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Presence/Absence Variation (PAVs) in Maize:
Phenotypic Variation, Heterosis, and
Domestication (with some final comments
about cassava)
GCP21-II
Kampala, Uganda
22 June 2012
Patrick S. Schnable
Iowa State University
China Agriculture University
Data2Bio, LLC
1
The $30M B73 Maize Genome
Sequencing Project
Schnable, Ware et
al., 2009
The Maize Genome
Sequencing Project,
Rick Wilson, PI
2
Genome Projects are Analogous to the Lewis &
Clark Expedition
•Expensive and require extensive planning/coordination
•Generates lots of information that requires subsequent analysis
•Exploration of the unknown; expect surprises
Outline
• CNV and Presence-Absence Variation
(PAVs)
• The origin of “recurrent de novo CNV”
• Revisit the domestication bottleneck in
light of SV
• Relevance to cassava?
4
Outline
• CNV and Presence-Absence Variation
(PAVs)
• The origin of “recurrent de novo CNV”
• Revisit the domestication bottleneck in
light of SV
Kai Ying
Yan Fu
• Relevance to cassava? 应开
傅延
5
Structural Variation (CNV & PAV)
CNV
PAV
•What is overall level of
(genic) SV?
•Does SV contribute to
phenotypic diversity?
Array-based Comparative Genome
Hybridization (CGH)
•Nimblegen’s HD2 Array (~2.1M probes)
•Probes designed using a “frequency masked” 200 bp tile-path
through the draft B73 genome sequence
•Genotypes: B73, Mo17 (different heterotic groups)
Springer et al., PloS Genetics, 2009
Several hundred intact, expressed, phylogenetically
conserved genes exhibit CNVs and PAVs
Segmentation Results
Springer et al., PloS Genetics, 2009
Beló A et al. Theor Appl Genet. (2010) (Rafalski Lab)
2 Mb deletion on Ch 6*
*Includes ~2 dozen genes, incl. resistance gene (Xu Mingliang, 徐明良); this large
deletion also identified by Antoni Rafalski (CGH) and Ed Buckler (Re-Seq)
CNV and PAV Loss (blue) & CNV
Gain (red) Intervals relative to B73
Outer to Inner rings:
Teosinte vs. B73
Tx303 vs. B73
Hp301 vs. B73
Mo17 vs. B73
~10,000 YBP
Re-sequencing Six Inbreds
Identified PAVs
5.4X coverage/inbred
~150 Genes Present
among Six Inbreds are
Missing from B73
Lai et al., 2010
Classical Models for Heterosis
Complementation
AA bb
aa BB
Over-dominance
Aa Bb
x
Zamir
Complementation of PAVs in pairs
of inbreds could contribute to
heterosis; PAVs could also play a
role in over-dominance
Deletions can be favorable
• Removal of traits lost during domestication
• Ion uptake machinery (Heavy metal resistance in
wheat)
• Cyanide release (chemical defense) in white
clover (Olsen et al., 2007; 2008)
• Favorable rice QTL are in some cases PAVs:
– qPE9-1, panicle erectness; Zhou et al. (2009)
Genetics
– semi-dwarf1, height; Ashikari et al. (2005) Science
– GW5, grain width; Weng et al. (2008) Cell Res
13
Outline
• CNV and Presence-Absence Variation
(PAVs)
• The origin of “recurrent de novo CNV”
• Revisit the domestication bottleneck in
light of SV
Sanzhen Liu
• Relevance to cassava?
刘三震
14
Detection of De Novo CNV in
Human Trios
Mom
(no CNV)
X
Dad
(no CNV)
“Kid”
(de novo CNV)
Detection of Recurrent De Novo
CNV in Human Trios
Mom
(no CNV)
X
Dad
(no CNV)
“Kid”
(de novo CNV)
Mom
(no CNV)
X
Dad
(no CNV)
“Kid”
(de novo CNV)
15
Novel CGH Patterns
Liu et al., Plant J, 2012
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Reciprocal Gene Loss Model
to explain speciation
• Lynch and Force, 2000
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Segregation of Non-Allelic Homologs (SNH)
Generates “Recurrent De Novo CNV”
Model Predicts:
Changes in “gene
complement” among RILs
(gains and losses)
Should affect multiple RILs
Affected genes should have
non-allelic positions in B73
and Mo17
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Segregation of Non-Allelic Homologs Generates
“Recurrent De Novo CNV” and Novel Phenotypes
•Consistent with model:
•Losses and gains in gene
content validated by SeqCapture experiments (~200
segments in 2 RILs)
•Specific losses (as
detected by PCR) observed
in multiple RILs (12/14
genes lost in 25% or 12.5%
of RILs)
•Affected genes are in nonallelic positions in the B73
and Mo17 genomes
•Inbreds can have different gene
complements than parents
•Strong statistical support for
association between gene loss
and yield component traits in
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IBM RILs
Association of Gene Loss with
Traits
• Losses of 2/14 (14.3%) tested segments are significantly
associated with phenotypic variation:
– Reduced yield component traits (adjusted pvalues=0.03 and 0.01).
– Increased tiller number (adjusted p-value=0.01).
• This rate (14.3%) is substantially higher than the 0.1%
(N=670) of 515,620 control pairs of unlinked SNP
markers that similarly exhibit associations with the same
set of traits, identified via a two-dimension genome-wide
scanning using a set of 1,016 SNP markers
Outline
• CNV and Presence-Absence Variation
(PAVs)
• The origin of “recurrent de novo CNV”
• Revisit the domestication bottleneck in
light of SV
• Relevance to cassava? Kai Ying Camile
应开
Rustenholz
21
Teosinte, the wild ancestor of
maize
Teosinte
Maize
Zea mays sp mays
Domestication ~ 10,000 years ago
Zea mays sp parviglumis
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Genes Selected During Domestication
Have a Molecular Signature
Yamasaki, M., et al. Plant Cell 2005;17:2859-2872
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Copyright ©2005 American Society of Plant Biologists
CNV and PAV Loss (blue) & CNV Gain
(red) Intervals relative to B73
Outer to Inner rings:
Teosinte vs. B73
Tx303 vs. B73
Hp301 vs. B73
Mo17 vs. B73
~10,000 YBP
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Hypothesis:
maize lacks some teosinte genes
B73
Mo17
Tx303
CML277
Oh7B
Maize inbreds
Domestication and crop improvement
Teo. 1
Teo. 2
Teo. 3
Teo. 4
Teo. 5
Teosinte
Genes conserved in
maize and teosinte
Non-B73 genes
Non-maize genes25
1,000s of expressed genes in teosinte are
missing from the B73 reference genome
B73 × Teosinte Ac3660
B73 × Teosinte Ac3662
F1 teosinte Ac3660
F1 teosinte Ac3662
RNA-Seq
190M reads,
14Gb
ABySS assembly
≥ 300bp
63,464 contigs
Alignment against B73 reference genome v2
(≥90% identity, ≥50% coverage)
59,690 contigs aligned with
B73 reference genome
3,774 contigs NOT aligned
with B73 reference genome
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Extensive validation (sequence capture & CGH)
identified 72 expressed teosinte genes that are
absent from all tested (N=92) maize genomes
92 diverse maize lines
Number of genes
2,836 potential
PAVs
Genotyping CGH array
Number of maize lines where the gene is present
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Map locations of T+ M- PAVs
N = 60/72
26 isolated genes
11 clusters of 2 genes or more
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Presence rate of 72 T+ M- PAVs in 91
teosinte accessions
100%
Genotyping teosinte
diversity panel via
PCR
% of teosinte accessions
90%
80%
70%
60%
Absent
50%
Questionable
Uncertain
40%
Présent
30%
20%
10%
0%
Low presence
rate (≤50%)
High presence
rate (≥75%)
Random model – low frequency PAVs
16/72 (22%) of
T+M- PAVs are
present in <50% of
tested teosintes.
Low frequency T+MPAVs could be lost via
random processes,
such as drift.
Genetic
bottleneck
Teosinte
Drift or
other
random
mechanisms
Maize
Direct Selection Against T+M- Genes
Direct
49/72 (68%) T+MPAVs are present in
more than 75% of
the tested teosintes
Genetic
bottleneck
Selection (direct or
indirect) can explain
the loss of high
frequency T+Mgenes
Teosinte
Selection
AGAINST a PAV;
multiple
haplotypes in
maize possible
(depending on LD
in teosinte)
Maize
Indirect Selection Against T+M- Genes
49 T+M- PAVs
(68%) are present
in more than 75%
of the tested
teosintes
Indirect
Selective
sweep
Teosinte
Selection (direct or
Selection FOR a
indirect) can
domestication allele
explain the loss of in LD and coupling
with the absence of a
high frequency
PAV indirectly selects
against the PAV
T+M- genes
27/49 highfrequency PAVs
(55%) co-localize
with low diversity
regions
Maize
Summary (Part I)
• Maize haplotypes exhibit extensive SV (CNV and PAVs) that
affects several hundred genes (supported by CGH, PCR, and resequencing results: both WGS and exome capture)
• SV provides a testable hypothesis for heterosis (potentially
making heterosis more predictive)
• SV may help explain extraordinary level of phenotypic diversity
in maize. CNVs and PAVs that are not in LD with SNPs could
contribute to some of “missing heritability” in GWAS
experiments.
• “Recurrent de novo CNVs” can arise via meiotic segregation
(SNH Model), yielding non-parental gene complements that
have phenotypic consequences (transgressive segregation?)
• Genetic variation arising from SNH model would NOT be
detected in typical genome scans
Summary (Part II)
• It is widely accepted that allelic diversity is reduced by
domestication. We now know that not only alleles but
entire genes can be lost during domestication
• ~2,000 expressed genes present in teosinte are missing from
the B73 genome. 72 of these genes are missing from all
other tested maize lines.
• Teosinte genes failed to pass through the domestication
bottleneck for a variety of reasons (selection for or against
haplotypes and random processes).
• Teosinte genes that were lost inadvertently during
domestication, may have potential in crop improvement
(e.g., biotic and abiotic stress resistance).
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Outline
• CNV and Presence-Absence Variation
(PAVs)
• The origin of “recurrent de novo CNV”
• Revisit the domestication bottleneck in
light of SV
• Relevance to cassava?
35
What About Cassava?
• Interesting questions:
– How common are PAVs among cassava CVs? (Steve
Rounsley)
– Do wild relatives of cassava contain genes that are
absent from breeding germplasm? (Wenquan Wang)
– Do these missing genes confer agronomically relevant
traits? (e.g., resistance to biotic or abiotic stresses)
(implications for positional cloning experiments)
– Does complementation of key PAVs contribute to
heterosis in cassava? (Ismail Rabbi)
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Srinivas Aluru
Dan Nettleton
Nathan
Springer
Jeffrey Jeddeloh
Jinsheng
Lai
Collaborators
Brad Barbazuk
The Maize Genome
Sequencing Project
Mike Scanlon (PI, Cornell)
Jianming Yu, M. Timmermans,
G. Muehlbauer,
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D. Jannick-Buckner
Sanzhen Liu
刘三震
Kai Ying
应开
Camile
Rustenholz
Yan Fu
傅延
Wei Wu
吳薇
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