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Species identification (taxonomy) - PowerPoint PPT Presentation


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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?

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Presentation Transcript
slide1

Uses of population genetic data

Species

identification

(taxonomy)

Population/stock

differentiation

Individual

identification

slide2

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?

slide3

A T

T A

G C

G C

C G

T A

A T

A T

DNA

sequence

DNA

strand

chromosome

protein

organism

slide4

morphometric

and meristic

counts

~ 40 characters

Often lethal

slide6

Morphometrics (measurements)

6-7 pelvic fin rays

meristics (counts)

slide7

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?
slide8

LEVELS OF VARIATION

morphometric

and meristic

counts

~ 40 characters

Often lethal

protein loci

~ 200 loci

Often lethal

protein electrophoresis
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 electrophoresis1
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
slide11

Protein electrophoresis

alleles

Population 1

+

a’

a

_

1 2 3 4 5 6 7 8 9 10

INDIVIDUALS

slide12

Protein electrophoresis

alleles

Population 1

a’’

a’

a

Population 2

a’’

a’

a

Population 3

a’’

a’

a

1 2 3 4 5 6 7 8 9 10

INDIVIDUALS

slide13

monomeric

protein

dimeric

protein

slide14

LEVELS OF VARIATION

morphometric

and meristic

counts

~ 40 characters

Often lethal

protein loci

~ 200 loci

Often lethal

chromosome

100s of

characters

May be lethal

slide15

Cytogenetics

– information from chromosome number, shape, banding patterns

slide16

LEVELS OF VARIATION

morphometric

and meristic

counts

~ 40 characters

Often lethal

protein loci

~ 200 loci

Often lethal

chromosome

100s of

characters

May be lethal

DNA strand

100s to 1,000s

of characters

Non-lethal

slide17
Mitochondrial

circular

small - 16,000-20,000 bp

many copies

maternal inheritance

moderate variation

mostly coded loci

Nuclear

linear

3 billion + base pairs

two copies per cell

bi-parental inheritance

high variation

much non-coded DNA

DNA
problems with 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
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
slide20

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

slide21

Polymerase Chain Reaction (PCR)

each replication cycle doubles amount of DNA

slide22

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)

slide24

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

slide25

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)
slide26

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 A A G C G T A G

T A A C T G A A T T C G C A T C

cleavage site (palindrome)

slide27

Restriction enzyme cleavage of mitochondrial DNA

Mitochondrial DNA DNA fragments gel

slide28

A T T G A C T T A A G C G T A G

T A A C T G A A T T C G C A T C

Single base pair substitution removes cleavage site recognition

A T T G A C T C A A G C G T A G

T A A C T G A G T T C G C A T C

microsatellite dna
Microsatellite DNA
  • tandem repeats of short DNA sequences

(e.g. ACACACACAC…)

  • number of repeats is highly variable – easy cross-over ‘mistake”
  • co-dominant
microsatellite dna1
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
slide31
SNPs (single nucleotide polymorphisms)
  • DNA sequencing
  • Genome sequencing
  • Barcode of Life Database (BOLD)
slide32

LEVELS OF VARIATION

A T

T A

G C

G C

C G

T A

A T

A T

morphometric

and meristic

counts

~ 40 characters

Often lethal

protein loci

~ 200 loci

May be lethal

chromosome

100s of

characters

May be lethal

DNA strand

100s to 1,000s

of characters

Non-lethal

DNA

sequence

millions…

Non-lethal

whole frozen live preserved or dried

organism tissue tissue tissue

slide33
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

slide34

Smelt isozymes, Lake Champlain

MDH-1

MPI

Main lake Mallets Bay Inland Sea

slide35

Comparison of Eurasian and N. American

yellow perch

XDH-1

LDH-1

Perca flavescens Perca fluviatilis

slide36

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)

slide37

Cheetah (Acinonyx jubatus):

O’Brien et al. 1983. The cheetah is depauperate in genetic variation

- using protein electrophoresis

# # % poly. av.

Species popns. N loci loci H

Drosophila 43 >100 24 43.1 0.14

Mus 2 87 46 20.5 0.088

Homo sapiens many >100 104 31.7 0.063

Felis catus 1 56 55 22 0.076

Cheetah 2 55 47 0.0 0.0

slide38

Cheetah (Acinonyx jubatus):

Menotti-Raymond and O’Brien et al. 1993.

- using protein electrophoresis, high-resolution PE, and mtDNA

# # % poly. av.

Species popns. N loci loci H

Drosophila 43 >100 24 43.1 0.14

Mus 2 87 46 20.5 0.088

Homo sapiens many >100 104 31.7 0.063

Felis catus 1 56 55 22 0.076

Cheetah 2 55 47 0.0 0.0

Drosophila 1 20 54 11.1 0.04

Mus 1 72 4.1 0.02

Homo sapiens 1 34 many 1.2 0.063

Cheetah 1 6 155 3.2 0.013

Felis catus 1 17 46.0

Cheetah 3 76 45.0

slide39

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

slide40

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