Discovering the role of CNV in populationsg p p

Joseph R. ShawThe School of Public and Environmental Affairs, and 

Th C f G i d Bi i f iThe Center for Genomics and Bioinformatics

A sea of CNV(Two people differ in copy number across 0.78% of their genomes)( p p py g )

“The genome is dynamic playing field on which new genes are g y p y g f gcontinually arising via the molecular processes that give rise to duplication events, with their fate (pseudogene or fixation –altered dosage new function sub‐function) regulated by thealtered dosage, new function, sub function) regulated by the population genetic forces that provide fuel for evolution.” 

Modified from Lynch 20072

Spontaneous deletion/insertion rates

• Phylogenetic comparisons of non‐functional DNA (psueudogeneloci) suggest excess of deletions over insertions but estimated half‐loci) suggest excess of deletions over insertions, but estimated half‐life’s are extremely long.

R t Si (BP)Rate Size (BP)ORGANISM Deletion Insertion Deletion Insertion Half‐lifeCaenorhabditis elegans 0.034 0.019 166 151 0.25Drosophila melanogastor 0.115 0.028 42 12 0.15Drosophila melanogastor 0.115 0.028 42 12 0.15Birds 0.043 0.007 12 4 2.31Mammals 0.033 0.017 5 6 6.93

• Unbiased estimates of nuclear mutations in C. elegans suggest a 15:4 excess of insertions over deletions and much higher rates.g

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Mutation influences gene copy number plasticity

SPECIES Birth Death B/DHomo sapiens 0 0049 0 081 0 0605

Rates of origin and loss of duplicate genes

Homo sapiens 0.0049 0.081 0.0605Mus musculus 0.0030 0.134 0.0224Fugu rubripes 0.0043 0.189 0.0228Caenorhabditis elegans 0.0028 0.136 0.0206Caenorhabditis elegans 0.0028 0.136 0.0206Drosophila melanogastor 0.0011 0.229 0.0048

Lynch and Conery 2003Scaled to 1% divergence at silent sites.

• For vertebrates, this results in a 2% rate of duplication per gene per 106 generations; estimated half‐life of 2.5 millions of generations.

• The balance between gene birth and death rates is fairly constant but genes present in redundant copies areconstant, but genes present in redundant copies are continually changing.

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Consequences of copy number plasticity(a framework for studying environmental contributions to CNV)(a framework for studying environmental contributions to CNV)

• CNV are common 

• CNV alter phenotypep yp

• CNV are associated with diseases

What is the evolutionary history of CNV and do these important structural attributes of the genome represent a source variation upon which 

l i ll / i t ll d d t l ti fecologically/environmentally dependent selective forces can act?

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Challenges in applying tests of selection to CNV

• Genotypes can be hard to ypestablish since CNV are often multi‐allelic within populations.populations.

• Test that rely on amino acid substitutions and linkage based tests are not applicablebased tests are not applicable to all CNV. 

• High identity duplicates are difficult to assemble and they are often underrepresented in reference genomes.  Iskow et al., 2012

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A genomic model for CNV and the environment

Control Metals

Cyclical Parthenogenesis

a a

Control Metals

Benign Conditions

Stressful Conditions

b b

Daphnia now has well characterized genome, rich in tandem gene duplications, and gene copy number variants (Science, 2011, 331:555‐561). 7

The Daphnia pulex sequence

Gene richness attributed to a compact genomeStructural features Daphnia Arthropods (6) Worm Mouse

Mean (SE)Genome size (Mbp) 200 318 (74) 100 3,450Genome size adj. (Mbp) 175 250 (48) 100 2,600No of protein coding genes 31 000 21 017 (3309) 20 100 27 600No. of protein coding genes 31,000 21,017 (3309) 20,100 27,600Gene length (bp) 2,300 3,650 (249) 3,000 32,000CDS size (bp) 1,360 1,433 (80) 1,300 2,140Exons/gene 6.6 5.2 (0.6) 6 8/g ( )Exon size (bp) 210 297 (35) 200 280Intron size (mean bp) 170 875 (179) 290 2,800Intr > Exon 10% 33% (3%) 33% 85%UTR size (bp) 370 490 (116) 260 --Intergenic size (bp) 4,000 9,160 (2476) 2,400 78,000

* Bold indicates values outside the mean ± 2SE of other arthropod genomes Bold indicates values outside the mean ± 2SE of other arthropod genomes(Science, 2011, 331:555‐561)

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Nearly half the Daphnia genes have no homologs

* Of 716 highly conserved single copy orthologs, Daphnia is missing only two(Science, 2011, 331:555‐561)

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Elevated gene count is from gene duplicationDaphnia specific genes are most duplicated

• 50% of Daphnia genes are duplicates (35% in fly) (Science, 2011, 331:555‐561)p g p ( y)• 20% of all genes are within tandem duplicated gene (TDG) clusters• Daphnia counts over 1,000 TDG clusters with 3+ paralogs

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Age distribution of duplicated genesDuplication rates are elevated in DaphniaDuplication rates are elevated in Daphnia

(and relatively unpunctuated)

D. Pulex C. elegans H. sapiens 

Single copy genes  16,285  13,768  15,002 Duplicated genes  14,655 (47%) 6,350 (31%)  7,678 (34%)Total genes 30,940 20,118 22,680Total genes  30,940 20,118  22,680 Birth rate  9.3 3.3 7.3

(Science, 2011, 331:555‐561)Rates scaled to:  duplicates/1,000 genes/1% divergence11

Duplicate gene families are functionally related

Pathways enriched with duplicated genes  provide evidence that geneevidence that gene families are functionally linked and maintained by selection

• 54 enzymes are lost in arthropods (BLUE)

• 38 enzymes are amplified in crustaceans plus insects (YELLOW)

• 32 enzymes are specifically amplified in Daphnia (RED)

(Science, 2011, 331:555‐561) 12

Man made selection experiment: Sudbury Ontario

• 170 years of industrial mining

• 6000 square miles of forest6000 square miles of forest damaged , over 100 square miles barren

• 7000 lakes decimated from acid and metal stress

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Some Sudbury Daphnia populations adapt to cadmium

Figures removed at presenters requestFigures removed at presenters request

Genotyping across 30 microsatellite loci reveals two monophyletic clades A (only populations from mining region) and C, and a paraphyleticfrom mining region) and C, and a paraphyletic clade (B) that is closer to to C. Clade A is adapted to cadmium stress, while populations from B and C show no fitness advantage whenfrom B and C show no fitness advantage when exposed to cadmium. There is variation in adaptation within clade A.

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Array CGH identifies large number of segregating CNV

Figure removed at presenters request

Comparison of genomic DNA from

Figure removed at presenters request

Comparison of genomic DNA from • 12 isolates from adapted 

populations• 12 isolates from non‐adapted 

populations• Each hybridized against clonal y g

isolates from the reference genome

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Correlation of CNV with expression and phenotype

• 5% of segregating CNV are significantly correlated with differential expression.

• Only half of these are positively correlated with dosage.

% of CNV  Phenotype Tolerance1.30% Assimilation rate Adapted1.20% Assimilation rate Non‐adapted1.20% Assimilation rate Non adapted4.30% Performance reduction (0.5 ug/L) Adapted4.50% Performance reduction (0.5 ug/L) Non‐adapted13.00% Performance reduction (1 ug/L) Adapted3.00% e o a ce educt o ( ug/ ) dapted3.20% Performance reduction (1 ug/L) Non‐adapted

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A closer look at metallothionein‐1

Figure removed at gpresenters request

Induced expression of the MT‐1 gene is greater inInduced expression of the MT 1 gene is greater in adapted isolates compared to non‐adapted isolates. This difference in transcript levels is due to increased basal expression of the gene in adapted animals. 

From Shaw et al, 2007 17

C and T‐variant differ in copy number

Figure removed at presenters requestFigure removed at presenters request

Increased copies of the T‐variant are observed in d t d i l t C i f th C i t t iadapted isolates. Copies of the C‐variant are greater in non‐adapted isolates, but these are lower than observed for the T‐variant in adapted animals.

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T‐variant located in more dynamic genome region

Scaffold 36adapted:    π = 0.028Non‐adapted: π = 0 027

MT1‐T

Non adapted: π = 0.027 

MT1‐C

Scaffold 549adapted:    π = 0.016Non‐adapted: π = 0.008 

MT1 C

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Access to past populations allows direct evaluation

g DaphniaDaphniaDaphniag Daphniag DaphniaDaphniaDaphniaB

Stressful Conditions

Cyclical Parthenogenesis

Benign Conditions

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CNV selected for adaptive advantage in cadmium

Figure removed at presenters request

Low and even copy number of the C and T variant are observed in adapted populations prior to mining stress (drift). However, following mining increased copies of the T‐variant (associated with adaptation) are swept to fixationcopies of the T variant (associated with adaptation) are swept to fixation. 

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Summary of findings in Daphnia

• Documented cadmium specific adaptation that associated with the phylogeographic structure.with the phylogeographic structure.

• Observed CNV that segregate among adapted and non‐adapted animals in what appears to complicated, yet f ti ll i ifi tfunctionally significant ways. 

• Shown that MT‐1 plays a role in the adaptive response via constitutive up‐regulation of the gene due to increased geneconstitutive up regulation of the gene due to increased gene copy number. 

• Demonstrated allele specific changes in MT‐1 copy number h d d f d d hthat segregate adapted from non‐adapted phenotypes through space and time.

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CollaboratorsJohn Colbourne (University of Birmingham)Susanne Paland (IU, CGB)( , )Don Gilbert (IU, Biology; CGB)Deenie Bugge (Dartmouth College)Stephen Glaholt (IU, SPEA)Norman Yan (York College)Norman Yan (York College)Bill Keller (Luarentian College)Mike Pfrender (Notre Dame)Jeff Dudycha (University of South Carolina)Michael Lynch (IU, Biology)Carol Folt (Dartmouth College)Celia Chen (Dartmouth College)Xin Zhou (BGI)

Our lab groupXin Zhou (BGI)

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This work benefits from and contributes to the Daphnia Genomics Consortium.24

Direct measure of CNV rates in MA lines

How does stress affect rates and spectra of mutation (including large scale structural variation)?How does stress affect rates and spectra of mutation (including large scale structural variation)?

With cadmium

Buck 4 (non‐adapted) 50 generations

50 generations Sequence 100 lines from each of four

Without cadmium

With cadmium

Buck 4 (non‐adapted)250 sisters per line

Simon 14 (adapted)of four conditions

With cadmium

Without cadmium50 generations

50 generations250 sisters per line

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