Using Genetics, Genomics, and Breeding to Understand Diverse Maize

Germplasm

Sherry A. Flint-Garcia United States Department of Agriculture–

Agriculture Research Service (USDA–ARS)

Corn breeding and genetic analysis of agronomic traits relies on phenotypic variation and genetic variation in the genes controlling these traits. My research program focuses on understanding genetic diversity in maize so we can mine beneficial alleles from the appropriate germplasm sources for continued corn improvement. The process of domestication that began 9000 years ago has had profound consequences on maize, where modern corn has moderately reduced genetic diversity across nearly all genes in the genome relative to teosinte, and severely reduced levels of diversity for key genes targeted by domestication. The question that remains is whether these reductions in genetic diversity have impacted our ability to make progress in corn breeding today. As an outcrossing species, maize has tremendous genetic variation compared to most other crops. The complementary combination of genome-wide association mapping (GWAS) approaches, large HapMap datasets, and germplasm resources are leading to important discoveries of the relationship between genetic diversity and phenotypic variation in inbred lines. However, among the traits targeted during domestication and breeding are many yield component traits, including number of ears, kernel row number, seed size, and kernel composition. Therefore, we must reintroduce variation from landraces and/or teosinte if we hope to learn how domestication has impacted these yield component traits, and yield itself.

Using Genetics, Genomics, and Breeding to Understand Diverse Maize Germplasm

Sherry Flint-Garcia USDA-ARS Columbia, MO

Outline Introduction to Maize Domestication & Diversity Inbred Lines Teosinte, the wild ancestor Breeding with Zea

Maize Domestication

Domesticated from Zea mays ssp. parviglumis

Single domestication event ~9,000 years ago in Mexico

Intermediate form of landraces; populations adapted across the Americas to specific microclimates and/or human uses

Artificial Selection

Teosinte (ssp parviglumis) Inbred Lines Landraces

Teosintes

Maize Landraces

Maize Inbred Lines

Unselected (Neutral) Gene Domestication Gene Improvement Gene

Artificial Selection

Plant Breeding

Domestication

98% (~49,000) maize genes

2% (~1,000) maize genes

Nested Association Mapping (NAM) Maize ATLAS Project

Teosinte NILs Teosinte Synthetic Zea Synthetic

Consequences of Artificial Selection

Germplasm Enhancement of Maize (GEM)

Inbred Lines

Linkage-Based QTL Mapping “Genome Scan” Identify genomic regions that contribute to

variation and estimate QTL effects

u162

2

u155

2

b227

7

m23

1

b224

8

b122

5

b207

7

b152

0

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)

LOD

Scor

e

Genotype Phenotype Statistics for Mapping

Parent 1

F1

F2 population

Parent 2

1.3m

1.5m

1.4m

1.8m

2.0m

2.5m

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

Line 1

Line 2

Line 3

Line 4

Line N …

Gene X

Association Analysis Utilize natural populations

Exploit extensive historical recombination

Candidate gene approach:

Ancestral chromosomes

Subject Population

Linkage vs. Association Analysis Linkage (QTL) Mapping Association Mapping Structured population BC, F2, RIL, etc

Analysis of 2 alleles High power Low resolution 10-20 cM (10-20 Mb in maize)

Genome scan Don’t know anything about the genetics

underlying the trait

Unstructured population Unrelated or distantly-related

Analysis of many alleles Low power High resolution 1000-5000 bp in maize *

* Candidate gene testing Pathway or candidates previously identified. Used as a validation method.

* Genome Scan Don’t know anything about the genetics underlying the trait. Used as a discovery method

* Depends on the species/population

Nested Association Mapping

NAM Founders

0.1

NC33

B115

I137TN

81-1

MEF 156-55-2

IL677A

Ia5125

IA2132

IL101

F2 EP1

F7

CO255

NC366

B52

SC213R

NC238

GT112

Mp339

GA209

D940Y

T232

U267Y

CI28A

B2

F2834T

Mo24W MS1334

IDS28

I-29

SA24

4722

SG18 Sg1533

IDS91 IDS69

F6

F44

Ab28A

CML328

Oh603

A441-5

CML323

SC55

NC264 NC370

NC320

NC318

NC334

NC332

CML92

CML220

CML218 A272

Tzi9

CML77

TZI10

CML311

CML349 CML158Q

CML154Q

CML281

CML91

parvi-14

parvi-36

parvi-03

parvi-49

parvi-30

NC356

TX601

NC340

NC304

NC338 NC302

NC354 TZI18

A6

Ki44

Ki43

Ki2007 Ki21

Ki2021

Ki14

CML238 CML321

CML157Q

CML322

CML331 CML261

CML341 CML45 CML11

CML10

Q6199

CML314

CML258

CML5 CML254

CML61

CML264

CML9

CML108

CML287 Tzi11

CML38 CML14

SC357 L578

T234

N6 E2558W

K64

CI64

CI44

CI31A NC230

CI66 K55

R109B MoG

L317

NC232

VaW6

CH701-30

Va85

CI90C M14

H99

Va99 PA91

Ky226

H95

Oh40B

VA26 Pa762

A619

Va22

Va17

Va14 Va59

Va102

Va35

T8

A654

Pa880

SD44

CH9

Pa875

H49

W64A

WF9

Mo1W

Os420

R168

38-11

NC260

Mo44

CI21E

Hy

A661

ND246

WD

A554

CO125

CO109

B75

DE-3

DE-2

DE1

B57

C123

C103

NC360

NC364

NC362

NC342

NC290A

NC262

NC344

NC258

CI91B

CI187-2

A682

K4

NC222

NC236

CI3A

K148

Mt42

MS153

W401

A556

B77

CM37

CMV3

W117HT

CM7

CO106

B103

R4

Ky228

B164

Mo46

Mo47

Mo45

Hi27

Yu796-NS

I205

Tzi16 Tzi25

B79

B105

N7A

SD40

N28HT

A641

DE811

H105W

A214N

H100

A635

A632

A634 B14A

H91 B68

B64

CM174 CM105

B104

B84

NC250

H84

B76

B37

N192

A679

B109 NC294

NC368 NC326

NC314

NC328 R229

A680

NC310

NC324 NC322 NC330

NC372

B73Htrhm

B73 NC308 NC312 NC306

NC268

B46 B10

A239

C49A C49

A188

W153R

A659

R177

W22

W182B

CI-7

33-16

4226

NC348

NC298

NC352

NC336

NC296

NC346 NC296A

NC292

SWEET

POPCORN TROPICAL- SUBTROPICAL

STIFF STALK

NON STIFF STALK

MIXED Based on 89 SSR loci

MO17

Oh43E

ssp. parviglumis

NC300

NC350

P39

M37W HP301 CML277

B97

CML103 CML69

CML52

CML228

CML247

CML332

IL14H

Ky21

Ki11

Ki3

MS71

Mo18W

OH7B

M162W

Tx303

TZI8 CML333

NC358

OH43

Flint-Garcia, et al. (2005) Plant J.

NAM Development

Yu, et al. (2008) Genetics; McMullen, et al. (2009) Science

Link

age

Map

ping

Association Mapping

Corn Tracker

Starch

Amylose

Amylopectin Fiber

Oil Fatty Acid

Profiles

Protein

Zeins

Amino Acid Profiles

Jason Cook

NAM Kernel Composition

NIR conducted by Syngenta Seeds, Inc.

Oil Composition QTL in NAM

0

10

20

30

40

50

60

70

80

90

- 500,000,000 1,000,000,000 1,500,000,000 2,000,000,000

LO

D

Joint Linkage Mapping - Oil

Physical Distance (bp)

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

LOD

Genetic Distance (cM)

Joint Linkage Mapping - Oil

Cook, et al. (2012) Plant Phys.

Chr. 1 10 9 8 7 6 5 4 3 2

Chr. 6 Oil Candidate: DGAT1-2

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 High allele has Phenylalanine insertion in C-terminus

Phenylalanine insertion

Oil 4.4% 5.3% 3.6% 3.9%

Cook, et al. (2012) Plant Phys.

NAM Genome Wide Association (GWAS)

1.6 Million HapMap.v1 SNPs projected onto NAM Bootstrap (80%) sampling to test robustness of models

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

- 500,000,000 1,000,000,000 1,500,000,000 2,000,000,000

Physical Distance (bp)

GWAS - Oil

RMIP

Cook, et al. (2012) Plant Phys.

DGAT 1-2 (Chr 6: 105,013,351-105,020,258)

Marker Trait Population Analysis Method BPP P-Value Effect M1 Oil 282 Assn. MLM (Q+K) - 1.2E-04 0.18 M2 Oil 282 Assn. MLM (Q+K) - 9.9E-04 0.16 M3 Oil NAM GWAS - Bootstrap 31 - 0.18 M4 Oil 282 Assn. MLM (Q+K) - 4.3E-05 0.19 M4 Starch NAM GWAS - Bootstrap 51 - -0.38 M5 Oil NAM GWAS - Bootstrap 67 - 0.13 M5 Starch NAM GWAS - Bootstrap 11 - -0.31

NAM Population: 24 HapMap.v1 SNPs in DGAT 281 Association Panel: 2 55K SNPs in DGAT (plus the 3 bp indel)

M1

M3 M5 M4

M2: Phe Insertion

Cook, et al. (2012) Plant Phys.

DGAT 1-2 (Chr6: 105,013,351-105,020,258)

Rare allele?

= B73 Genotype = Non-B73 Genotype

Cook, et al. (2012) Plant Phys.

M2 (indel)

M3 M5 M4 M1

Summary – NAM Kernel Composition Genetic Architecture of Kernel Composition Traits

Governed by many QTL (N = 21-26) with small to moderate effects

GWAS results confirm many QTL DGAT is our favorite gene, but we still don’t have the

complete story!

NAM In General We can identify the common alleles in maize, but still

have problems with rare alleles.

Cook, et al. (2012) Plant Phys.

Ames Plant Introduction Station Inbreds 2,815 inbred lines from the Ames PI Station Genotyping-by-sequencing (GBS) - 681,257 SNPs

Romay, et al. (2013) Genome Biology

Ames Plant Introduction Station Inbreds More than half of the SNPs in collection are rare!

Romay, et al. (2013) Genome Biology

Expired PVPs

77% 48%

42%

302 Association panel = 75%, NAM founders = 57%.

Teosinte - The Wild Ancestor

Development of Teo Introgression Libraries

B73 × teosinte (parviglumis)

10 accessions

F1 : B73 × teosinte

BC1 BC2 BC3 BC4 B73 F1 teosinte

Library Development 10 libraries of 887 BC4S2/DH Near Isogenic Lines (NILs) BC4S4 NILs: GBS & RAD sequencing = 33,000–600,000 SNPs Lines to be released in 2013

Z031E0035

Z035E0012

Illumina GoldenGate

768 SNP

assay

Library Coverage

c1 c2 c3 c4 c5 c6 c7 c8 c9 c10

10 maize-teosinte libraries 804 BC4S2 NILs and 83 BC4DH NILs Each line: 2.3 chromosomal segments 4.1% of the teosinte genome 3.3X genome coverage

KEY

0123456789

BC4S2 vs BC4DH from PI384071 donor

KEY

0123456789

c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH

. . 2 2 . . 3 1 7 2 . . 2 1 2 0 3 5 2 03 0 2 3 6 2 . . 6 2 . . 1 1 4 0 5 7 . .3 0 . . 7 3 3 2 7 2 . . . . 3 0 4 5 . .3 0 . . 6 2 3 0 . . . . 1 1 . . 4 5 2 03 0 3 3 6 2 . . . . 3 2 2 1 2 1 4 5 . .. . . . 6 2 2 0 5 0 4 2 2 1 1 1 5 5 . .. . 3 4 4 3 . . 5 0 . . 2 1 . . 4 3 3 03 0 3 4 4 3 . . . . 4 2 . . 1 2 . . . .. . 3 3 4 3 3 0 2 0 4 2 1 1 . . 3 3 . .. . 3 3 . . 2 0 3 0 3 2 2 1 1 2 4 3 1 0. . 2 3 5 4 3 0 . . . . 1 1 . . 3 1 . .1 0 2 3 5 4 . . 2 0 3 2 2 1 . . . . 1 02 0 2 2 . . 2 1 1 0 3 2 3 2 1 0 . . . .2 0 . . 6 1 4 1 1 0 4 2 4 2 1 0 . . 1 02 0 . . 7 1 3 1 1 0 . . 3 2 1 0 1 0 0 02 0 . . 7 1 4 1 1 0 4 2 4 2 1 0 2 1 1 02 0 3 2 . . 4 1 1 0 4 3 3 2 0 0 1 1 . .1 0 3 2 . . . . 2 0 3 2 2 2 2 0 . . 1 01 0 3 2 7 1 4 1 2 0 3 2 2 2 1 0 1 1 1 01 0 . . 8 1 . . 2 0 5 0 2 2 1 0 . . . .. . 5 1 8 1 4 1 3 0 4 1 . . 1 0 . . 2 02 0 5 2 . . 4 1 3 0 5 0 2 2 1 1 1 0 1 12 0 3 2 . . 4 1 3 0 . . 2 2 1 1 1 0 1 02 0 5 2 8 1 4 1 3 0 3 0 3 1 0 1 1 0 1 11 0 . . 8 1 4 1 2 1 . . 4 2 0 2 . . . .. . 5 2 . . 4 1 3 1 4 0 4 2 . . 1 0 . .2 0 5 1 8 1 3 1 3 1 4 0 3 2 0 1 2 0 2 1. . 5 2 7 1 . . 2 1 5 0 . . 1 2 2 0 2 12 0 . . 7 1 4 1 3 1 . . 3 1 0 2 1 0 2 1. . 5 2 . . 5 1 2 2 5 0 2 3 0 2 1 0 2 12 0 4 1 6 1 . . 3 2 5 0 4 3 . . 2 0 . .. . 2 1 7 2 5 1 3 2 7 0 3 2 . . 2 0 2 12 0 1 1 5 2 4 1 3 2 7 0 . . . . 1 0 2 13 0 3 2 . . . . . . . . 4 2 0 2 1 1 . .2 0 . . . . 5 1 4 2 5 1 4 2 1 3 . . . .3 0 . . 6 3 4 1 4 2 6 0 . . 1 3 . . . .2 0 3 2 . . 3 0 4 2 5 2 4 2 1 3 1 1 4 04 0 2 3 6 3 . . 4 2 6 2 3 1 1 3 1 1 4 04 0 3 3 5 4 5 1 4 2 6 2 4 1 1 4 1 1 . .4 0 2 2 . . 6 2 3 1 5 4 4 1 . . . . 4 03 0 3 2 6 3 . . 4 2 4 3 2 2 1 4 1 1 4 03 0 3 2 6 4 . . 3 0 6 2 2 2 1 4 1 0 3 03 0 3 2 6 4 . . . . 4 2 1 1 . . 1 1 . .. . 2 2 2 5 1 1 4 1 . . 0 1 4 4 2 0 2 13 0 3 2 6 4 1 1 4 1 5 2 . . 4 4 2 1 . .3 0 2 2 7 4 1 2 4 1 5 2 1 1 5 4 2 1 2 13 0 2 1 . . 1 1 4 1 5 2 5 3 2 2 4 1. . 2 1 6 4 . . 4 1 5 1 . . 2 2 . .3 0 3 1 6 4 1 3 4 1 5 1 5 4 2 2 4 13 0 3 1 6 6 3 3 4 1 4 0 5 4 . . 4 13 0 3 1 6 7 3 2 4 1 . . 5 4 3 23 0 3 1 6 7 3 3 4 1 2 0 4 4 2 22 0 3 0 5 6 3 3 3 1 . . 5 4 0 03 0 3 1 5 7 3 3 4 1 . . . . . .3 1 4 2 5 7 3 3 4 0 3 1 5 4 1 03 1 4 2 4 3 3 3 2 2 3 1 5 4 2 13 1 . . 4 3 . . . . 6 33 1 4 2 . . . . 2 1 4 23 1 3 1 4 1 3 2 2 2 4 33 1 4 2 . . 3 2 3 2 4 13 1 . . 4 2 3 3 . . 5 13 1 5 2 3 1 4 3 . . 6 2. . 5 2 2 1 3 3 2 2 6 22 3 4 2 2 1 . . 3 2 6 22 3 5 0 1 1 3 3 3 2 . .. . 6 3 1 2 1 2 3 1 . .. . 5 3 1 2 1 2 3 1. . . . . . 1 2 3 12 3 . . . . 1 3 2 12 3 3 3 . . 1 2 2 12 3 . . 3 5 . . 3 12 2 3 6 3 4 . . 6 1. . 2 6 2 4 3 3 . .. . 3 7 3 4 3 3 6 13 1 4 6 3 4 5 13 1 . . 3 4 5 0. . . . 3 4 5 1. . 4 7 4 4 6 13 2 3 7 4 4 5 13 2 5 6 . . 2 24 2 5 5 5 4 5 10 0 . . . . 6 04 2 4 4 5 4 . .5 1 8 3 3 2 6 06 1 8 3 3 2 7 05 1 6 3 7 06 3 . .6 3 . .5 3 7 04 3 7 04 2 7 0. . . .3 2 8 0. . . .3 2 7 1. . . .2 0 . .. . 6 23 1 5 2. . . .2 1 2 03 13 14 13 13 1. .5 14 14 15 1. .5 15 1. .5 12 1

Applications of Teosinte NILs

Application 1. Empirical Genetics 1000 Selected Genes

What do these selected genes do? What traits were targeted by artificial selection during

domestication/breeding? Are selected genes important?

Auxin response factor, ARF1

0

0.01

0.02

1000 2000 3000 1 (bp)

Dive

rsity

(π)

Inbreds Teosinte

Tillering/branching?

Application 2. QTL Mapping Phenotyped at up to 18 reps: Maturity, plant & ear height, kernel row number, kernel weight, kernel shape, leaf length-width,

Grain composition (protein, starch, oil)

Zhengbin Liu

Avi Karn

0102030405060708090

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

Days to Anthesis

Teosinte NILs

NAM RILs

Kernel Row Number Pop Add. Eff.(rows)Z029 -3.4Z030 -1.8Z031 -0.6Z032 -1.4Z033 -1.0Z034 -1.1Z035 -1.1Z036 -0.3Z037 0.2Z038 -0.9

Pop Add. Eff.(rows)Z029 ---Z030 -1.4Z031 -1.4Z032 -1.1Z033 -2.1Z034 -2.3Z035 -1.7Z036 -0.6Z037 -0.7Z038 -1.4

Seed Weight (50 kernels) Pop Add.Eff.(g)Z029 -1.2Z030 -0.9Z031 -0.6Z032 -1.2Z033 -0.4Z034 -1.3Z035 -2.3Z036 -1.2

Pop Add.Eff.(g)Z029 -0.9Z030 0.2Z031 -0.6Z032 0.8Z033 -1.5Z034 3.3Z035 2.4Z036 -0.2

Pop Add.Eff.(g)Z029 3.7Z030 0.0Z031 -1.2Z032 -1.2Z033 -0.6Z034 -1.1Z035 0.6Z036 -0.3

β γ

α family

δ

Application 3. Reintroduce Variation

Biological hypothesis: A loss of genetic variation results in a loss of phenotypic variation.

Breeding hypothesis: We can improve modern traits.

0

10

20

30

40

50

60

70

80

Carbohydrate Protein Fat

Perc

ent o

f Ker

nel W

eigh

t

Teosinte (N = 11) Landraces (N = 17) Inbred Lines (N = 27)

Flint-Garcia et al. (2009) TAG

seed size

Zein Profile

Embryo Endosperm

Zeins

Teosinte NIL Collaborations Hibbard –

Corn Rootworm Resistance

Buckler – Flowering

Brutnell – Shade Avoidance

Balint-Kurti – SCLB & GLS

Smith – Corn Smut

Tracy – Germination

AgReliant – Agronomic

Traits

Nelson – NCLB

Hoekenga – Iron Bioavailability

Harmon – Circadian

Clock

Dallo – Mal de Rio Cuarto Virus

Turlings – Terpenes & Armyworms

Baxter– Ionomics

Breeding With Zea

Synthetic Populations

Zea Synthetic

B73 & NAM parents are inbreds,

teosinte has NEVER been inbred

NAM Synthetic Teosinte Synthetic ×

Zea Synthetic Inbreeding Depression

Ginnie Morrison

Selfed (S1) F ≈ 0.5

Full Sib (FS) F ≈ 0

S0 Male S0 Female

924 pairs

38% B73, 2% each

NAM parent, 12% teosinte

Zea Synthetic Doubled Haploids

38% B73, 2% each

NAM parent, 12% teosinte

Anna Selby

Goal: 2000 DH First 800+ in 2013 Another 700 in Puerto Rico AgReliant producing more 2014 trial: MO, NC, NY, IA

Doubled Haploids (DH)

Per se Hybrids

Genotype by GBS

Identity by Descent (IBD)

Association Analysis &

Genomic Selection

“Crazy?” Idea

2014

Agronomics Fertilizer Density

Mechanization

?

Select only on yield

ideotype

7000 BC

Teosinte Synthetic 75% B73 (SS), 25% teosinte

Acknowledgements

www.panzea.org

NSF Maize Diversity Project

Syngenta Seeds AgReliant Genetics

Susan Melia-Hancock, Kate Guill, Jason Cook, Zhengbin Liu, Ginnie

Morrison, Christopher Bottoms, Avi Karn,

Anna Selby