Factors Influencing the Composition/Metabolic Profile of Food from Plants

Sherry Flint-Garcia, PhDUSDA Agricultural Research ServiceSherry.Flint-Garcia@ars.usda.gov

Conflict of Interest Statement

As a federal employee, I have no conflicts of interest to declare.

Outline

Domestication Plant Breeding and Biotechnology Maize Case Study

– Domestication and Diversity– Genetics of Kernel Composition– Composition and Metabolites

Appendix Slides– Overview of Genetics, Terms, and Methods

1. Domestication

Domestication: “Adaptation of plants and animals to intimate association with human beings”

Domestication Syndrome

Domesticates:– More robust plants– Synchronous flowering– Increased apical dominance– Non-shattering– Loss of seed dormancy– Larger fruits/grains

Peanut

Corn

Rice

CoffeeSoybean

Hops

Pistachio

Sorghum

DomesticatesProgenitors

USDA-ARS

Wheat

Complex evolutionary history due to polyploidizationand distinct market classes

USDA-ARS

Einkornwheat

Emmer wheat

Breadwheat

Durum (pasta) wheat

Ae. speltoides (BB)

T. tauschii(DD)

T. turgidum(AABB)

T. urartu (AA)

T. spelta(AABBDD)

T. aestivum(AABBDD)

Tomato

Details of domestication are unclear: likely Ecuador/Peru and Mexico. “Pre-domesticate” was a cherry tomato. Brought to Europe by Columbus or conquistadors <1544. Model system for genetic studies.

USDA-ARSPI 647555

USDA-ARS

PI 347239PI 634844

Wild Ancestor: Solanumpimpinellifolium Pre-domesticate:

S. lycopersicumssp cerasiforme

Domesticate: S. lycopersicumssp lycopersicum

2. Plant Breeding and Biotechnology

Plant Breeding: “The art and science of plant improvement”Biotechnology: “The use of living systems and organisms to develop or

make useful products”

First Plant Breeders

Thousands of years ago– Likely women who selected plants that they liked.

Hundreds of years ago– Farmers had their favorite “family” variety = “heirloom.”

1926 Pioneer Hi-Bred was founded– First commercial hybrid corn seed company.

Plant Breeding

Selection (artificial) of plants with desirable traits.– Often done without knowledge of the genes controlling the trait(s).

Decreasing Unfavorable Alleles.– Susceptibility to diseases and insects, susceptibility to drought,

unadaptedness. Increasing Favorable Alleles.

– Increased yield and better quality, resistance to disease, insects and environmental stresses.

This process has typically produced safe food for over 100 years.

Plant Breeding

• Changing the phenotypic mean of a population; two methods

Select the “best”

individuals to form

the next generation

Original population

200

Progeny population

218

Parent 1Low yield

Insect resistant

F1

Parent 2High yieldSusceptible

F2

Select the “best” individuals to form the next generation

New Inbred LineHigh yieldInsect resistant

Plant Biotechnology

• Requires knowledge of the gene underlying the trait.• Gene(s) excised from donor species, combined with promoter of

choice to drive gene expression.• The construct is transformed into recipient plant.• Backcrossed six times to recover 99.2% recurrent parent.

Commercial Variety

Donor Plant

NewVariety

Traditional Plant BreedingDNA is a strand of genes, much like a strand of pearls.Traditional plant breeding transfers many genes at once.

Desired gene Many genes are transferred

Plant BiotechnologyUsing plant biotechnology, you can add a single gene to the strand without the extra baggage (linkage drag).

Donor Commercial Variety

Desired gene Desired gene

× +A single gene is transferred

Improved Commercial

Variety

3. Maize Case Study

What Can We Learn From Maize Diversity Using Breeding, Genetics, and Genomics?

Maize is an important crop! Domestication and Diversity Inbred Lines Teosinte, the Wild Ancestor

Maize Diversity Project

www.panzea.org

Maize diversity is greater than the difference between human and chimps

Silent Diversity; Tenallion, et al. (2001) PNAS

1.42% 0.09%1.34%

Maize Domestication

Domesticated from Zea mays ssp. parviglumis ~9,000 years ago in SW Mexico.Intermediate form of landraces, i.e., populations adapted to specific microclimates

and/or human uses.

Artificial Selection

Teosinte (ssp parviglumis) Inbred LinesLandraces

The Maize Genome

Ten chromosomes. Ancient tetraploid – whole genome duplication ~ 5-12 mya. B73 is the reference inbred for the genome. 32,500 predicted genes in B73 – more likely ~40,000. Nearly 85% of the B73 genome sequence is annotated as

transposable elements. B73 may capture only ~70% of the low-copy gene fraction of all maize

inbred lines.

B73

Schnable, et al. (2009) Science

Genome-Wide Diversity Maize HapMap.v1, HapMap.v2, HapMap.v3

– 83 million SNPs Two historically important inbred lines in breeding (B73 and Mo17)

differ by more than 20 million SNPs! Related species exhibit synteny (same gene order) along

chromosomes. However, we see lots of non-collinearity in maize!

Gore, et al. (2009) Science; Chia, et al. (2011) Nature Genetics; Bukowski, et al. (2018) GigaScience; Fu and Dooner (2002) PNAS

GCCATC ---CGA

GCGATCGGGCGA

SNP In/Del

Domestication Genes in Maize

• teosinte branched 1 – plant branching• teosinte glume architecture 1 - hard fruitcase

• Whole genome analysis of HapMap.v2– Stronger selection during domestication than improvement– > 1000 genes experienced selection

Wang, et al. (2005) Nature; Hubbard, et al. (2002) GeneticsHufford, et al (2012) Nature Genetics

Impact of Artificial Selection on Diversity

Teosintes

MaizeLandraces

MaizeInbred Lines

Unselected (Neutral) Gene Domestication Gene Improvement Gene

Artificial Selection

Plant Breeding

Domestication

98% (~49,000)maize genes

2% (~1,000) maize genes Commercial Corn Breeding

?

Diverse Maize Inbred LinesNested Association Mapping (NAM)

Linking Phenotype to Genotype

Can we identify the genes controlling phenotypic variation?

QTL (Quantitative Trait Locus) Mapping– Identify genomic regions that contribute to variation– Very loose definition of a large region

Association Mapping– Test specific genes for their involvement in the trait– Much more precise way to test (if done correctly!)

Association Panel

302 Inbred Linesfrom around theworld

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

0.1

NC33

B115

I137TN

81-1

MEF 156-55-2

IL677A

Ia5125

IA2132

IL101

F2EP1

F7

CO255

NC366

B52

SC213R

NC238

GT112

Mp339

GA209

D940Y

T232

U267Y

CI28A

B2

F2834T

Mo24WMS1334

IDS28

I-29

SA24

4722

SG18Sg1533

IDS91IDS69

F6

F44

Ab28A

CML328

Oh603

A441-5

CML323

SC55

NC264NC370

NC320

NC318

NC334

NC332

CML92

CML220

CML218A272

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

NC354TZI18

A6

Ki44

Ki43

Ki2007Ki21

Ki2021

Ki14

CML238CML321

CML157Q

CML322

CML331CML261

CML341CML45CML11

CML10

Q6199

CML314

CML258

CML5CML254

CML61

CML264

CML9

CML108

CML287

Tzi11

CML38

CML14

SC357L578

T234

N6E2558W

K64

CI64

CI44

CI31ANC230

CI66

K55

R109BMoG

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

Tzi16Tzi25

B79

B105

N7A

SD40

N28HT

A641

DE811

H105W

A214N

H100

A635

A632

A634B14A

H91B68

B64

CM174

CM105B104

B84

NC250

H84

B76

B37

N192

A679

B109

NC294

NC368NC326

NC314

NC328 R229

A680

NC310

NC324

NC322NC330

NC372

B73Htrhm B73NC308

NC312 NC306

NC268

B46B10

A239

C49AC49

A188

W153R

A659

R177

W22

W182B

CI-7

33-16

4226

NC348

NC298

NC352

NC336

NC296

NC346NC296A

NC292

SWEET

POPCORNTROPICAL-SUBTROPICAL

STIFF STALK

NON STIFF STALK

MIXED Based on 89 SSR loci

MO17

Oh43E

teosinte

NC300

NAM Founders

25 Diverse Lineschosen to maximize diversity

0.1

NC33

B115

I137TN

81-1

MEF 156-55-2

IL677A

Ia5125

IA2132

IL101

F2EP1

F7

CO255

NC366

B52

SC213R

NC238

GT112

Mp339

GA209

D940Y

T232

U267Y

CI28A

B2

F2834T

Mo24WMS1334

IDS28

I-29

SA24

4722

SG18Sg1533

IDS91IDS69

F6

F44

Ab28A

CML328

Oh603

A441-5

CML323

SC55

NC264NC370

NC320

NC318

NC334

NC332

CML92

CML220

CML218A272

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

NC354TZI18

A6

Ki44

Ki43

Ki2007Ki21

Ki2021

Ki14

CML238CML321

CML157Q

CML322

CML331CML261

CML341CML45CML11

CML10

Q6199

CML314

CML258

CML5CML254

CML61

CML264

CML9

CML108

CML287

Tzi11

CML38

CML14

SC357L578

T234

N6E2558W

K64

CI64

CI44

CI31ANC230

CI66

K55

R109BMoG

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

Tzi16Tzi25

B79

B105

N7A

SD40

N28HT

A641

DE811

H105W

A214N

H100

A635

A632

A634B14A

H91B68

B64

CM174

CM105B104

B84

NC250

H84

B76

B37

N192

A679

B109

NC294

NC368NC326

NC314

NC328 R229

A680

NC310

NC324

NC322NC330

NC372

B73Htrhm B73NC308

NC312 NC306

NC268

B46B10

A239

C49AC49

A188

W153R

A659

R177

W22

W182B

CI-7

33-16

4226

NC348

NC298

NC352

NC336

NC296

NC346NC296A

NC292

SWEET

POPCORNTROPICAL-SUBTROPICAL

STIFF STALK

NON STIFF STALK

MIXED Based on 89 SSR loci

MO17

Oh43E

teosinte

NC300

NC350

P39

M37WHP301CML277

B97

CML103CML69

CML52

CML228

CML247

CML332

IL14H

Ky21

Ki11

Ki3

MS71

Mo18W

OH7B

M162W

Tx303

TZI8CML333

NC358

OH43

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

Nested Association Mapping

NAM combines the statistical power and mapping resolution from twodifferent QTL mapping methods– Explore diversity from around the

world Used to study many different

traits in maize

25 DL SSD

. . .

B97

CML103

CML228

CML247

CML277

CML322

CML333

CML52

CML69

Hp301

Il14H

Ki11

Ki3

Ky21

M162W

M37W

Mo18W

MS71

NC350

NC358

Oh43

Oh7B

P39

Tx303

Tzi8

⊗ ⊗ ⊗

⊗ ⊗ ⊗

⊗ ⊗ ⊗

Asso

ciat

ion

Linkage = QTL mapping

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

NAM Kernel Composition

Jason CookStarch

Amylose

Amylopectin

Fiber

Oil

Fatty Acid Profiles

Protein

Zeins

Amino Acid Profiles

Cook, et al. (2012) Plant Phys.

Kernel Composition QTL Mapping in Maize

0

10

20

30

40

50

60

70

80

LOD

StarchProteinOil

Chr. 1 2 3 4 5 7 8 9 10

“QTL for kernel oil content somewhere in here.”

Cook, et al. (2012) Plant Phys.

Genome Wide Association (GWAS)

1.6 Million HapMap.v1 SNPs projected onto NAM

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

RM

IP

Getting closer to saying “This genecontrols oil content”Cook, et al. (2012) Plant Phys.

Teosinte - The Wild Ancestor

Development of Teo Introgression Libraries

B73 parviglumis

BC1 BC2 BC3 BC4

F1 Hybrid: B73×parviglumis

Backcrossing of the F1 with B73 to create BC1, BC2, BC3, BC4;Each backcross removes 50% of the teosinte genome.

Liu, et al. (2016) Plant Genome

Kernel Composition QTL in Teosinte

Karn et al. (2017) G3

Avi KarnTeosinte NILs

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

Allelic effects (% starch)

Cook et al. (2012) Plant Phys. & Karn et al. (2017) G3

Teosinte NILs

NAM RILs

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

Application: Reintroduce Variation

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

Breeding hypothesis: We can improve modern corn by introducing variation.

Sucrose

UDP Glucose

ADP Glucose

Amylose Amylopectin

Sh1 : sucrose synthase

Sh2, Bt2 : ADP-glucose pyrophosphorylase

Su1 : DBEAe1 : SBE-IIB

Wx1 : GBSS

Selected Genes in Maize Starch Pathway; Whitt et al. (2002)

Gene ID Gene Name Possible Target TraitGRMZM2G348551 Su2; Sugary 2 StarchGRMZM2G394450 Ivr1; Invertase 1 StarchGRMZM2G089836 Ivr2; Invertase 2 StarchGRMZM2G110175 Bm1; Brown midrib 1 StarchAC196475.3_FG004 Bm3; Brown midrib 3 StarchGRMZM2G098298 Ccp1; Cysteine protease 1 ProteinGRMZM2G138727 Zp27; 27-kDa zein protein Protein & Amino AcidsGRMZM2G087612 SDP1; Sugar dependent1 Oil

Hufford et al. (2012) Nature GeneticsSchnable and Freeling (2011) Plos One

Do Teosinte Alleles Have Value for Improving Today’s Corn?

Traits that were relevant 9000, 1000, or 100 years ago may not be as relevant today.

Therefore the alleles selected 9000, 1000, or 100 years ago that persist in modern germplasm may not be optimal today.

Conclusions: Impact of Selection in Maize

Selection on the genome – Thousands of years during domestication– Over one hundred years during plant breeding– 25+ years during plant biotechnology

Modification of seed composition– Traditional breeding– Biotechnology

Never identified a deadly/toxic variety of corn– Despite tremendous genetic and phenotypic variation– Long history of safe use

Surveys of Metabolites in Diverse Maize Germplasm

27 Nested Association Mapping (NAM) founder inbreds27 Landrace (LR) inbreds created by selfing landracesBoth sets crossed with B73 to make hybrid seed

B73 Genome Inbred

Genetic Relationships Among the Maize Lines

Text in this font and color• First level bullet

− Second level dash

NAM Hybrids

Landrace Hybrids

LandracesNAM Inbreds

Venkatesh et al. (2015) J. Agric. Food Chem.

Compositional Variation in Diverse Maize

Venkatesh et al. (2015) J. Agric. Food Chem.

Compositional Variation in Diverse Inbred Lines

Wide range inproximatesamong inbredlines

Total Fat Total Carbohydrate

Venkatesh et al. (2015) J. Agric. Food Chem.

Compositional Variation in Diverse Inbred Lines

Two folddifference in some fatty acidsamong inbred lines

C18:3 (Linolenic Acid) C18:1 (Oleic Acid)

Venkatesh et al. (2015) J. Agric. Food Chem.

Inbred lines versus landraces: Gamma-Tocopherol

Nearly six-folddifferencein gamma-tocopherolacross inbred lines andlandraces

LandracesInbred Lines

Venkatesh et al. (2015) J. Agric. Food Chem.

Inbred lines versus landraces: Beta-Carotene

Huge rangeof variationfor beta-carotene

LandracesInbred Lines

Venkatesh et al. (2015) J. Agric. Food Chem.

Metabolite Profiles by GC-MS and NMR

Same samples as previous study GC-MS and NMR were used to examine metabolite profiles 675 total metabolites collected by GC-MS

– 504 met QC checks; 174 were annotated 33 total metabolites collected by NMR

– 17 amino acids, 7 organic acids, 5 sugars, 4 others

Partial least-squares discriminant analysis (PLS-DA) of the GC-MS data

Venkatesh et al. (2016) J. Agric. Food Chem.

Another source of variation: Xenia Effect (Pollen)

Corn is a cross-pollinated species (pollen travels in the air)

R-locus

Y-locus

Sh2-locus (sweet corn)

Ornamental Corn

Xenia Effect (Pollen Donor) on Amino Acids

β γα family

δ

Flint-Garcia et al. (2009) Theor App Genet Unpublished data, Flint-Garcia (USDA) and Angelovici (University of Missouri) labs

EmbryoEndosperm

Zeins

Zein Profiles Xenia ExperimentZeins are the storage proteins in the endosperm.

To Summarize

Maize is extremely genetically diverse. Maize is extremely phenotypically diverse.

– Plant-level metabolite level Genetics, Environment, GxE contribute to phenotypes. Many genes contribute to grain composition. Even pollen source affects grain composition.

To Summarize

For geneticists and breeders, the more variation the better!

When a farmer plants their corn, every hybrid seed is genetically the same. When the farmer harvests the seed, every seed is different.

References Bukowski, et al. (2018) GigaScience; 7:1–12 Chia, et al. (2011) Nature Genetics 40:803-

807 Cook, et al. (2012) Plant Phys. 158:824-834 Flint-Garcia, et al. (2005) Plant J. 44:1054–

1064 Flint-Garcia, et al. (2009) Theor App Genet

119:1129-1142 Fu and Dooner (2002) PNAS 99:9573–9578 Gore, et al. (2009) Science 326:1115–1117 Hubbard, et al. (2002) Genetics 162: 1927–

1935 Hufford, et al (2012) Nature Genetics 44:808-

811

Karn, et al. (2017) G3 7:1157-1164 Liu, et al. (2016) Plant Genome 9 Schnable, et al. (2009) Science 326:

1112−1115. Schnable and Freeling (2011) PLOS ONE 6:

e17855. Tenaillon, et al. (2001) PNAS 98:9161-9166 Venkatesh, et al. (2015) J. Agric. Food

Chem. 63:5282−5295 Venkatesh, et al. (2016) J. Agric. Food

Chem. 64:2162−2172 Wang, et al. (2005) Nature 436:714–719 Whitt, et al. (2002) PNAS 20:12959–12962

Appendix Slides: Overview of Genetics & Breeding

Genetics: “The study of heredity and the variation of inherited characteristics”

Breeding: “The art and science of the genetic improvement of plants”

Genetics Terms

Genotype - The genetic constitution of an individual organism Phenotype - The set of observable characteristics of an individual

resulting from the interaction of its genotype with the environment Trait - A genetically determined characteristic Allele - An alternative form of a gene Genetic Variation - Naturally occurring genetic differences among

individuals of the same species

Genetics 101

TranscriptionDNA RNA

TranslationProtein

Glutamine|

Glycine|

Leucine|

Threonine |

Histidine |

Arginine|

Serine

Nucleotides

CAGGGACUGACUCAUCGAUCA

Sugar-phosphatebackbone

(a) primary structure -amino acids in

linear order

Base Pairs

(b) secondary structure – helices or sheets of amino acids(c) tertiary structure – folding of the protein into a shape(d) quaternary structure – multiple proteins form a complex

Where Does Genotypic Variation Come From?

Mutations– Point mutations – Change or delete base pairs– Chromosomal – Polyploidy and larger scale mutations

Transposable Elements – “Jumping Genes”– Can completely disrupt gene function

Hybridization– Two different genomes come together

Recombination – Shuffling of genes during meiosis

AATCGCGTTAAATCCCGTTA

DNA Sequence Variation

SNP: Single Nucleotide Polymorphism

InDel: Insertion/Deletion

Where Does Phenotypic Variation Come From?

Genotype– Why fraternal twins look different

Environment– Why identical twins look different

Genotype x Environment– Complex interactions between

genetics and the environment

How Does Variation Change?

Usually variation (allele frequencies) is balanced, i.e., “in Hardy-Weinberg Equilibrium.”

Genetic Drift– Sampling error – Rarer alleles can disappear completely and thereby reduce

genetic variation. Selection – Natural or Artificial

– Increased survival and reproduction of some individuals Gene Flow

– Migration of individuals, bringing new alleles together

Types of Variation

Qualitative = Discrete Quantitative = Continuous

dwarf 8

Male sterile 8

Albino seedling

Cob Color

NativeRootworm Resistance

Seed Composition

Carotenoid (proviatmin A)

Yield

Quantitative Trait Locus (QTL) Mapping

• “Genome Scan”• Identify genomic regions that contribute to variation and estimate QTL

effects Parent 1

F1

Parent 2

F2

u162

2

u155

2

b227

7

m23

1

b224

8

b122

5

b207

7

b152

0

0

1

2

3

4

56

7

8

9

0 10 20 30 40 50 60 70 80 90 100

110

120

130

140

Chr 1, Position (cM)

LOD

Sco

reGenotypePhenotype

Composite Interval Mapping

Association Analysis

“Genome Scan” Utilizes natural populations

− Exploit extensive ancestral recombination

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 300…

Gene ABC

Association Analysis

“Genome Scan” Utilizes natural populations

− Exploit extensive ancestral recombination

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 300…

Gene ABC

Nested Association Mapping (NAM)

Genome scanStructured populationHigh powerLow resolutionAnalysis of 2 alleles

Association Mapping Candidate gene testing

Unstructured populationLow power

High resolutionAnalysis of many alleles

Nested Association Mapping (NAM)Structured families nested within an unstructured population

High powerHigh resolution

Analysis of many alleles

Linkage (QTL) Mapping