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?

• 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)

An Introduction to Mobile Elements

1. Short INterspersed Elements2. <500 bp3. No open reading frame (non-coding)4. Copy-and-paste mobilization5. Derived from tRNA or 7SL RNA6. Alu (primates), B1 (rodents), SmaI

(salmon), AFC (cichlids), etc…7. Very high copy numbers

(>100,000 copies/genome)

What are SINEs?

SINE Mobilization – “Copy and Paste”

Pol III transcription

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

Genomic Impacts:SINE Insertions and Mutation

Genomic Impacts:SINE/SINE Recombination

Mammalian SINE Distribution

Genome

Mobile element subfamily evolutionT

ime

Subfamily 1

Subfamily 2

Subfamily 3G

enom

e

Organismal speciation event

Gen

ome

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?

1. Identical by descent2. Known ancestral state 3. Simple evolutionary model4. Neutral5. “Low-tech”6. Bi-allelic markers7. Essentially homoplasy-free8. “Two markers in one”

Presence/absence andDNA sequence

Mobile elements as tools for conservation biologists

Alu Insertion PCR AssayAlu Insertion PCR Assay

400400 bpbp

100100 bpbp

332211

400400 bpbp BandBand 100100 bpbp BandBand400 & 100400 & 100 bpbp BandsBands

AA

BB

Ray et al., Forensic Sci. Intl. 2005

Identity by Descent

Species A

Species A

Species A’ Species B

Identity By Descent

time

Identity By State

Species AATGGTCC

Species BATGATCC

Species AATGGTCC

Species BATGGTCC

insertion

mutation

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

Capuchins and squirrel monkeys

Owl monkeys Saki monkeysAtelids Titi monkeys

Platyrrhine Primates

16 genera described as six “natural groups”

Callitrichids

Capuchins and squirrel monkeys

Owl monkeys Saki monkeysAtelids Titi monkeys

Family Cebidae:Callithrix, Cebuella, Leontopithecus, Saguinus, Callimico, Cebus, Saimir, Aotus

Platyrrhine Primates

16 genera described as six “natural groups”

Callitrichids Atelids

Capuchins and squirrel monkeys

Owl monkeys Saki monkeysTiti monkeys

Family Atelidae:

Ateles, Lagothrix, Brachyteles, Alouatta

Platyrrhine Primates

16 genera described as six “natural groups”

Callitrichids Saki monkeys

Capuchins and squirrel monkeys

Owl monkeys Atelids Titi monkeys

Family Pitheciidae:

Pithecia, Chiropotes, Cacajao, Callicebus

Cebidae

Pitheciidae

Atelidae

Previous Molecular Analyses

G6PD - Parsimony G6PD - Likelihood G6PD - Bayesian

E-globin - ParsimonyIRBP - Parsimony

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 elementsAdvantages – Quick and inexpensive, no biasDisadvantages- 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 taxaCallithrix, Saguinus, Saimiri, Aotus,

Pithecia, Callicebus, Ateles, Alouatta, Lagothrix

Computational – 94 usable loci from three taxaCallithrix, Pithecia, Saimiri

Results – Insertion Polymorphisms

New World Monkey Phylogeny Resolved with 185 Alu Insertion Polymorphisms

Consistency index = 1.00Homoplasy index = 0.00

Mol. Phyl. Evol. (2005)G6PD - Parsimony G6PD - Likelihood G6PD - Bayesian

E-globin - ParsimonyIRBP - Parsimony

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 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

Alu evolution in New World Primates

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)

AluSp-like

AluSc-like

A unique mode of Alu subfamily evolution

Ray and Batzer - BMC Evol Biol (2005)

A unique mode of Alu subfamily evolutionT

ime

AluSc

AluT

AluTa10, AluTa15

AluSp

Ancestral primate genome

Ancestral platyrrhine genome

Cebid-Atelid genomes

A

B

C. k

uh

lii

C. p

ygm

aeu

s

Sag

uin

us

Sai

mir

i

Ao

tus

wat

er

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Platyrrhini (n=58) Cebidae-Atelidae (n=14)

Cebidae (n=6) Callitrichinae(n=40)

Callithrix (n=40)

Sc

Sg

Sp

Sq

Sx

Ta10

Ta15

Ta7

Y

A

B

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) Trace DNA detection 2) Species identification3) Human DNA quantitation4) Human gender typing5) Inferring geographic affiliation

Human Population Biology and Investigative Forensics

1) Trace DNA detection 2) Species identification3) Human DNA quantitation4) Human gender typing5) Inferring geographic affiliation

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

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 - LSUBatzer Lab - LSUMark A. Batzer Scott Herke Jinchuan Xing Michael A. HallDale J. Hedges Meredith E. LabordeJerilyn Walker Bridget A. AndersAbdel-Halim Salem Brittany R. WhiteRandy Garber Nadica StoilovaRichard Cordaux Justin D. FowlkesGail Kilroy Cheney Huang

Jorde Lab – University of UtahJorde Lab – University of UtahLynn Jorde David Witherspoon

Center for Reproduction of Endangered Species – San Diego ZooCenter for Reproduction of Endangered Species – San Diego Zoo

Oliver A. Ryder Leona G. Chemnick

Texas Tech UniversityTexas Tech UniversityLou Densmore

University of FloridaUniversity of FloridaGinger Clark

Savannah River Ecology LaboratorySavannah River Ecology LaboratoryTravis Glenn

WVUWVUJeffrey Wells, Zena Urban,

Heather Campbell

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.

Allele freq = 0.9

Allele freq = 0.2Allele freq = 0.3

Allele freq = 0.3

Population-indicative Loci

Little variation to exploit at subpopulation levels

Subpopulation-indicative loci are needed