Mobile Element Applications for Molecular Ecology David A. Ray West Virginia University 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 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? 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 Terminalrepeat Elements) What are SINEs? 1. 2. 3. 4. 5. 6. 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… 7. Very high copy numbers (>100,000 copies/genome) SINE Mobilization – “Copy and Paste” Reverse transcription and insertion Pol III transcription 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 Genomic Impacts: SINE Insertions and Mutation Genomic Impacts: SINE/SINE Recombination Mammalian SINE Distribution Mobile element subfamily evolution Subfamily 1 Subfamily 2 Organismal speciation event Genome Genome Genome Time Subfamily 3 SINE Subfamilies – Diagnostic Mutations SINE Subfamilies – Diagnostic Mutations 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? Mobile elements as tools for conservation biologists 1. 2. 3. 4. 5. 6. 7. 8. Identical by descent Known ancestral state Simple evolutionary model Neutral “Low-tech” Bi-allelic markers Essentially homoplasy-free “Two markers in one” Alu Insertion PCR Assay A 400 bp 100 bp B 1 400 bp Band 2 3 400 & 100 bp Bands 100 bp Band Presence/absence and DNA sequence Ray et al., Forensic Sci. Intl. 2005 Identity by Descent Identity By Descent Identity By State Species A Species A ATGGTCC Species B ATGATCC insertion Species A’ Species B time Species A mutation Species A ATGGTCC Species B ATGGTCC SINE analysis of phylogeny Misleading and homoplasy-inducing events in a SINE analysis. Rates of misleading events in SINE analysis 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? Platyrrhine Primates 16 genera described as six “natural groups” Callitrichids Owl monkeys Capuchins and squirrel monkeys Atelids Titi monkeys Saki monkeys Platyrrhine Primates 16 genera described as six “natural groups” Callitrichids Owl monkeys Atelids Titi monkeys Saki monkeys Capuchins and squirrel monkeys Family Cebidae: Callithrix, Cebuella, Leontopithecus, Saguinus, Callimico, Cebus, Saimir, Aotus Platyrrhine Primates 16 genera described as six “natural groups” Callitrichids Owl monkeys Capuchins and squirrel monkeys Atelids Titi monkeys Saki monkeys Family Atelidae: Ateles, Lagothrix, Brachyteles, Alouatta Platyrrhine Primates 16 genera described as six “natural groups” Callitrichids Owl monkeys Capuchins and squirrel monkeys Atelids Titi monkeys Saki monkeys Family Pitheciidae: Pithecia, Chiropotes, Cacajao, Callicebus Previous Molecular Analyses Atelidae Pitheciidae Cebidae IRBP - Parsimony G6PD - Parsimony E-globin - Parsimony G6PD - Likelihood G6PD - Bayesian 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 bias Computational – 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) Alu Recovery – Experimental (9 taxa) Mol. Phyl. Evol. (In press) Alu Recovery – Computational (3 taxa) (NISC comparative vertebrate sequencing program; http://www.nisc.nih.gov/) Query sequence - Callithrix Human ortholog 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 Results – Insertion Polymorphisms New World Monkey Phylogeny Resolved with 185 Alu Insertion Polymorphisms Consistency index = 1.00 Homoplasy index = 0.00 IRBP - Parsimony E-globin - Parsimony Mol. -Phyl. Evol. (2005) G6PD - Likelihood G6PD Parsimony G6PD - Bayesian 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 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 Three (possibly five) new platyrrhinespecific 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) A unique mode of Alu subfamily evolution AluSc-like AluSp-like Ray and Batzer - BMC Evol Biol (2005) A unique mode of Alu subfamily evolution AluSc Time AluT AluSp AluTa10, AluTa15 Ancestral Ancestral platyrrhine primate Cebid-Atelid genomes genome genome A B 0.9 0.8 Sc Sg Sp Sq Sx Ta10 Ta15 Ta7 Y 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Platyrrhini (n=58) A B CebidaeAtelidae (n=14) Cebidae (n=6) Callitrichinae (n=40) Callithrix (n=40) 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? SINEs, Biology, and Investigative Forensics 1) 2) 3) 4) 5) Trace DNA detection Species identification Human DNA quantitation Human gender typing Inferring geographic affiliation Human Population Biology and Investigative Forensics 1) 2) 3) 4) 5) Trace DNA detection Species identification Human DNA quantitation Human gender typing Inferring geographic affiliation 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? 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. Inferring Geographic Affiliation 1) Series of genetic markers (100 Alu loci) 2) Database of human variation (currently 715 individuals of known ancestry) 3) Genotype unknown sample(s) 18 samples from two forensic laboratories 4) Analytical approach (Structure analysis) Forensic Sci. Intl. (In press) Forensic Sci. Intl. (In press) Identifying 18 Unknown DNAs Forensic Sci. Intl. (In press) 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? 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 Contributors Batzer Lab - LSU Mark A. Batzer Jinchuan Xing Dale J. Hedges Jerilyn Walker Abdel-Halim Salem Randy Garber Richard Cordaux Gail Kilroy Scott Herke Michael A. Hall Meredith E. Laborde Bridget A. Anders Brittany R. White Nadica Stoilova Justin D. Fowlkes 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. Ryder Leona G. Chemnick Future Directions Additional Loci: Tiered approach to subpopulation affiliations. Initial screen for broad population affiliation Secondary screen for subpopulation affiliation Loci good for the first screen unlikely to be useful in the second. Population-indicative Loci Allele freq = 0.9 Allele freq = 0.3 Subpopulation-indicative Little variation to exploit at subpopulation loci are needed levels Allele freq = 0.3 Allele freq = 0.2