Mobile Element Applications for Molecular Ecology

1. Mobile Element Applications for Molecular Ecology David A. Ray West Virginia University

2. Outline • An introduction to mobile elements • Classification, distribution, evolution, impacts • SINEs as phylogenetic tools • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Current work

3. Outline • An introduction to mobile elements • Classification, distribution, evolution, impacts • SINEs as phylogenetic tools • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Where to go from here?

4. An Introduction to Mobile Elements • Present in most eukaryotes • Very high copy numbers • 10,000 – 1 million+ copies/genome • Retrotransposons • SINEs (Short INterspersed Elements) • LINEs (Long INterspersed Elements) • Transposons and derived elements • hAT (hobo, Activator, Tam) elements • MITEs (Miniature Inverted Terminal-repeat Elements)

5. What are SINEs? • Short INterspersed Elements • <500 bp • No open reading frame (non-coding) • Copy-and-paste mobilization • Derived from tRNA or 7SL RNA • Alu (primates), B1 (rodents), SmaI (salmon), AFC (cichlids), etc… • Very high copy numbers • (>100,000 copies/genome)

6. Pol III transcription SINE Mobilization – “Copy and Paste” Reverse transcription and insertion 1. Usually a single or a few ‘master’ elements 2. Pol III transcription to an RNA intermediate 3. Target primed reverse transcription (TPRT) – enzymatic machinery provided by LINEs

7. Genomic Impacts:SINE Insertions and Mutation

8. Genomic Impacts:SINE/SINE Recombination

9. Mammalian SINEDistribution

10. Subfamily 2 Subfamily 3 Organismal speciation event Genome Genome Mobile element subfamily evolution Subfamily 1 Genome Time

11. SINE Subfamilies – Diagnostic Mutations

12. SINE Subfamilies – Diagnostic Mutations

13. Outline • An introduction to mobile elements • Classification, distribution, evolution, impacts • SINEs as genetic markers • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Where to go from here?

14. Mobile elements as tools for conservation biologists • Identical by descent • Known ancestral state • Simple evolutionary model • Neutral • “Low-tech” • Bi-allelic markers • Essentially homoplasy-free • “Two markers in one” • Presence/absence and • DNA sequence Ray et al., Forensic Sci. Intl. 2005

15. Species A Species B ATGGTCC ATGATCC insertion time mutation Species A Species B ATGGTCC ATGGTCC Identity by Descent Identity By Descent Identity By State Species A Species A Species A’ Species B

17. SINE analysis of phylogeny

18. Misleading and homoplasy-inducing events in a SINE analysis.

19. Rates of misleading events in SINE analysis

20. Outline • An introduction to SINEs • SINEs as genetic markers • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Where to go from here?

21. Titi monkeys Saki monkeys Platyrrhine Primates • 16 genera described as six “natural groups” Atelids Callitrichids Owl monkeys Capuchins and squirrel monkeys

22. Owl monkeys Capuchins and squirrel monkeys Titi monkeys Saki monkeys Atelids Platyrrhine Primates • 16 genera described as six “natural groups” Callitrichids Family Cebidae: Callithrix, Cebuella, Leontopithecus, Saguinus, Callimico,Cebus, Saimir, Aotus

23. Owl monkeys Capuchins and squirrel monkeys Titi monkeys Saki monkeys Atelids Platyrrhine Primates • 16 genera described as six “natural groups” Callitrichids Family Atelidae: Ateles, Lagothrix, Brachyteles, Alouatta

24. Owl monkeys Capuchins and squirrel monkeys Titi monkeys Saki monkeys Atelids Platyrrhine Primates • 16 genera described as six “natural groups” Callitrichids Family Pitheciidae: Pithecia, Chiropotes, Cacajao, Callicebus

25. G6PD - Likelihood G6PD - Bayesian IRBP - Parsimony E-globin - Parsimony G6PD - Parsimony Previous Molecular Analyses Atelidae Pitheciidae Cebidae Β2m - Parsimony

26. Investigating Platyrrhine Phylogenetics with Alu Elements • Two complementary methodologies:Experimental – Use “wet-bench” techniques to find Alu elements in nine New World monkey genomes Advantages – Nine whole genomes to search, no chromosomal limitations Disadvantages – Nine whole genomes to search, cost, selection biasComputational – Use available sequence databases to identify and characterize Alu elements Advantages – Quick and inexpensive, no bias Disadvantages- Limited to available sequence data and organisms (three)

27. Alu Recovery – Experimental (9 taxa) Mol. Phyl. Evol. (In press)

28. Alu Recovery – Computational (3 taxa)(NISC comparative vertebrate sequencing program; http://www.nisc.nih.gov/) Query sequence - Callithrix Human ortholog

29. Results – Alu Loci • Alu insertions were considered “usable” if they amplified as discreet bands in 5 of the 9 available platyrrhine genera and at least one Catarrhine primate. • Experimental – 89 usable loci from nine taxa • Callithrix, Saguinus, Saimiri, Aotus, Pithecia, Callicebus, Ateles, Alouatta, Lagothrix • Computational – 94 usable loci from three taxa • Callithrix, Pithecia, Saimiri

30. Results – Insertion Polymorphisms

31. Consistency index = 1.00 Homoplasy index = 0.00 G6PD - Likelihood G6PD - Bayesian IRBP - Parsimony E-globin - Parsimony G6PD - Parsimony New World Monkey Phylogeny Resolved with 185 Alu Insertion Polymorphisms Mol. Phyl. Evol. (2005)

32. Finding mobile element loci for a group • Mobile element subfamilies tend to have finite life spans • The first step in identifying polymorphic and/or species/taxon specific markers is to identify the appropriate subfamily

33. Alu evolution in New World Primates • Alu evolution in New World primates • Bayesian analysis of 60 Alu sequences found exclusively in platyrrhine primate genomes • Well-supported clades were analyzed for diagnostic sites • Ray and Batzer, BMC Evol Biol – 2005

34. Three (possibly five) new platyrrhine-specific subfamilies were characterized – AluTa5, AluTa10, AluTa15 • Two subfamilies are exclusive to Cebid and Atelid monkeys • In addition, we identified a unique mode of Alu subfamily evolution Ray and Batzer - BMC Evol Biol (2005)

35. AluSc-like AluSp-like A unique mode of Alu subfamily evolution Ray and Batzer - BMC Evol Biol (2005)

36. AluT AluSp A unique mode of Alu subfamily evolution AluSc Time AluTa10, AluTa15 Ancestral platyrrhine genome Ancestral primate genome Cebid-Atelid genomes

37. A C. pygmaeus Saguinus Saimiri Aotus water C. kuhlii B

38. A B

39. Outline • An introduction to SINEs • SINEs as genetic markers • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Where to go from here?

40. SINEs, Biology, and Investigative Forensics • Trace DNA detection • Species identification • Human DNA quantitation • Human gender typing • Inferring geographic affiliation

41. Human Population Biology and Investigative Forensics • Trace DNA detection • Species identification • Human DNA quantitation • Human gender typing • Inferring geographic affiliation

42. Human Population Biology and Investigative Forensics • FBI CODIS loci are great for matching a single unknown DNA sample to a single individual. • Must have a suspect. • Early in an investigation, can we narrow the field of suspects using information from Alu polymorphisms?

43. Human Population Biology and Investigative Forensics Mobile elements continue to propogate in the human genome. Many elements are polymorphic and occur at variable frequencies in human populations. Display-based PCR methods can be used to “extract” recent, population-indicative elements.

44. Forensic Sci. Intl. (In press) Forensic Sci. Intl. (In press) Inferring Geographic Affiliation • Series of genetic markers (100 Alu loci) • Database of human variation (currently 715 individuals of known ancestry) • Genotype unknown sample(s) 18 samples from two forensic laboratories • Analytical approach (Structure analysis)

45. Identifying 18 Unknown DNAs Forensic Sci. Intl. (In press)

47. Outline • An introduction to SINEs • SINEs as genetic markers • Primate phylogeny • SINEs as tools in population genetics and forensics • Population-indicative insertions and narrowing a criminal investigation • Where to go from here?

48. Where Do We Go from Here?Population Biology and Phylogenetics • Expand the use of mobile elements as phylogenetic and population genetic markers to additional organisms • Genome exploration • Mobile element characterization • Techniques for identifying “useful” loci • Species identification and DNA quantitation • Bats, blowflies, fish, and crocodilians

49. Contributors Batzer Lab - LSU Mark A. Batzer Scott Herke Jinchuan Xing Michael A. Hall Dale J. Hedges Meredith E. Laborde Jerilyn Walker Bridget A. Anders Abdel-Halim Salem Brittany R. White Randy Garber Nadica Stoilova Richard Cordaux Justin D. Fowlkes Gail Kilroy Cheney Huang Jorde Lab – University of Utah Lynn Jorde David Witherspoon Texas Tech University Lou Densmore University of Florida Ginger Clark Savannah River Ecology Laboratory Travis Glenn WVU Jeffrey Wells, Zena Urban, Heather Campbell Center for Reproduction of Endangered Species – San Diego Zoo Oliver A. RyderLeona G. Chemnick