Download U.S. Fish and Wildlife Service, Developing

Developing Microsatellite Loci for Alligator Gar
and Their Usefulness in Other Gar Species.
Greg Moyer
U.S. Fish and Wildlife Service - Warm Springs, GA
Brian Kreiser
University of Southern Mississippi
OR
Where Are You From
?
&
Who’s Your Daddy
A Brief Introduction
Moyer & Colleagues
Kreiser & Colleagues
Brian Sloss – USGS
Kevin Feldheim - The Field Museum
Josh Rousey – Valdosta State
Wilfredo Matamoros - USM
Justin Sipiorski - SIU
Jake Schaefer - USM
Samples in Hand
MS Gulf Coast Fishing Rodeo (2002+) - Dennis Riecke (MDWFP)
St. Catherine Creek NWR - Ricky Campbell (UWFWS)
Vicksburg - Jan Hoover (Army Corp)
Oklahoma - Kerry Graves (USFWS)
Louisiana - Allyse Ferrara (Nichols State U.)
Texas - Mark Malfa (bowfishing guide)
Choctawhatchee - Frank Parauka (USFWS)
Other gar species (spotted, longnose, shortnose, Florida)
Overview
1. Background on conservation genetics, molecular tools and
microsatellites
2. Summary of work to date
3. Future directions - your input
Conservation Genetics
Fields of - Ecology, Population Genetics & Systematics
Tools of - Molecular Biology & Mathematical Modeling
Overlapping Questions
What is genetic variation and why is
it important?
• All the variation due to differences in alleles and genes in
an individual, population, or species
• Raw material for adaptive evolutionary change
– Genetic diversity is required for populations to evolve in response
to environmental changes1
– Heterozygosity levels are linked directly to reduced population
fitness via inbreeding depression2
1McNeely
et al. 1990; 2Reed and Frankam 2003
What is genetic variation and why is
it important?
• Conservation plans
– maintain self sustaining populations
– . . . long-term viable populations
• What does viability and self-sustaining actually
mean?
• A viable population must be large enough to
maintain sufficient genetic variation for adaptation
to environmental changes
The Molecular Markers
Species Boundaries
Mitochondrial DNA
Population Structure
Microsatellites
Within Populations
Screening for Genetic Variation
Microsatellite gel run
Not Really That Complicated
Kreiser Lab
Not the Kreiser Lab
Why Mitochondria?
“Powerhouse of the cell”; with its own genome
Small, circular genome
High mutation rate
Variation for population studies
Clonal
Maternal inheritance
Microsatellites - DNA Fingerprinting
Many loci in genome
Highly polymorphic
Microsatellites - DNA Fingerprinting
Example:
- (CA) repeat
- 6 alleles
#8 Genotype = 144 bp / 156 bp - six repeat units difference
#2 Genotype = 148 bp / 146 bp - one repeat unit difference
#5 Genotype = same
Microsatellites - DNA Fingerprinting
Multilocus genotype = the DNA fingerprint
Individual
#2
#5
#8
Locus 1
148/146
148/146
144/156
Locus 2
160/156
158/156
160/158
Locus 3
220/212
218/210
224/220
Locus 4
138/130
140/136
142/132
Microsatellite Marker Development
• Collaborative effort among agencies,
universities and laboratories
• Goal
– Develop a suite of 12-16 markers for estimating
population genetic parameters
Microsatellites - Isolating Loci
DNA
Extraction
Sequence
Clones
Enrichment
(GCC)
(AT)
(GATA)
Clone
Select
Loci Bearing
Clones
Microsatellites - Isolating Loci
Atsp 84 - primer design
TATTCCAAGGTGCAGCTGTAAGAATGCCATACAAACAAACAAACAAACAAACAAAC
AAACAAACAAACAAACAAACAACTCACTCTTCTGAGCTAAAATTCTGTGCTGTCTGTT
TTGGGTGAAAACTAGGGAGTTTGCAGAACTCTTTGAGAGTTTTTTTAAGGTGCACATAA
AAACTTCATCAGGATCTGAAACACCGTCACTGTGCTGGCTTCCCATTAACCAATATCTG
TTTCCTC
Atsp 159
4 alleles
16 alleles
Atsp 12
1 allele
Results
• Moyer lab (lots of work and nothing to show for it!)
– Two libraries constructed (enriched for di and tri repeats)
– 24 primers sets developed and optimized
– 14 of 24 loci -- successful and consistent amplification of alligator gar
DNA
– Limited variation
• 5 loci limited to 1 allele
• 7 loci had 2 alleles
• 2 loci had 3 alleles
– Cross species amplification with L. osseus, oculatus, platostomus,
tropicus, and platyrhincus
• Similar results
Results
Kreiser lab - Alligator gar
19 individuals - MS Gulf Coast fishing rodeo
30 loci tested
14 - not resolved
16 - amplified
5 - monomorphic (one allele)
11 - polymorphic (no deviation from HWE or LD)
Loci Testing
Locus
# Alleles
Ho
He
Atsp 7
4
0.263
0.238
Atsp 35
3
0.750
0.627
Atsp 40
4
0.684
0.620
Atsp 54
4
0.316
0.614
Atsp 57
2
0.053
0.051
Atsp 66
4
0.842
0.658
Atsp 84
16
0.947
0.898
Atsp 95
3
0.444
0.545
Atsp 122
3
0.389
0.329
Atsp 159
4
0.526
0.597
Atsp 341
3
0.688
0.506
Average
4.5
0.537
0.517
Loci Testing
Other gar
L. oculatus (n=14) & L. osseus (n=13) - Pascagoula
30 loci tested
12-13 - not resolved
7-8 - amplified
2-4 - monomorphic
4-5 - polymorphic (no deviation from HWE or LD)
Loci Testing
# Alleles
# Alleles
L. oculatus
L. osseus
Atsp 12*
3
2
Atsp 40
1
3
Atsp 54
1
1
Atsp 57
6
7
Atsp 66
13
10
Atsp 95
8
8
Atsp 324*
1
NR
Atsp 339*
1
1
Locus
Where do we go from here?
Restoration Goals
• Ecological
– Supplement existing populations
– Establish new populations
– Ecological functions
• Genetic
– Maintain/restore adaptive diversity and evolutionary
processes to promote population persistence.
• Adaptive genetic variation within populations
• Genetic structure among populations
• Historical
– Restore the past?
– Ensure the future?
Strategies for ecological restoration
• Wait and see
• Restore habitat
– Goal is simply to enhance natural recruitment
– No intended or unintentional genetic impact
• Hatchery-based enhancement
– Goal is to increase numbers
– No intentional genetic impacts
– Unintentional impacts depending on the source of brood stock,
how it’s managed, and the natural genetic structure
• Genetic Rehabilitation
– Goal is to “improve” the genetics of populations
• Manipulate gene flow
• Selectively bred or genetically engineered brood stock
Conservation Genetics & Hatchery Propagation
• Best choice is local brood stock
• Non local
– Risk – outbreeding depression (relative fitness of hybrids and back
crosses < natural population)
– Does genetic similarity = adaptive similarity?
– Can have high gene flow but local adaptation
• Recommendation
– Avoid non-local brood stock
– Test for adaptive vs. neutral genetic variation
Conservation Genetics & Hatchery Propagation
• Local brood stock
– Genetic diversity
• Hatchery ≈ natural
• Risk: inbreeding depression
– Lower relative fitness of hatchery stock
• Recommendation – Genetic baseline data
– Large number of unrelated founders – number depends on generation
time of organism
– Spawn unrelated
– Avoid spawning brood stock more than once
– Use brood held in captivity < 1 generation1
– Equalize parent contributions
– Rearing conditions
• Hatchery ≈ natural
• Risk: artificial selection
– Lower relative fitness of hatchery stock
• Recommendation
– Equalize parent contributions
1Araki
et al. 2007 Science
Questions/Suggestions?