Development and Characterization of Maize-Teosinte Introgression Libraries

Sherry Flint-GarciaUSDA-ARS Columbia, MO

OutlineIntroductionDevelopment of Teo NIL LibrariesApplications of Teo NIL LibrariesLofty Ideas

Evolutionary Genetics of Maize

Domesticated from Zea mays ssp. parviglumis, existed/exists in an intermediate form of landraces

Single domestication event in Mexican highlands

6,000~9,000 years ago

Selection

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

GENOMICS Nested Association Mapping (NAM)

BREEDINGGermplasm Enhancement

of Maize (GEM)

Teosinte Introgression LibrariesGENETICS

Impact of Artificial Selection

Development of Teo NIL Libraries

B73 × teosinte(parviglumis)

10 accessions

F1 :B73 × teosinte

teosinte BC1 BC2 BC3 BC4B73 F1

Library Development161 BC4S2 NILs: 2 libraries (done in 2009)643 BC4S2 NILs: 8 libraries (done in 2008) 83 BC4DH NILs: duplicated library887 Near Isogenic Lines (NILs)

Z031E0035

Z035E0012

Illumina GoldenGate

768 SNP assay

Introgression Stats# Avg. # Avg. % Avg. % Avg. %

Population Lines Regions Teosinte Het. TeozygousCoverage

Ames 21785 96 2.5 4.5 2.9 1.6 4.3 Ames 21786 87 2.3 4.4 2.9 1.5 3.8 Ames 21789 93 2.5 3.9 2.9 1.1 3.6 Ames 21814 77 2.1 3.8 2.5 1.3 2.9 Ames 21889 91 2.7 4.7 3.3 1.3 4.2 Ames 21809 79 2.7 4.9 3.1 1.7 3.8 Ames 21812 82 2.1 4.6 3.1 1.5 3.8 PI 384065 53 2.5 4.2 2.4 1.7 2.2 PI 384066 64 2.2 4.0 2.5 1.5 2.6 PI 384071 82 2.3 4.1 2.6 1.5 3.3 PI 384071 - DH 83 1.4 1.9 0.1 1.8 1.6

Expected Genotype Ratios for BC4S2 (sibbed): 95% BB, 3% BT het., 1.5% TT

Library Coverage

c1 c2 c3 c4 c5 c6 c7 c8 c9 c10

10 maize-teosinte libraries804 BC4S2 NILs and 83 BC4DH NILsEach line:

2.3 chromosomal segments4.1% of the teosinte genome

3.3X genome coverage

KEY

0123456789

BC4S2 vs BC4DH Coverage

KEY

0123456789

c1 c2 c3 c4 c5 c6 c7 c8 c9 c10S2 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

1. Empirical Genetics Questions1000 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 30001 (bp)

Div

ersi

ty (π

)

InbredsTeosinte

Tillering/branching?

?Auxin-mediatedProtein

Degradation

Could be any trait!

2. Evaluate Allele Series

Examine the range of allele effects of maize versus teosinte.

Validate QTL identified by NAM.

Trait ValueB73

MaizeAlleles

TeosinteAlleles

A Brief DigressionNested Association Mapping (NAM)

Flowering Time

Ed Buckler, Jim Holland, Mike McMullen, et al

Buckler, et al. (2009) Science

Flowering Genetic ArchitectureAt least 39 QTL explain flowering architectureGenetic architecture of maize is very complex

and very different than Arabidopsis.

Buckler, et al. (2009) Science

Many QTL with small Effects

333 alleles significant at P = 0.05.

Only 7 alleles had more than 24 hour effect.

Buckler, et al. (2009) Science

-150

-100

-50

0

50

100

150

Freq

uenc

y of

QTL

12h

Significant QTL

24h 36h

Increase Flowering Time

Decrease Flowering Time

Additive Allelic Effects

Allele Series

69% of QTLs had both alleles with both positive and negative effects.

Buckler, et al. (2009) Science

1 39……………….………………. QTL……………………….……….

Additive Allelic Effects (days)

“Gene Stacking”

Large differences in parental lines (32 day span) are the product of stacking large numbers of modest effect QTL.

Buckler, et al. (2009) ScienceEarliest Latest

Back To Teosinte

Flowering Evaluation~850 NILs from 10 librariesDays to Anthesis (DTA) from:

Ithaca, NY (2 reps), Rebecca Nelson & Oliver OttIthaca, NY (2 reps), Buckler labRaleigh, NC (8 reps), Peter Balint-Kurti Madison, WI (1 rep), Loren TrimbleSt. Paul, MN (1 rep), Nathan SpringerColumbia, MO (4 reps)

TOTAL 18 reps

0102030405060708090

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

0

10

20

30

40

50

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

Teosinte Introgressions vs. NAM

Additive Effect(days)

Teosinte NILs

1.1

NAMPhoto-period Gene?

-1.01.6

-1.13.2

-2.64.2

-0.92.3 1.9

-2.91.3

-0.72.2

-2.32.4

-1.20.8

1.03.8

-4.12.2

-1.02.8

0.74.5

0.7 0.7

Sucrose

UDP Glucose

ADP Glucose

Amylose Amylopectin

sh1 : sucrose synthase

sh2, bt2 : ADP-glucose pyrophosphorylase

su1 : debranching enzymeae1 : SBE-IIB

wx1 : g.b starch synthase

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

3. Reintroduce Genetic Variation

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

Breeding hypothesis: We can improve modern traits.

Kernel 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

β γ

α family

δ

Zein Profile

Teosinte NIL Evaluations

Hibbard – Corn Rootworm

Resistance

Buckler – Flowering

Brutnell – ShadeAvoidance Response

Balint-Kurti – SCLB and GLS

Smith – Smut

Tracy – Germination

AgReliant – Agronomic

TraitsNelson – NCLB

Hoekenga – Iron Bioavailability

Harmon – Circadian

Clock

Dallo – Mal de Rio Cuarto Disease

Flint-Garcia –Starch, Protein, Oil

Zein ProfileAmino Acid Profile

Lofty Ideas

2010

AgronomicsFertilizerDensity

Mechanization

?

Select only on yield

ideotype

7000 BC

Teosinte Synthetic75% B73 (SS), 25% teosinte

Acknowledgements

www.panzea.org

NSF Maize Diversity Project

SyngentaAgReliant