Uses of population genetic data
Species
identification
(taxonomy)
Population/stock
differentiation
Individual
identification
Genetics questions relevant to conservation biology:
are x and y different species?
are populations genetically different?
how much variation is present in a population?
how much variation has been lost?
which parents contributed to the breeding population?
how much migration is occurring between populations?
A
T
G
G
C
T
A
A
T
A
C
C
G
A
T
T
DNA
sequence
DNA
strand
chromosome
protein
organism
morphometric
and meristic
counts
~ 40 characters
Often lethal
Morphometrics (measurements)
6-7 pelvic fin rays
meristics (counts)
Issues with morphometric and meristic data
• Are they different enough?
• What is the significance of the differences?
(do they represent genetic differences?)
• Do the differences indicate reproductive isolation?
LEVELS OF VARIATION
morphometric
and meristic
counts
~ 40 characters
protein loci
Often lethal
Often lethal
~ 200 loci
Protein electrophoresis
Basis:
• alleles result from changes in base pairs in DNA code
• base pair change results in different amino acid in protein
• resulting proteins may vary in size, shape, net electric charge
+
+
+
+
+
+
+
+
+
Protein electrophoresis
Basis:
• electrophoresis separates proteins on the basis of their net
electric charge, and size/shape
• thus, alleles coding for proteins with different size or charge
will be detected as different
• genotype is deduced from protein types
• co-dominant, low variability
Protein electrophoresis
alleles
Population 1
+
a’
a
_
1
2
3
4
5
6
INDIVIDUALS
7
8
9
10
Protein electrophoresis
alleles
Population 1
a’’
a’
a
Population 2
a’’
a’
a
Population 3
a’’
a’
a
1
2
3
4
5
6
INDIVIDUALS
7
8
9
10
monomeric
protein
dimeric
protein
LEVELS OF VARIATION
morphometric
and meristic
counts
~ 40 characters
protein loci
chromosome
~ 200 loci
Often lethal
Often lethal
100s of
characters
May be lethal
Cytogenetics
– information from chromosome number, shape, banding patterns
LEVELS OF VARIATION
morphometric
and meristic
counts
~ 40 characters
protein loci
Often lethal
Often lethal
~ 200 loci
chromosome
100s of
characters
May be lethal
DNA strand
100s to 1,000s
of characters
Non-lethal
DNA
Mitochondrial
Nuclear
•
•
•
•
•
•
•
•
•
•
•
•
circular
small - 16,000-20,000 bp
many copies
maternal inheritance
moderate variation
mostly coded loci
linear
3 billion + base pairs
two copies per cell
bi-parental inheritance
high variation
much non-coded DNA
‘Problems’ with DNA
• not very much of it (2-100 copies per cell)
- need to ‘amplify’ it
• too much of it (3 billion base pairs)
- need to look at small pieces at a time
• different areas of DNA have different variation
Amplification of DNA
PCR - polymerase chain reaction:
–
–
–
–
split DNA strand into two strands
bind primers on either side of segment to be amplified
allow new matching DNA strands to assemble on each side
repeat as often as needed
Polymerase Chain Reaction (PCR)
- DNA replication in a test tube
Requires ingredients:
DNA template
DNA polymerase
free nucleotide bases
DNA template must be separated (denatured, unwound)
must be a primer for DNA polymerase to add free nucleotides to
Polymerase Chain Reaction (PCR)
each replication cycle doubles amount of DNA
Polymerase Chain Reaction (PCR)
Requires ingredients:
DNA template
DNA polymerase
free nucleotide bases
DNA template must be separated (denatured, unwound)
heat will denature DNA, but deactivates protein enzymes
use Taq DNA polymerase
(from bacterium Thermus aquaticus from thermal vents)
Polymerase Chain Reaction (PCR)
Requires ingredients:
DNA template
DNA polymerase
free nucleotide bases
DNA template must be separated (denatured, unwound)
must be a primer for DNA polymerase to add free nucleotides to
short sequence of DNA that binds ‘upstream’ of area to be replicated
DNA analyses
• mtDNA (mitochondrial DNA analysis)
• RFLP (restriction fragment length polymorphism)
• RAPD (randomly amplified polymorphic DNA)
• AFLP (amplified fragment length polymorphism)
• microsatellites (SSR, simple sequence repeats)
• single nucleotide polymorphisms (SNPs)
Restriction Fragment Length Polymorphism (RFLP)
- cut DNA with restriction enzymes
- isolate cut fragments based on length (electrophoresis)
- deduce length of fragments
- individuals differ based on mutations at restriction sites
A T T G A C T T AA G C G T A G
T AA C T G AA T T C G C A T C
cleavage site (palindrome)
Restriction enzyme cleavage of mitochondrial DNA
Mitochondrial DNA
DNA fragments
gel
A T T G A C T T AA G C G T A G
T AA C T G AA T T C G C A T C
Single base pair substitution removes cleavage site recognition
A T T G A C T C AA G C G T A G
T AA C T G A G T T C G C A T C
Microsatellite DNA
• tandem repeats of short DNA sequences
(e.g. ACACACACAC…)
• number of repeats is highly variable – easy cross-over ‘mistake”
• co-dominant
Microsatellite DNA
• tandem repeats of short DNA sequences
(e.g. ACACACACAC…)
• number of repeats is highly variable – easy cross-over ‘mistake”
• co-dominant
• isolate portions of DNA with primers (time-consuming to
develop)
• separate fragments by length ~ number of repeats
• SNPs (single nucleotide polymorphisms)
• DNA sequencing
• Genome sequencing
• Barcode of Life Database (BOLD)
LEVELS OF VARIATION
A
T
G
G
C
T
A
A
morphometric
and meristic
counts
~ 40 characters
Often lethal
whole
organism
protein loci
chromosome
100s of
characters
May be lethal May be lethal
~ 200 loci
frozen
tissue
live
tissue
T
A
C
C
G
A
T
T
DNA strand
DNA
100s to 1,000s sequence
of characters
millions…
Non-lethal
Non-lethal
preserved or dried
tissue
Step 1: find markers
‘survey’ species/population for polymorphisms
before conducting full study
= # of loci, # alleles/locus
Step 1(b): find/develop primers for PCR
often available on web databases from related taxa
Step 2: determine how many markers are needed
depends on question(s) of interest
Smelt isozymes, Lake Champlain
MDH-1
MPI
Main lake
Mallets Bay
Inland Sea
Comparison of Eurasian and N. American
yellow perch
XDH-1
LDH-1
Perca flavescens
Perca fluviatilis
Cheetah (Acinonyx jubatus):
O’Brien et al. 1983. The cheetah is depauperate in genetic variation
- using protein electrophoresis
- assumed to be result of small N, bottleneck, then inbreeding
- highly vulnerable to disease outbreaks (50% mortality in one
captive population)
Cheetah (Acinonyx jubatus):
O’Brien et al. 1983. The cheetah is depauperate in genetic variation
- using protein electrophoresis
Species
Drosophila
Mus
Homo sapiens
Felis catus
Cheetah
#
popns.
N
43
>100
2
87
many >100
1
56
2
55
#
loci
24
46
104
55
47
% poly.
loci
43.1
20.5
31.7
22
0.0
av.
H
0.14
0.088
0.063
0.076
0.0
Cheetah (Acinonyx jubatus):
Menotti-Raymond and O’Brien et al. 1993.
- using protein electrophoresis, high-resolution PE, and mtDNA
Species
Drosophila
Mus
Homo sapiens
Felis catus
Cheetah
#
popns.
N
43
>100
2
87
many >100
1
56
2
55
#
loci
24
46
104
55
47
% poly.
loci
43.1
20.5
31.7
22
0.0
av.
H
0.14
0.088
0.063
0.076
0.0
Drosophila
Mus
Homo sapiens
Cheetah
1
1
1
1
20
54
72
many
155
11.1
4.1
1.2
3.2
0.04
0.02
0.063
0.013
Felis catus
Cheetah
1
3
17
76
34
6
46.0
45.0
Merola, 1994. A reassessment of homozygosity ….
- carnivores tend to show low levels of genetic variation
(several have lower levels of H and P than cheetah)
- measures of fluctuating asymmetry indicate cheetah is not
suffering from low homozygosity or genetic stress
- sperm deformities – do not affect fertility, may be normal in felids
- low litter sizes – in captivity (high in wild)
- susceptibility to disease – may be due to captive contact (in wild,
cheetahs avoid conspecifics)
Concluded that conservation is better directed at habitat
Spotted owls vs. barred owls
(Haig et al. 2004, Cons. Biol. 18:1347-1357)
Northern spotted owl – endangered
Barred owl – rapid range expansion has led to
overlap with spotted owl
- potential competition
- potential for hybridization
mtDNA and AFLP analysis:
- species are distinct
- no evidence of previous gene flow
- hybrids occur with male spotted and female barred owls
- hybrids can be identified until F2 generation
e-DNA (environmental DNA) as a method for detection
of rare/elusive species