Genetic Diversity and the Effects of Artificial

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Genetic Diversity and the Effects of Artificial
Selection in Maize
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Maize Diversity Project Team
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Molecular Diversity

• How 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

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Teosinte
Landraces
Inbreds/Hybrids
Photos courtesy J. Doebley
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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.
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Can we develop genomics screens to identify genes
that have undergone selection?
Invariant SSR approach (Vigouroux et al. 2002
PNAS 999650) Directly contrast sequence
diversity among teosintes and inbreds (Wright et
al. 2005 Science 3081310) Are genes with low
inbred diversity enriched for selected genes?
(Yamasaki et al. 2005 Plant Cell
172859) mcmullenm_at_missouri.edu for .pdfs
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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 inbreds 774 alignments 3463
SNPs MPZ inbreds 6136 SNPs in teosintes.
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Sequence statistics for 1095 genes for diverse
maize inbred lines. N L Total L S Total
S p 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, p average
number of pairwise differences per bp.
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Inbred-Teosinte Sequence Summary

• Number of alignments gt5 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

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Diversity in maize inbreds vs. teosinte
0.07
0.06
0.05
0.04
q inbreds
0.03
0.02
0.01
0
0
0.02
0.04
0.06
0.08
q teosintes
Average q.inbred/q.teosinte 0.57 Excluding
q.inbred0 values 0.63
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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

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Two models To be considered selected need to
fail the neutral model and be accepted by the
selected model.
selected
neutral
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Genes significant for selection
Locus S inb. S teo. Probability of being in 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
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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

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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.

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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.
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p
Nucleotide position (bp)
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Inbreds Inbreds Inbreds Inbreds Inbreds Teosintes Teosintes Teosintes Teosintes Teosintes
Unigene N L S P value in HKAtotal P value in HKAsilent   N L S P value in HKAtotal P value in HKAsilent Candidate status Homology search
AY108876 14 1,055 1 lt 0.0068 lt 0.0120 16 1,026 13 lt 0.0433 lt 0.1849 Selected Gene Amino acid transporter
AY107195 14 3,119 1 lt 0.0058 lt 0.0087 lt 0.0087 11 3,097 81 lt 0.5889 lt 0.6761 Selected Gene Auxin response factor
AY110109 14 1,466 1 lt 0.0051 lt 0.0054 lt 0.0054 14 1,355 43 lt 0.3613 lt 0.5321 Selected Gene GTP-binding protein
AY105060 14 1,090 0 lt 0.0041 lt 0.0053 lt 0.0053 15 1,112 59 lt 0.7005 lt 0.7631 Selected Gene
AY108178 14 1,259 0 lt 0.0054 lt 0.0082 lt 0.0082 13 1,224 54 lt 0.3233 lt 0.4719 Selected Gene Circadian clock
AY106616 14 2,745 84 lt 0.4395 lt 0.2205 7 2,619 97 lt 0.6859 lt 0.7214 - Ankyrin repeat-like protein
AY107952 14 2,469 23 lt 0.1193 lt 0.0927 14 2,599 38 lt 0.1453 lt 0.1678 - Putative fruit protein, Oxidoreductase
AY106371 14 1,574 4 lt 0.0094 lt 0.0061   15 1,615 65 lt 0.4603 lt 0.4047 Selected Gene Putative methyl-binding domain protein
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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

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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

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Trans-cinnamic acid
Lignin
PAL
Glucose
Tyrosine
Phenylalanine
Serine
Glycine
O-Acetylserine
3-Phospho- glycerate
Prephenate
Cysteine synthase
Erythrose 4-P
Chorismate mutase
Cysteine
Phosphoenol pyruvate
2-isopropyl- malate synthase
Chorismate
Shikimate
DAHP
Leucine
Anthranilate Synthase ß
Pyruvate
Alanine
Pyruvate
Anthranilate
Valine
Acetyl-CoA
Acetohydroxy acid synthase
Indole-3-glycerol phosphate
Asparagine
Isoleucine
Tryptophan Synthase ß1
Aspartate Amino- transferase
Asparagine synthetase
Tryptophan
2-Ketobutyrate
Aspartate
Oxalo- acetate
TCA Cycle
Threonine deaminase
Aspartate kinase
Glutamate
Threonine
Aspartate 4-seminaldehyde
NH4
Arginine
a-Keto- glutarate
DHDP synthase
Proline
Homoserine 4-phosphate
Glutamate dehydrogenase
Proline dehydrogenase
2,3-Dihydro- dipicolinate
Cysteine
Cystathionine ?-synthase
Glutamate
Cystathionine
Homocysteine
Lysine
Glutamine
NH4
NO3
NO2
Methionine
Nitrate Reductase
Histidine
SAM synthetase II
SAM synthetase I
Hexokinase (NC sensing)
S-Adenosyl- methionine
ntl1 -- nitrogen regulating protein
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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

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100
80
60
38,000 genes
1,000 genes
1,000 genes
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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

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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.