Genetic Dissection of Compositional &

Anatomical Characteristics Associated

with Biofuel Production in Maize

Natalia de Leon

Department of Agronomy – Univ. of Wisconsin, Madison

Collaborators and Sponsors

University of Wisconsin: Shawn Kaeppler

• Marlies Heckwolf• German Muttoni• Jillian Foerster• James Johnson

Edgar Spalding

• Svern Heckwolf

Michigan State University:

Robin Buell

Kevin Childs

Brieanne Vaillancourt

University of Minnesota:

Candy Hirsch

Nathan Springer

UW Graduate SchoolDupont-PioneerMonsanto

OverviewBiofuels and the Role of Maize

Cell Wall Composition and Stalk Anatomical Characteristics

Tackling the Complexity of the Maize Genome

Final Remarks

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Maize as a Model

Species

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Wheat

Brachypodium

Rice

Adapted from Hilu et al., 1999

SorghumMaize

Switchgrass

C4 photosynthesis

Maize54%

Soybean12%

Wheat10%

Hay23%

Sorghum1%

Percentage from total field crop area harvested (~300,000,000 acres)

Maize29%

Soybean25%

Wheat16%

Hay19%

Others10%

Sorghum1%

Field Crops Biomass Production in the US

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5

Field Crops Area Harvested 2011 (acres) Biomass Production Estimates 2011 (tons)

Percentage of biomass produced by these major field crops

Source: USDA - National Agricultural Statistics Service

What Can Be Learned From Forage Breeding?

Biorefineries Ethanol

Lorenz and Coors (2008)

Maize Stover Composition

Component % dry basis

Cellulose [β-1,4-glucan] 34-37 %

Hemicellulose 22-28 %

Xylan 80%

Arabinan 12%

Galactan 8%

Lignin 17-18 %

p-hydroxyphenyl (H) 4%

guaiacyl (G) 35%

syringyl (S) 61%

Aden et al. 2002; Humbird et al. 2011;

Grabber et al. 2004

Biomass Distribution on a Corn Plant

Stalk46%

Cob13%

Leafblade12%

Leafsheath12%

Husk-shank10%

Midrib6%

Tassel1%

Hansey et al, 2010

Plant Compositional and

Anatomical Data CollectionMarlies Heckwolf

WiDiv Population and Genotypic

(RNASeq) Data CollectionWiDiv association panel (~800 diverse lines)

Lines of Midwest origin, ex-PVPs, GEM and exotic lines

Restricted flowering – matures in northern latitudes

Whole seedling (higher number of expressed genes)

Targeted ~20M reads per genotype

Goal: SNPs, non-reference genes, expression

Sugar Release - WiDiV

Population

Glucose Release

(%/mg of dry biomass)

Pentose Release

(%/mg of dry biomass)

Two years

Two field reps per year

GWAS Sugar Release WiDiv

Glucose

Pentose

CBS domain containing protein

Transcription Factor

Transcription Factor Not

annotatedCBS domain

containing protein

Stalk Anatomy

Rind Thickness

Vascular Bundle Density

Long axis

Short axis

Stalk Diameter

Traits Analyzed:

WiDiv Anatomical Traits

Bundle Density

(number/cm2)

Rind thickness

(mm)

Long-axis

(cm)

GWAS WiDiv Anatomical TraitsATP synthase

subunit

ABC Transporter

Metabolite Transporter

Vascular Bundle Density

Rind Thickness

Stalk Anatomy in Genotypes with

Extreme Sugar Yieldhighlow

Pentose Release (%/mg of dry biomass)

Glucose Release (%/mg of dry biomass)

highlow

Stalk Anatomy in Genotypes with

Extreme Sugar Yield

Glucose Release in Genotypes

with Extreme Anatomical Traits

n= 12-15 per groups

p= 1*10-8

p= 2.78*10-10

p= 5.88*10-8

p= 3.1*10-4

Pan Genome/Transcriptome

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Pan genome – full complement of genes in a speciesCore genome – genes present

in all individualsDispensable genome – genes

found in only a subset of the individuals Dispensable and unique

genomePan, core and dispensable

transcriptome

Line1

Line2

Line3

Line4

Line5

Line6

Line7

Line8

Understanding Phenotypic Diversity

Associate genetic factors with traits of interest

Need to characterize all forms of genetic diversity that can underlie phenotypic diversity

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Sequence Level Variation (SNPs,

small Insertions/Deletio

ns)

Structural Variation (Copy Number Variation

– CNV & Presence/Absence

Variation – PAV)

Gene Expression Variation –Gene and Isoform level variation (quantitative expression differences)

RNA allows the study of most of these forms of variation

Candice Hirsch

Novel Transcript Discovery

Line1

Line2

Line3

Map all reads from all lines to the reference

sequence

Identified 3.2 billion unmapped reads

B73 Reference Genome

Assemble 400k unmapped reads per inbred (201.2M total)

Assembled transcript 1Assembled transcript 2Assembled transcript 3

>60% of reads mapped for each line

(85% identify and coverage)

• 31,398 loci containing 102,017 transcripts• 884 bp average size• Used longest transcript within a loci

• representative transcript assembly (RTA)

12.5 billion reads

Hirsch et al., submitted

8,681 high confidence novel transcripts(about 50% of those supported by sequence alignments)

Karyotype of the maize

chromosomes with

3,396 placed joint RTAs

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= RTAs expressed in

every line

= RTAs not expressed

in every inbred line but

expressed in B73

= RTAs not expressed

in every inbred line and not

expressed in B73

~77% of the RTAs with at

least one SNP - placed to a

single place in the reference

Transcriptome PAV

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0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0

1-10

11-5

0

51-1

00

101-

150

151-

200

201-

250

251-

300

301-

350

351-

400

401-

450

451-

490

491-

502

503

Nu

mb

er

of

Ge

ne

s

Number of Genotypes

Reference 5b Annotated Genes

Assembled Contigs

16,393

essential/cor

e transcripts

25,510 dispensable transcripts

Genotype-specific variants

Missing Heritability?

Juvenile to Adult Transition

www.glbrc.org

Poethig 2003. Phase Change and the Regulation

of Developmental Timing in Plants. Science

301:334-336

Phenotype: Last leaf with

juvenile (dull) wax -

TRANSITION

Jillian Foerster

Juvenility has been associated with lower recalcitrance & increased starch content

QTL and GWAS of Transition

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Novel gene

glossy15

A.

GRMZM2G362718

B.

GRMZM2G096016

GRMZM2G104610

C.Pairwise LD − Chr 2 Region

GRMZM2G362718

GRMZM2G362718.1

GRMZM2G362718.2

GRMZM2G362718.3

GRMZM2G362718.4

GRMZM2G362718.5

GRMZM2G362718.6

GRMZM2G362718.7

GRMZM2G362718.8

GRMZM2G362718.9

GRMZM2G362718.10

GRMZM2G362718.11

GRMZM2G362718.12

GRMZM2G362718.13

GRMZM2G362718.14

GRMZM2G362718.15

GRMZM2G362718.16

GRMZM2G362718.17

GRMZM2G362718.18

GRMZM2G362718.19

GRMZM2G362718.20

GRMZM2G362718.21

GRMZM2G362718.22

GRMZM2G362718.23

GRMZM2G362718.24

GRMZM2G362718.25

GRMZM2G362718.26

GRMZM2G362718.27

GRMZM2G362718.28

GRMZM5G851654

GRMZM5G851654.1

GRMZM5G851654.2

GRMZM5G851654.3

GRMZM5G851654.4

GRMZM5G851654.5

GRMZM5G851654.6

GRMZM5G851654.7

GRMZM5G851654.8

GRMZM5G851654.9

GRMZM5G851654.10

GRMZM5G851654.11

GRMZM5G851654.12

GRMZM5G851654.13

GRMZM5G851654.14

GRMZM5G851654.15

GRMZM2G095969

GRMZM2G095969.1

R2 Color Key

0 0.2 0.4 0.6 0.8 1

PhysicalDistance:36.5Kb

?

-lo

g10

(p

-val

ue

)

FROM

PREVIOUS

WORK:

NAM Popn.

WiDiv

Panel

glossy15

A.

GRMZM2G362718

B.

GRMZM2G096016

GRMZM2G104610

C.Pairwise LD − Chr 2 Region

GRMZM2G362718

GRMZM2G362718.1

GRMZM2G362718.2

GRMZM2G362718.3

GRMZM2G362718.4

GRMZM2G362718.5

GRMZM2G362718.6

GRMZM2G362718.7

GRMZM2G362718.8

GRMZM2G362718.9

GRMZM2G362718.10

GRMZM2G362718.11

GRMZM2G362718.12

GRMZM2G362718.13

GRMZM2G362718.14

GRMZM2G362718.15

GRMZM2G362718.16

GRMZM2G362718.17

GRMZM2G362718.18

GRMZM2G362718.19

GRMZM2G362718.20

GRMZM2G362718.21

GRMZM2G362718.22

GRMZM2G362718.23

GRMZM2G362718.24

GRMZM2G362718.25

GRMZM2G362718.26

GRMZM2G362718.27

GRMZM2G362718.28

GRMZM5G851654

GRMZM5G851654.1

GRMZM5G851654.2

GRMZM5G851654.3

GRMZM5G851654.4

GRMZM5G851654.5

GRMZM5G851654.6

GRMZM5G851654.7

GRMZM5G851654.8

GRMZM5G851654.9

GRMZM5G851654.10

GRMZM5G851654.11

GRMZM5G851654.12

GRMZM5G851654.13

GRMZM5G851654.14

GRMZM5G851654.15

GRMZM2G095969

GRMZM2G095969.1

R2 Color Key

0 0.2 0.4 0.6 0.8 1

PhysicalDistance:36.5Kb

Association Mapping with SNP Markers

glossy15

A.

GRMZM2G362718

B.

GRMZM2G096016

GRMZM2G104610

C.Pairwise LD − Chr 2 Region

GRMZM2G362718

GRMZM2G362718.1

GRMZM2G362718.2

GRMZM2G362718.3

GRMZM2G362718.4

GRMZM2G362718.5

GRMZM2G362718.6

GRMZM2G362718.7

GRMZM2G362718.8

GRMZM2G362718.9

GRMZM2G362718.10

GRMZM2G362718.11

GRMZM2G362718.12

GRMZM2G362718.13

GRMZM2G362718.14

GRMZM2G362718.15

GRMZM2G362718.16

GRMZM2G362718.17

GRMZM2G362718.18

GRMZM2G362718.19

GRMZM2G362718.20

GRMZM2G362718.21

GRMZM2G362718.22

GRMZM2G362718.23

GRMZM2G362718.24

GRMZM2G362718.25

GRMZM2G362718.26

GRMZM2G362718.27

GRMZM2G362718.28

GRMZM5G851654

GRMZM5G851654.1

GRMZM5G851654.2

GRMZM5G851654.3

GRMZM5G851654.4

GRMZM5G851654.5

GRMZM5G851654.6

GRMZM5G851654.7

GRMZM5G851654.8

GRMZM5G851654.9

GRMZM5G851654.10

GRMZM5G851654.11

GRMZM5G851654.12

GRMZM5G851654.13

GRMZM5G851654.14

GRMZM5G851654.15

GRMZM2G095969

GRMZM2G095969.1

R2 Color Key

0 0.2 0.4 0.6 0.8 1

PhysicalDistance:36.5Kb

Association Mapping with Quantitative Expression

?-l

og1

0 (

p-v

alu

e)

-lo

g10

(p

-val

ue

)

WiDiv

Panel

Final RemarksConsiderable natural variation observed for sugar release and anatomical traits

Genotypes with low sugar yields have wider stalks, a thicker rind & less bundles

Ability to characterize different source of genetic diversity is critical to explain the observed phenotypic diversity

RTA are not represented in the reference and therefore appropriate sampling of those variants is missed in QTL and association analysis

Transcriptome level PAV is extensive and can predict genomic level PAV, in the case of maize

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Thank you

Questions?