Hexokinase(N:C sensing)

Trans-cinnamic acid Lignin

PAL

TyrosinePhenylalanine

Chorismate mutase

Prephenate

AnthranilateSynthase β Anthranilate

Tryptophan

Indole-3-glycerolphosphate

TryptophanSynthase β1

ChorismateShikimateDAHP

Erythrose 4-P

SAM synthetase I

Alanine

Adapted from Buchanan et al 2000

0

10

20

30

40

50

60

70

80

Ash Carbohydrate Crude Fat Crude Fiber Moisture Crude Protein

Per

cen

t

LR vs Inb

T vs LR**

T vs Inb****

******

ns

****

*****

ns

****

******

Teosintes

Landraces

Inbred Lines

Testing for Phenotypic Effects

Teosintes

Inbred Lines

Unselected (Neutral) Gene

Domestication/Improvement Gene

Selection

Mu transposon insertion knockout

B73

B73

B73

B73

B73Teosinte-B73

NILs

B73

Effect of teo background & teo allele

Effect of B73 allele if additive gene action

8-7-06

B73 x teosinte BC1

8-7-06

Teosinte (ssp. parviglumis)

7-24-06

B73 x teosinte BC1

6’ tall

7-24-06

3’ tall

12’ tall

9’ tall

7-24-06

Ames21814

7-24-06

PI384063

7-24-06

PI384065

7-24-06

PI384066

Teosinte BC2

7-24-06

Ames21889

8-7-067-24-06

PI384071

8-7-06

7-24-06

Ames21785

8-7-06

Ames21786

8-7-06

Ames21789

8-7-06

Landraces

Guirua Nal-tel

Conico

Costeno CristalinoNorteno Chalqueno

Chapalote Bolita

Cateto Sulino

Dzit Bacal

Gordo Pissccotunto Sabanero Serrano

Tuson

8-7-067-24-06

B73 x Landraces

NC33

B115I137TN

81-1MEF 156-55-2IL677A

Ia5125IA2132P39IL14H

IL101

F2EP1F7

CO255NC366

B52

SC213R

NC238

GT112

Mp339GA209

M37W

D940Y

T232

U267YCI28A

B2

F2834TMo24WMS1334

IDS28

I-29

SA24

4722

SG18Sg1533HP301

IDS91IDS69

F6F44

Ab28A

CML328Oh603

A441-5

CML323

SC55

Mo18WNC264NC370

NC320NC318NC334NC332

CML92Tx303

CML220

CML218A272

Tzi9

CML77

TZI8

TZI10

CML311

CML349CML333CML158Q

CML154Q

CML281

CML91

parvi-03

NC358NC356

TX601

NC340NC300

NC350NC304

NC338NC302

NC354TZI18

A6

Ki44Ki43

Ki11

CML228Ki3

Ki2007Ki21

Ki2021Ki14CML238CML321

CML157QCML247CML322

CML332CML331CML261

CML277

CML341CML45CML11CML10Q6199

CML314CML258

CML5CML254CML61

CML264

CML9CML108CML103

CML287Tzi11CML38CML69CML14

CML52SC357L578T234

N6E2558WM162WK64

CI64CI44

CI31ANC230CI66K55R109BMoG

L317NC232VaW6CH701-30

Va85CI90C M14

H99Va99 PA91

Ky226

H95Oh40BOh43E

VA26Pa762OH43

A619

Va22Va17Va14 Va59Va102

Va35

T8A654

Pa880SD44CH9

Pa875H49

W64A

WF9

Mo1WOs420

R168MS7138-11

NC260Mo44

CI21E

Hy Ky21A661

ND246

WDA554

CO125CO109

B75

DE-3

DE-2

DE1

B57C123

C103

NC360

NC364NC362

NC342NC290A

NC262

NC344

NC258CI91B

CI187-2

A682MO17K4

NC222

NC236

CI3A

K148

Mt42

MS153

W401A556B77

CM37

CMV3OH7B

W117HT

CM7

CO106

B103B97

R4Ky228

B164

Mo46Mo47Mo45

Hi27Yu796-NS

I205

Tzi16Tzi25

B79

B105

N7ASD40N28HT

A641DE811

H105W

A214NH100

A635A632

A634B14AH91B68

B64

CM174CM105B104B84

NC250H84

B76

B37

N192

A679B109 NC294

NC368 NC326NC314

NC328

R229A680

NC310

NC324NC322NC330NC372

B73HtrhmB73

NC308NC312NC306NC268

B46 B10A239

C49AC49A188

W153RA659R177

W22W182B

CI-7

33-16

4226

NC352NC336

NC296

NC346NC296A

NC292

SWEET CORN

POPCORNTROPICAL-SUBTROPICAL

STIFF STALKNON STIFF STALK

MIXED

Linkage Mapping Association Mapping Structured population

High power

Low resolution

Analysis of 2 alleles

Moderate marker density

Genome scan

Unstructured population

Low power

High resolution

Analysis of many alleles

High marker density

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.)

TropicalCML103 CML228CML247CML277CML322CML333CML52CML69

Ki11Ki3

NC350NC358

Tzi8

Non-Stiff StalkB97 Ky21

M162WMS71Oh43Oh7B

Mo17 (IBM)

MixedM37W

Mo18WTx303

OtherHP301 IL14HP39

Nested Association Analysis

Yu, Holland, McMullen, and Buckler (in progress)

25 DL

× B73

F1s

SSD

NAM

1

2

200

B97

CM

L103

CM

L228

CM

L247

CM

L277

CM

L322

CM

L333

CM

L52

CM

L69

Hp3

01

Il14H

Ki1

1

Ki3

Ky2

1

M16

2W

M37

W

Mo1

8W

MS

71

NC

350

NC

358

Oh4

3

Oh7

B

P39

Tx3

03

Tzi

8

NC33

B115I137TN

81-1MEF 156-55-2IL677A

Ia5125IA2132P39IL14H

IL101

F2EP1F7

CO255NC366

B52

SC213R

NC238

GT112

Mp339GA209

M37W

D940Y

T232

U267YCI28A

B2

F2834TMo24WMS1334

IDS28

I-29

SA24

4722

SG18Sg1533HP301

IDS91IDS69

F6F44

Ab28A

CML328Oh603

A441-5

CML323

SC55

Mo18WNC264NC370

NC320NC318NC334NC332

CML92Tx303

CML220

CML218A272

Tzi9

CML77

TZI8

TZI10

CML311

CML349CML333CML158Q

CML154Q

CML281

CML91

parvi-03

NC358NC356

TX601

NC340NC300

NC350NC304

NC338NC302

NC354TZI18

A6

Ki44Ki43

Ki11

CML228Ki3

Ki2007Ki21

Ki2021Ki14CML238CML321

CML157QCML247CML322

CML332CML331CML261

CML277

CML341CML45CML11CML10Q6199

CML314CML258

CML5CML254CML61

CML264

CML9CML108CML103

CML287Tzi11CML38CML69CML14

CML52SC357L578T234

N6E2558WM162WK64

CI64CI44

CI31ANC230CI66K55R109BMoG

L317NC232VaW6CH701-30

Va85CI90C M14

H99Va99 PA91

Ky226

H95Oh40BOh43E

VA26Pa762OH43

A619

Va22Va17Va14 Va59Va102

Va35

T8A654

Pa880SD44CH9

Pa875H49

W64A

WF9

Mo1WOs420

R168MS7138-11

NC260Mo44

CI21E

Hy Ky21A661

ND246

WDA554

CO125CO109

B75

DE-3

DE-2

DE1

B57C123

C103

NC360

NC364NC362

NC342NC290A

NC262

NC344

NC258CI91B

CI187-2

A682MO17K4

NC222

NC236

CI3A

K148

Mt42

MS153

W401A556B77

CM37

CMV3OH7B

W117HT

CM7

CO106

B103B97

R4Ky228

B164

Mo46Mo47Mo45

Hi27Yu796-NS

I205

Tzi16Tzi25

B79

B105

N7ASD40N28HT

A641DE811

H105W

A214NH100

A635A632

A634B14AH91B68

B64

CM174CM105B104B84

NC250H84

B76

B37

N192

A679B109 NC294

NC368 NC326NC314

NC328

R229A680

NC310

NC324NC322NC330NC372

B73HtrhmB73

NC308NC312NC306NC268

B46 B10A239

C49AC49A188

W153RA659R177

W22W182B

CI-7

33-16

4226

NC352NC336

NC296

NC346NC296A

NC292

SWEET CORN

POPCORNTROPICAL-SUBTROPICAL

STIFF STALKNON STIFF STALK

MIXED

Linkage Mapping

GenotypePhenotype

Composite Interval Mapping

Quantitative Trait Locus (QTL) Mapping of Stalk Traits

• Identify genomic regions that contribute to variation and estimate QTL effects

Flint-Garcia et al. (2003) Crop Sci. 43: 13-22

Parent 1

F1

F2 population

Parent 2

u1622 u1552 b2277 m231 b2248 b1225 b2077 b1520

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90 100

110

120

130

140

Position (cM)

LO

D S

core

Association Mapping

• Utilize natural populations– Exploit extensive ancestral recombination

• Evaluate several alleles simultaneously

1.3m

1.5m

1.4m

1.8m

2.0m

2.0m

T

C

C

T

T

C

A

G

G

A

G

G

G

G

T

T

T

G

A

A

A

C

A

A

A

A

A

G

G

G

Association Analysis

Linkage Disequilibrium (LD)

• Correlation between polymorphisms• Extent of LD in the population determines

the resolution of association analysis

MaizeLandraces < 1 kb

Diverse Inbreds 1.5 kb

Elite Inbreds > 100 kb

Arabidopsis 250 kb

Flint-Garcia et al. (2003) Ann. Rev. Plant Biol. 54: 357-374.

0 10 kb 20 kb 30 kb 40 kb 50 kb

dwarf3

Association Analysis

T = Q + C + ε

IdentifyPolymorphisms

(C)

Association Analysis Methodology

Choose Candidate Gene

PCR Amplify& Sequence

Contig and AlignSequences

Obtain Independent Genome-wide Marker Data

Choose TargetTrait

Choose Germplasm

Estimate Population

Structure (Q)

Evaluate Traitin Replicated

Trials (T)

100

120

140

160

180

200

Andes U.S.

P = 0.04

GT80

Pla

nt

Hei

gh

t

Non-functional association

Population Structure and Associations

G TG G G G TT T G T T

P << 0.001

T G0

2

4

6

8

10

Ker

nel

Hu

e

True association