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Masters thesis presentation
The document discusses the significance of the 4R genome duplication in salmonine fishes and its implications for vertebrate evolution. It explores the role of conserved non-coding elements (CNEs) as tools for understanding polyploidy, presenting hypotheses about the phylogenetic relationships among salmonids. The findings suggest potential evolutionary timelines and unresolved phylogenetic relationships within the salmoninae subfamily, highlighting the utility of CNEs in studying whole genome duplications.
Masters thesis presentation
- 1. The 4R GenomeDuplicationThe 4R Genome Duplication in Salmonine Fishes:in Salmonine Fishes: Insights from ConservedInsights from Conserved Non-Coding ElementsNon-Coding Elements by Aníbal H. Castilloby Aníbal H. Castillo February 5th, 2008February 5th, 2008
- 2. Outline- •Introduction •Polyploidy •Salmonids: a recentlypolyploid 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
- 3. •Gene duplications andpolyploidization 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
- 4. •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
- 5. Introduction - Modified fromModified from Froschaueret 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 fish BichirBichir BowfinBowfin EelEel Ray finned fishRay finned fish 450 MYA450 MYA Whole Genome Duplications in Vertebrates
- 6. Introduction- Approaches toWGDs •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
- 7. •Individual genes, chromosomesegments, 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
- 8. •Require a map-baseddataset with the position of markers in the species’ genome •Substantial effort added for each species included •Inherently expensive Introduction- Limitations of usual approaches
- 9. Introduction- WGD canalso be studied through a phylogenetic approach WGD event time duplicated genes outgroup Sp. A Sp. B Sp. C Sp. D Sp. 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. C Sp. C Sp. D
- 10. Modified from Froschauer etal. 2006 Mammals Fishes Fishes b Fishes a Fishes a Fishes b Fishes a Fishes b Mammals Mammals Birds Mammals Birds Outgroup Introduction- WGD pattern in Vertebrates
- 11. Introduction- -Ancestrally tetraploid -Monophyletic; Phylogenyunresolved -Intermediate development as a genomic model -Genetic maps -Sequencing projects (O. mykiss & S. salar) Subfamily Salmoninae, family Salmonidae Rainbow troutRainbow trout OncorhynchusOncorhynchus mykissmykiss Atlantic salmonAtlantic salmon Salmo salarSalmo salar www.lofotakvariet.nowww.lofotakvariet.no Arctic charrArctic charr Salvelinus alpinusSalvelinus alpinus www.fishbase.orgwww.fishbase.org www.fishbase.orgwww.fishbase.org
- 12. Introduction - Modified fromModified from Froschaueret 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 fish BichirBichir BowfinBowfin EelEel Ray finned fishRay finned fish 450 MYA450 MYA Whole Genome Duplications in Vertebrates- 4R
- 13. •Comparing the genomeof 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)
- 14. •CNEs in Salmonids •Evolutionof 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
- 15. Hypotheses: 1. Members ofa 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-
- 16. -Members of aCNE 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
- 17. -The date ofinferred CNE duplications will be consistent with the range of 25-100Mya (Allendorf & Thorgaard 1984) time 25-100Mya I II Hypothesis 2- Prediction
- 18. -Salmoninae phylogeny: areOncorhynchus and Salvelinus sister groups? Crespi & Fulton (2003) Hypothesis 3- Prediction Salvelinus Salmo Outgroup Salvelinus Salmo Oncorhynchus Outgroup Oncorhynchus
- 19. Methods- Marker development •Conservation •Polymorphism PCR amplification, cloningand sequencing Alleles CNE 1 CNE 2 reverse primer forward primer•6 pairs of CNEs
- 20. • Tree building •Bayesian analyses • Maximum Likelihood • Maximum Parsimony • Model selection • Bayesian Information Criterion • Akaike Information Criterion • Likelihood Ratio Test Methods- Phylogenetic analyses
- 21. •Strict and relaxedclocks •Calibration points: •Oncorhynchus fossils 6MY •Salmonine fossils 20MY Methods- Molecular clock time I II
- 22. Results- 1. CNE7060-7061phylogeny- 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)
- 23. Results- 1. WGDpattern WGD event time duplicated genes outgroup Sp. A Sp. B Sp. C Sp. D Sp. 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. C Sp. C Sp. D
- 24.
- 25. 0.960.96 1.001.00 0.990.99 0.860.86 1.001.00 1.001.00 1.001.00 0.690.69 0.650.65 0.550.55 0.840.84 1.001.00 0.680.68 Results- 1. CNE6699-6700phylogeny- 4R Interpretation: •Basal polytomy •Moderate statistical support
- 26.
- 27. •Three markers hadsignal 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
- 28. 6 Mya 20 Mya 38- 47 Mya 20 Mya Oncorhynchu s 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
- 29. •Stochastic nature ofthe 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…
- 30. •First estimates forthe 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
- 31. Results- 3. Salmoninephylogeny: CNE7060-7061 OncorhynchusOncorhynchus I- and SalmoSalmo OncorhynchusOncorhynchus II- 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 OncorhynchusOncorhynchus OncorhynchusOncorhynchus OncorhynchusOncorhynchus OncorhynchusOncorhynchus SalvelinusSalvelinus SalvelinusSalvelinus SalvelinusSalvelinus OncorhynchusOncorhynchus SalmoSalmo SalmoSalmo SalmoSalmo SalmoSalmo SalvelinusSalvelinus SalvelinusSalvelinus SalvelinusSalvelinus SalvelinusSalvelinus OncorhynchusOncorhynchus OncorhynchusOncorhynchus OncorhynchusOncorhynchus SalmoSalmo SalmoSalmo SalmoSalmo SalmoSalmo SalmoSalmo SalvelinusSalvelinus
- 32. Results- 3. Salmoninephylogeny: 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 OncorhynchusOncorhynchus OncorhynchusOncorhynchus OncorhynchusOncorhynchus OncorhynchusOncorhynchus SalmoSalmo SalmoSalmo SalmoSalmo SalvelinusSalvelinus SalvelinusSalvelinus SalmoSalmo SalmoSalmo SalvelinusSalvelinus SalvelinusSalvelinus SalvelinusSalvelinus OncorhynchusOncorhynchus SalmoSalmo and SalvelinusSalvelinus
- 33. Discussion- 3. Salmoninephylogeny •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
- 34. •Phylogeny showing abasal 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-
- 35. 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
- 36. Atlantic salmonAtlantic salmon www.fishbase.orgwww.fishbase.org Questions? www.fhwa.dot.govwww.fhwa.dot.gov AtlanticsalmonAtlantic salmon www.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
Editor's Notes
- #21 Speech: -I took many approaches both for the tree-building itself and for the selection of the models of molecular evolution upon which the trees were built. -The ones shown are those done by Bayesian Analyses, obtained with models chosen by BIC. -These agree in all major points with the ones obtained by the other methods. -I’d be happy to discuss this in more detail after the talk…