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