Masters thesis presentation

The 4R Genome Duplication The 4R Genome Duplication

in Salmonine Fishes:in Salmonine Fishes:

Insights from Conserved Insights from Conserved

Non-Coding ElementsNon-Coding Elements

by Aníbal H. Castilloby Aníbal H. Castillo

February 5th, 2008February 5th, 2008

Outline-•Introduction

•Polyploidy•Salmonids: a recently polyploid taxon•How to study polyploidy•CNEs: a potential tool for studying genome evolution•Hypotheses & predictions

•Methods•Marker development•Phylogenetic analyses•Molecular clock

•Results and Discussion•Phylogeny and 4R•Dating 4R•Salmoninae phylogeny

•Gene duplications and polyploidization events are important in the evolution of vertebrates (Ohno 1970; 1999)

•Recent outburst of genomic data provides an opportunity for innovative testing of established hypotheses

•Potential to extend new resources to organisms of biological but non-commercial interest

Introduction- Polyploidy

•Common in plants, fungi and animals

•Among vertebrates, fishes, reptiles and amphibians

• Within fishes:

•non-teleosts: paddlefish, sturgeon and spotted gar

•teleosts: carps and Salmonids

Introduction- Polyploid taxa

Introduction-

Modified from Modified from Froschauer et al. 2006Froschauer et al. 2006

4R4R

MedakaMedaka

PlatyfishPlatyfish

PufferfishPufferfish

CichlidsCichlids

TilapiaTilapia

SticklebackStickleback

SalmonidsSalmonids

3R3R

2R2R

1R1R

CatfishCatfish

ZebrafishZebrafish

SturgeonsSturgeons

TeleostsTeleosts

TetrapodsTetrapods

OutgroupOutgroup

Cartilaginous fishCartilaginous fish

Jawless fishJawless fish

Lobe finned fishLobe finned fishBichirBichir

BowfinBowfinEelEelRay finned fishRay finned fish

450 MYA450 MYA

Whole Genome Duplications in Vertebrates

Introduction- Approaches to WGDs

•Genomic approaches:

•chromosomal and genome evolution,•gene regulation•genetic architecture of phenotypic variation

• Patterns of gene and genome duplications:

•how many duplication events•when they occurred•mechanisms behind these events

•Individual genes, chromosome segments, entire chromosomes and finally entire genomes

•The most rigorous way of testing genome doublings is identifying paralogous chromosomal regions or block duplications

•Once multiple conserved syntenic blocks are identified, a WGD can be inferred

Introduction- Approaches to WGDs

•Require a map-based dataset with the position of markers in the species’ genome

•Substantial effort added for each species included

•Inherently expensive

Introduction- Limitations of usual approaches

Introduction- WGD can also be studied through a phylogenetic approach

WGD event

time

duplicated genes

outgroup

Sp. A

Sp. B

Sp. C

Sp. DSp. A

Sp. B

Sp. C

Sp. D

Sp. D

time

Individual duplication events

outgroup

duplicated genes

Sp. A

Sp. A

Sp. B

Sp. B

Sp. CSp. C

Sp. D

Modified from Froschauer et al. 2006 Mammals

Fishes

Fishes b

Fishes a

Fishes a

Fishes b

Fishes a

Fishes b

Mammals

MammalsBirds

MammalsBirds

Outgroup

Introduction-

WGD pattern in Vertebrates

Introduction-

-Ancestrally tetraploid-Monophyletic; Phylogeny unresolved-Intermediate development as a genomic model

-Genetic maps-Sequencing projects (O. mykiss & S. salar)

Subfamily Salmoninae, family Salmonidae

Rainbow troutRainbow troutOncorhynchusOncorhynchus mykissmykiss

Atlantic salmonAtlantic salmonSalmo salarSalmo salar

www.lofotakvariet.nowww.lofotakvariet.no

Arctic charrArctic charrSalvelinus alpinusSalvelinus alpinus

www.fishbase.orgwww.fishbase.org www.fishbase.orgwww.fishbase.org

Introduction-

Modified from Modified from Froschauer et al. 2006Froschauer et al. 2006

4R4R

MedakaMedaka

PlatyfishPlatyfish

PufferfishPufferfish

CichlidsCichlids

TilapiaTilapia

SticklebackStickleback

SalmonidsSalmonids

3R3R

2R2R

1R1R

CatfishCatfish

ZebrafishZebrafish

SturgeonsSturgeons

TeleostsTeleosts

TetrapodsTetrapods

OutgroupOutgroup

Cartilaginous fishCartilaginous fish

Jawless fishJawless fish

Lobe finned fishLobe finned fishBichirBichir

BowfinBowfinEelEelRay finned fishRay finned fish

450 MYA450 MYA

Whole Genome Duplications in Vertebrates- 4R

•Comparing the genome of humans and Japanese pufferfish (Fugu rubripes), 1373 CNEs were identified

•~90% conserved

•Average of 199bp, maximum length of 736bp

•Occur throughout the human genome; regulating developmental genes

•Unique to vertebrates

Introduction- Conserved Non-coding Elements, CNEs (Woolfe et al. 2005)

•CNEs in Salmonids

•Evolution of CNEs•Relative importance of 4R vs. other processes

•Potentially useful via both mapping and phylogenetic approaches

•Test CNEs in Salmonids application to non-classic organisms:

• e.g., South American rodents, Xenopus sp.

Introduction- CNEs: a tool to study genome evolution

Hypotheses:

1. Members of a CNE family will show a symmetric phylogeny consistent with the 4R hypothesis in Salmonid fishes

2. The date of inferred CNE duplications will be consistent with the range of 25-100Mya (Allendorf & Thorgaard 1984)

3. Salmoninae phylogeny: are Oncorhynchus and Salvelinus sister groups? Crespi & Fulton (2003)

Introduction-

-Members of a CNE family will show a symmetric phylogeny consistent with the 4R hypothesis in Salmonid fishes

time

WGD

CNE

duplicate I

CNE

duplicate II

Sp. A

Sp. A

Sp. B

Sp. B

Sp. C

Sp. C

Outgroup

Hypothesis 1- Prediction

-The date of inferred CNE duplications will be consistent with the range of 25-100Mya (Allendorf & Thorgaard 1984)

time

25-100MyaI

II


-Salmoninae phylogeny: are Oncorhynchus and Salvelinus sister groups? Crespi & Fulton (2003)


Salvelinus

Salmo

Outgroup

Salvelinus

Salmo

Oncorhynchus

Outgroup

Oncorhynchus

Methods- Marker development

•Conservation•Polymorphism

PCR amplification, cloning and sequencing

Alleles

CNE 1 CNE 2

reverse primer

forward primer•6 pairs of CNEs

• Tree building

• Bayesian analyses

• Maximum Likelihood

• Maximum Parsimony

• Model selection

• Bayesian Information Criterion

• Akaike Information Criterion

• Likelihood Ratio Test

Methods- Phylogenetic analyses

•Strict and relaxed clocks

•Calibration points:

•Oncorhynchus fossils 6MY

•Salmonine fossils 20MY

Methods- Molecular clock

time

I

II

Results- 1. CNE7060-7061 phylogeny- 4R

Interpretation:

•Basal bifurcation

•Strong statistical support

1.001.00

1.001.00

1.001.00

0.940.94

1.001.00

0.970.97

1.001.00

1.001.00

0.980.98

1.001.00

1.001.00

1.00

RT = Rainbow trout(Oncorhynchus mykiss)

AS = Atlantic salmon(Salmo salar)

AC = Arctic charr(Salvelinus alpinus)

Results- 1. WGD pattern

WGD event

time

duplicated genes

outgroup

Sp. A

Sp. B

Sp. C

Sp. DSp. A

Sp. B

Sp. C

Sp. D

Sp. D

time

Individual duplication events

outgroup

duplicated genes

Sp. A

Sp. A

Sp. B

Sp. B

Sp. CSp. C

Sp. D

1.001.00

1.001.00

1.001.00

1.001.00

0.910.91

1.001.00

1.001.00

1.001.00

0.970.97

0.960.96

0.980.98

0.750.75

0.990.99

0.960.96

1.001.00

1.001.00

1.001.00

Results- 1. CNE6820-6816 and CNE7061-7063

0.960.961.001.00

0.990.99

0.860.861.001.00

1.001.00

1.001.00

0.690.690.650.65

0.550.55

0.840.84

1.001.00

0.680.68

Results- 1. CNE6699-6700 phylogeny- 4R

Interpretation:

•Basal polytomy

•Moderate statistical support

0.530.53

0.560.56

1.001.00

0.540.54

1.001.00

0.990.990.950.95

1.001.00

1.001.00

0.540.54

1.001.00

1.001.00

0.580.58

0.530.53

0.780.78

0.970.97

0.760.76

Results- 1. CNE6700-6697 and CNE6739-6741

•Three markers had signal at the 4R level

•Basal bifurcation with symmetric topology

consistent with the 4R hypothesis

•Three markers had no signal; these do not

refute 4R hypothesis

•Suggestive evidence that one duplicate from

CNE7061-7063 was lost in Atlantic salmon

Discussion- 1. Phylogeny at the 4R level

6 Mya

20 Mya

38 - 47 Mya

20 Mya

Oncorhynchus

Salvelinus + Salmo

Subfamily

Salmoninae

CNE duplicate I

Subfamily

Salmoninae

CNE duplicate II

Oncorhynchus +

Salvelinus + Salmo

47

43

38

Mean

strict

relaxed

relaxed

Clock type

CNE6820-6816

CNE7061-7063

CNE7060-7061

Results- 2. Dating 4R

•Stochastic nature of the molecular clock

•Uncertainty of the assigned fossil dates and classification

•The correspondence between fossils and nodes in the tree

•Only the subfamily Salmoninae is included; including more subfamilies within Salmonidae (e.g., Thymallinae) would yield more robust results

Discussion- 2. Molecular clock- Uncertainties…

•First estimates for the date of the 4R since Allendorf & Thorgaard (1984)

•First ones ever based on nucleotide sequence data

•Narrower estimate of the date of the 4R, 38Mya to 47Mya

Discussion- 2. Molecular clock

Results- 3. Salmonine phylogeny: CNE7060-7061

OncorhynchusOncorhynchus I- and

SalmoSalmo

OncorhynchusOncorhynchusII- and

SalvelinusSalvelinus

1.001.00

1.001.00

1.001.00

0.940.94

1.001.00

0.970.97

1.001.00

1.001.00

0.980.98

1.001.00

1.001.00

1.00

OncorhynchusOncorhynchusOncorhynchusOncorhynchusOncorhynchusOncorhynchus

OncorhynchusOncorhynchusSalvelinusSalvelinusSalvelinusSalvelinusSalvelinusSalvelinus

OncorhynchusOncorhynchusSalmoSalmo SalmoSalmoSalmoSalmo

SalmoSalmoSalvelinusSalvelinusSalvelinusSalvelinusSalvelinusSalvelinusSalvelinusSalvelinus

OncorhynchusOncorhynchus OncorhynchusOncorhynchusOncorhynchusOncorhynchusSalmoSalmo

SalmoSalmoSalmoSalmo SalmoSalmo SalmoSalmoSalvelinusSalvelinus

Results- 3. Salmonine phylogeny: CNE6820-6816

0.970.97

0.960.96

0.980.98

0.750.75

0.990.99

0.960.96

1.001.00

1.001.00

1.001.00

OncorhynchusOncorhynchusOncorhynchusOncorhynchus

OncorhynchusOncorhynchus


SalmoSalmo

SalmoSalmo

SalmoSalmo



SalmoSalmo

SalmoSalmo





SalmoSalmo

and


Discussion- 3. Salmonine phylogeny

•No conclusive evidence supporting a sister relationship between Oncorhynchus and Salvelinus

•Duplicates within one locus support alternative phylogenies

•One locus suggests a sister relationship between Salmo and Salvelinus, never reported before

•Hard polytomy, reticulation

•Phylogeny showing a basal bifurcation and symmetric topology in some CNE pairs, consistent with 4R

•The estimated date of CNE duplication is consistent with the reported range of 25-100Mya

•However, the phylogenetic relationships within Salmoninae remain unresolved

•CNEs are a suitable tool for preliminary approaches to the study of Whole Genome Duplications

Conclusions-

Acknowledgements-

•My advisors, Dr. Moira M. Ferguson and Dr. Roy G. Danzmann•Past and current lab members, specially Hooman, Janet and Michael•From my advisory committee: Dr. T. Ryan Gregory•From my examination committee: Dr. J. Ballantyne and Dr. R. Hanner•Dr. Tom Nudds•Members of the honourable Zoology House, John Urquhart, Joe Crowley, Emilia Argue, Han Xu, Renji Lu, Jackie Porter, Alison Fischer and Liyan Qing•Derek Wong, Dan Noble, Vitali Rosen, Momina Mir and last but not least, Jessica-Margaret Paige

Atlantic salmonAtlantic salmon

www.fishbase.orgwww.fishbase.org

Questions?

www.fhwa.dot.govwww.fhwa.dot.gov

Atlantic salmonAtlantic salmonwww.lofotakvariet.nowww.lofotakvariet.no

Arctic charrArctic charr Rainbow troutRainbow trout

www.fishbase.orgwww.fishbase.org

englishriverwebsite.comenglishriverwebsite.com

Rainbow troutRainbow trout

www.altosarca.itwww.altosarca.it

Arctic charrArctic charr

Science

Masters thesis presentation