Genetic Diversity and the Effects of Artificial Selection in Maize

Maize Diversity Project Team

Molecular DiversityHow has selection shaped molecular diversity in maize?

What is the relationship of selected genes to agronomic traits?

Goal: Identify genes exhibiting selection– Domestication, agronomic improvement, and local

adaptation

Community resource: SNP marker collection

Teosinte Landraces Inbreds/HybridsPhotos courtesy J. Doebley

Major predictions for the model

Those genes have contributed most to maize improvement, i.e. have experienced the strongest history of selection have the least genetic variability left to contribute to crop improvement by classical breeding.

These genes will not be detected in standard QTL experiments because all lines will contain similar alleles.

Can we develop genomics screens to identify genes that have undergone selection?

Invariant SSR approach (Vigouroux et al. 2002 PNAS 99:9650)

Directly contrast sequence diversity among teosintes and inbreds (Wright et al. 2005 Science 308:1310)

Are genes with low inbred diversity enriched for selected genes? (Yamasaki et al. 2005 Plant Cell 17:2859)

mcmullenm@missouri.edu for .pdfs

Summary of Sequencing on Random Genes(Irie Vroh Bi, Masanori Yamasaki, Kate Houchins)

MPZ inbreds – (temperate) B73(2), Mo17(2), Hp301, Il14H, Ky21, M37W, Oh43, (tropical) CML69, CML247, CML322, CML333, KUI3, KUI11, NC350.1095 alignments - 6169 SNPs. MPZ inbreds + 16 teosinte partial inbreds774 alignments – 3463 SNPs MPZ inbreds – 6136 SNPs in teosintes.

p for 1095 genes in maize inbreds

0

30

60

90

120

0 0.01 0.02 0.03p

Average 0.0067

≥

Sequence statistics for 1095 genes for diverse maize inbred lines.

N L Total L S Total S π

All Maize 13.1 280.4 307034 5.6 6169 0.0067 Temperate 6.7 292.2 310306 4.3 4560 0.0065 Tropical 6.6 290.8 308816 4.2 4427 0.0061

N = number of sequences, L = length of alignment, S = number of segregating sites, π average number of pairwise differences per bp.

Inbred-Teosinte Sequence Summary

• Number of alignments >5 in both sets 774• Average sample size inbreds 12.0• Average sample size teosinte 12.7• Average alignment length 294• Total SNPS in inbreds 3463• Total SNP in teosintes 6136

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 0.02 0.04 0.06 0.08

teosintes

in

bred

s

Average .inbred/.teosinte 0.57Excluding .inbred=0 values 0.63

Diversity in maize inbreds vs. teosinte

To identify the selected genes we need new statistical approaches

• There are two models: a selection model and a bottleneck model

• We must estimate the size of the bottleneck• For each model, we estimate the probability of the

model given the data (the likelihood) for each gene• This is very simulation and computer intensive!• This approach allows us to estimate the proportion

of genes under selection and to identify the candidates

t1t2

Na

Nb

Np

t1

t2

Na

Nb

Np

Two models: To be considered selected need to fail the neutral model and be accepted by the selected model.

neutral selected

Locus S inb. S teo. Probability of beingin selected class

Annotated BLAST hit

scl394_p3** 0 27 0.74 Arabidopsis thaliana L28 ribosomal protein

scl491_p3** 0 13 0.62 Maize dihydrodipicolinate synthase

scl405_p3** 0 12 0.59 Unknown expressed protein

scl427_p2* 0 16 0.54 A. thaliana DNAJ heat shock protein

scl526_p3** 1 16 0.54 Maize hexokinase

scl499_p5** 0 12 0.51 Unknown expressed protein

scl512_p1** 0 16 0.51 Triticum adenylosuccinate synthetase

scl536_p4** 0 17 0.49 Oryza sativa putative acetyl transferase

scl531_p4** 0 11 0.46 Oryza sativa putative auxin-induced protein

scl457_p4* 0 7 0.45 Oryza sativa putative growth factor

Genes significant for selection

On a genomic scale….

• Assume 40,000 genes in maize• 40,000 x 0.04 = 1600 selected genes• Before genome scans, 11 genes had been

identified as selected by population genetic approaches

• By sequencing 1000 genes, have ~30 novel candidates

• These genes need to be divided between domestication and improvement

What genes show evidence of selection?

• Genes involved in amino acid synthesis or metabolism

• Genes involved in growth response.• Transcription factors and signal transduction

components.• Unique genes with no significant BLAST

homologies.

Are genes with low inbred diversity enriched for domestication and improvement candidates?

(Masanori Yamasaki)

Chose 35 genes with no diversity among the MPZ inbred set.

Sequenced same region in 16 haploid landrace samples, 16 teosinte partial inbreds and a Tripsacum dactyloides sample. Performed Hudson-Kreitman-Aguadé (HKA) (tests for selection) on inbreds, landraces and teosintes against the neutral genes adh1, glb1, fus6 and bz2.

Performed coalescent simulations of domestication (CS) of inbreds vs. teosintes and landraces vs. teosintes.

Nucleotide position (bp)

1 500 10000

0.01 Amino Acid Transporter

1 500 10000

0.02

0.01

1500

GTP-binding Protein

1 500 10000

0.02

0.01

1500

Unknown

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0.01

1 1000 2000 3000

0.02 Fruit protein

1 500 1000

0.02

0.01

15000

0.03Chromatin remodeling

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0.03 F-box (circadian clock)

1 1000 2000 1 1000 20000

0.04

0.02

3000

0.06

0.08 Ankyrin repeat

1 1000 20000

3000

ARF0.02

0.01

p

Inbreds Teosintes

Unigene N L SP value in HKAtotal

P value in HKAsilent

  N L SP value in HKAtotal

P value in HKAsilent

Candidate status Homology search

AY108876 14 1,055 1 < 0.0068 ** < 0.0120 * 16 1,026 13 < 0.0433

* < 0.1849 Selected Gene Amino acid transporter

AY107195 14 3,119 1 < 0.0058 ** < 0.0087 ** 11 3,097 81 < 0.5889 <

0.6761 Selected Gene Auxin response factor

AY110109 14 1,466 1 < 0.0051 ** < 0.0054 ** 14 1,355 43 < 0.3613 <

0.5321 Selected Gene GTP-binding protein

AY105060 14 1,090 0 < 0.0041 ** < 0.0053 ** 15 1,112 59 < 0.7005 <

0.7631 Selected Gene

AY108178 14 1,259 0 < 0.0054 ** < 0.0082 ** 13 1,224 54 < 0.3233 <

0.4719 Selected Gene Circadian clock

AY106616 14 2,745 84 < 0.4395 < 0.2205 7 2,619 97 < 0.6859 < 0.7214 - Ankyrin repeat-like protein

AY107952 14 2,469 23 < 0.1193 < 0.0927 14 2,599 38 < 0.1453 < 0.1678 - Putative fruit protein, Oxidoreductase

AY106371 14 1,574 4 < 0.0094 **

< 0.0061 **   15 1,615 65 < 0.4603 <

0.4047 Selected Gene Putative methyl-binding domain protein

Do genes exhibiting signatures of selection control agronomic traits?

(Sherry Flint-Garcia)

• Hypothesis: manipulation of the expression of domestication and improvement genes will alter key agronomic traits

• Methods: use genetic and transgenic approaches to examine teosinte, exotic, and inbred alleles

• Test case: amino acid composition in kernels• Evidence for selection for cysteine synthase,

chorismate mutase, dihydrodipicolinate synthase and hexokinase

To what extend has diversity in amino acid synthesis genes been reduced by

selection? (Sherry Flint-Garcia)

• Whitt et al., 2002 demonstrated that 3 of 6 genes in starch synthesis pathway in maize show solid evidence of artificial selection

• Evidence for selection for cysteine synthase, chorismate mutase, dihydrodipicolinate synthase and hexokinase from random sequencing

• Chose 16 additional genes for important steps in amino acid synthesis, sequenced in teosintes, landraces and inbreds and conducted tests of selection

0

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Per

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acid

Teosinte (n = 7)Landraces (n = 11)Maize (n = 27)

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of K

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l Wei

ght

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Tota

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cid

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artic

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tein

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ic A

cid

Gly

cine

His

tidin

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Isol

euci

ne

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nyla

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**** **** **** **** **** **** ns** **** ns**Teosinte vs. Landraces

Teosinte vs. Inbred Lines**** **** **** **** **** **ns nsns **** **ns **

**

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 synthase

Cystathionine 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

HistidineNitrate

Reductase

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

ntl1 -- nitrogen regulating protein

Sequencing candidate genes• Goal is to sequence 1000 candidate genes in all

inbreds for the 25DL, 16 teosintes, 2 Tripsacum, and W22 R-std

• Shared responsibility by E. Buckler and M. McMullen laboratories

• Develop SNP (or sequence) based assays for association analysis

• Develop a mechanism to accept candidate gene suggestions for outside the project

• www.panzea.org

100%

80%

60%

38,000 genes 1,000 genes1,000 genes

Implications for GEM

• For the vast majority of genes inbreds lines retain on average 60% of common diversity of teosinte and 80% of the diversity of landraces. Therefore the problem of loss of diversity is a specific problem to particular genes and traits rather than a general problem

• Most of the diversity lost in unselected genes is in rare alleles and therefore hard to capture

Implications for GEM

• Our studies to date have not addressed specific adaptation, possibly a more important justification for GEM than limited diversity per se

• It is hard for me to think about how to tap diversity for specific adaptation without considering diversity in a trait context.