The molecular toolbox

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The molecular toolbox
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First

• Plans for rest of class
• Today
• Lecture
• Thursday
• Guest lecture by Dr Gabrielle Rocap
• Genomics
• Class Evaluations
• Labs
• Review Sessions
• Bring questions
• Location to be determined
• Watch your e-mail and the webpage
• Return term paper
• Exam
• Monday, June 9, 1030, room 107
• cumulative

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The wide range of molecular applications

• Individuals
• Parentage
• Relatedness
• Genomics
• Populations
• Population structure
• Mixed Fishery Analyses
• Phylogeography
• Species
• Hybridization
• Phylogeny
• Taxonomy
• Communities
• Species diversity
• Phylogenetic diversity
• Community function

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The molecular toolbox
Toolbox
Molecular markers

• Materials
• Nails
• Screws
• Wire

• Materials
• MtDNA
• Nuclear DNA
• RNA

• Tools
• Hammer
• Drill
• Screwdriver

• Tools
• RFLP
• Sequencing
• PCR

• Essential
• Measuring tape

• Essential
• Primers

• Important lessons
• Youd only consider a nail if you have a hammer
• Without a measuring tape, there are only some
  things you can make

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The history of the molecular toolbox
1960
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The explosion in molecular markers
DGGE
Microarrays
ITS
SNPs
RAPD
Differential display
AFLP
RNA
VNTRs
scnDNA
RFLP
cpDNA
mtDNA

• Allozymes

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The molecular toolbox
Toolbox
Molecular markers

• Materials
• Nails
• Screws
• Wire

• Materials
• MtDNA
• Nuclear DNA
• RNA

• Tools
• Hammer
• Drill
• Screwdriver

• Tools
• RFLP
• Sequencing
• PCR

• Essential
• Measuring tape

• Essential
• Primers

• Decide what you want to make before you start
• Soft chair or bird house Population structure or
  phylogeny
• Chose the appropriate materials and tools
• Nails and hammer MtDNA and sequencing
• Check if you have the essentials
• Measuring tape Primers

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Considerations in choosing molecular makers

• Marker properties
• Mutation rates
• Detectable variability
• Homoplasy / Saturation
• Selection
• Mode of inheritance
• Bi- / uniparental
• Dominant/codominant
• Single locus / multilocus

• Logistics
• Primer availability
• Sample availability
• Funds Facilities
• Expertise

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Mutation rates

• Introduction of new variants
• Ultimate source of genetic variability
• Nucleotide substitutions or addition/deletion of
  repeats
• Rates differ - for example
• Allozymes 10-6-10-8
• Microsatellites 10-3-10-5
• MtDNA 2 / my
• Varies between mtDNA genes
• Determines levels of detectable variation
• Power of statistical tests
• Also determines the rate of back-mutation
• Homoplasy identity by state, not by descent

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Mutation rates - DNA sequences

• Mutation rates vary between genes
• mtDNA 0.5 2 10-7 (fish)
• S14 rDNA 210-11 (mammals)
• Coding or non-coding sequence
• Mutation rate higher in non-coding sequences
• E.g. mtDNA D-loop
• Important which gene to sequence
• Slow for deep phylogenies
• Fast for closely related taxa
• Important which part of variability to use
• Transitions / transversions
• Codon positions

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Variabilityshould correspond to the question

• Individuals
• Relatedness, dispersal
• The more variable the better
• Populations
• Population structure, adaptation, speciation
• Can be too variable
• More alleles than individual
• Difficult to estimate allele frequencies
• Homoplasy
• Species
• Hybridization, phylogeny, systematics
• Do not want too much variability within OTUs
• OTU operational taxonomic unit
• Homoplasy and mutation saturation a serious
  problem
• More conserved genes for deeper phylogeny
• E.g. mtDNA for intra-family
• rDNA for tree of life

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Allozymes

• Often few polymorphic loci
• 5-10
• Limited by available stains
• Usually few alleles
• Mostly 2-3
• Varies between taxa
• Higher in invertebrates
• Studies using parasites of vertebrates
• Low variability often main problem with allozymes
• Not true in some taxa
• e.g. mollusks

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For example rockfish

• Which marker is better for
• Species identification
• Population differentiation
• Why?

PGI-1
PGI-2
PGM
common name
scientific name
Abbrev.
A
B
A
B
A
B
Pacific Ocean Perch
S. alutus
POP
1
0
1
0
0.55
0.45
Rougheye rockfish
S. aleutianus
RE
1
0
0
1
0.91
0.09
Shortraker rockfish
S. borealis
SR
1
0
0
1
0.29
0.71
Black rockfish
S. melanops
B
0
1
0
1
0.96
0.04
Yelloweye rockfish
S. ruberrimus
YE
1
0
0
1
1
0
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Mutation ratesVNTRs (microsatellites)

• Mutate by insertion / deletion of repeats
• High mutation rates
• 10-3-10-5
• Higher in dinucleotides
• Higher levels of variability
• Many alleles gt 50
• High heterozygosity
• Prop of heterozygous individuals
• Mutations can mask previous mutations
• Size homoplasy
• Problem for distantly related populations

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Example population differentiation in Atlantic
herring

• Used allozymes and microsatellites
• Higher variability in microsatellites
• Greater power of tests

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Comparison Allozymes - Microsatellites

• Closely related populations
• FST often similar or higher
• tests more powerful

• Distantly related populations
• FST often smaller
• not very useful to reconstruct evolutionary
  distances

• Different species
• FST similar to conspecific populations
• not always useful for species ID

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Selection

• Most applications assume selective neutrality
• All genotypes have equal survival and
  reproduction
• Divergence caused by mutation, drift and
  migration
• Two effects of selection
• Homogenizing
• Similar environments favor similar genotypes
• Genetic distances underestimated
• Diversifying
• Different environments favor different genotypes
• Genetic distances overestimated
• May sometimes be useful for identifying closely
  related populations in different environment
• MHC (Major Histocompatibility Complex)

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MHC genes

• Major Histocompatibility Complex
• Presents antigens to T-cells
• a chains very polymorphic
• Up to 200 alleles
• Diversifying selection
• Heterozygote advantage
• Disassortative mating mate choice
• Mice
• Salmon
• Mutation patterns
• dns gtgt ds
• Why?
• Several statistical tests
• Gene trees vs species trees?

8
7
6
5
Substitutions (10-9)
NS
4
S
3
2
1
0
MHC
Insulin
Albumin
Prolactin
Histone H4
Apolipoprotein E
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Example population differentiation in Atlantic
salmon

• MHC vs microsatellites
• Landry Bernatchez 2001

• Main problem
• Strength of selection generally unknown
• Difficult to conclude on exchange / gene flow
• When is a population a population?

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Mode of inheritance

• Uniparental inheritance
• mtDNA, cpDNA maternal
• Y-chromosome paternal
• Excellent marker for sex biased dispersal
• Mainly mtDNA useful for species with
• Migrating males
• Philopatric females
• No recombination
• Inherited clonally
• Good for phylogeny reconstructions
• mtDNA Eve out of Africa
• Smaller population size (Ne)
• Only in one sex
• Haploid (instead of diploid)
• More prone to genetic drift
• Faster population differentiation

• Huge disadvantage
• Inherited as a single locus
• Small and no recombination
• Lower statistical power than several loci

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Mode of inheritance

• Co-dominance
• All genotypes identifiable
• E.g. allozymes, microsatellites
• Dominance
• E.g. RAPD, AFLP
• Presence / absence data
• Cannot distinguish heterozygotes from one of the
  homozygotes
• Problem to estimate allele frequencies
• Specific assumptions
• Random mating, no selection, etc.
• underestimates proportion of polymorphic loci
• Co-dominant loci preferable

AA
AB
BB
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Single locus / multilocus

• Single locus markers
• Allozymes 5-10 (limited by enzymes)
• Microsatellites unlimited but usually 5-15
• MtDNA inherited as single locus
• scnDNA single loci
• Multilocus
• RAPD, AFLP 100s
• Multilocus minisatellites gt50
• Problems
• difficult to assign alleles
• Analyzed as presence / absence data
• Some loci may be selected
• Advantages
• Large coverage of the genome
• Lots of independent loci
• Powerful genealogy reconstruction

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Example Littorina in the UK

• Exist as 2 morphotypes
• H high shore, thin shell
• M mid shore, thick shell (crab predation)
• AFLP study (Wilding et al. 2001)
• 306 loci
• 15 loci higher differentiation than expected

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Considerations in choosing molecular makers

• Marker properties
• Mutation rates
• Detectable variability
• Homoplasy / Saturation
• Selection
• Mode of inheritance
• Bi- / uniparental
• Dominant/codominant
• Single locus / multilocus

• Logistics
• Primer availability
• Sample availability
• Funds Facilities
• Expertise

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Primer Availability

• Methods needing specific primers
• Microsatellites
• MHC
• Primer sources
• Check databases
• Heterologous primers
• Primer isolation
• Lengthy expensive
• Alternatives
• mtDNA
• Universal primers
• RAPD, AFLP
• Artificial primers
• Allozymes

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Facilities and Costs

• Facilities
• Can be a sandwich box or a 300,000 sequencer
• Can dictate what can be done
• Costs
• Salary
• Need expertise
• Expensive staff
• Getting things to work may take time
• Isolation of primers
• Consumables
• 5-20 / ind.
• But different methods need different sample sizes
• Sequencing few individuals
• Allozymes 50 ind.
• Microsatellites 100 ind.

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Availability of Samples

• Sample quality
• Preservation
• Allozymes fresh or frozen
• DNA ethanol or frozen
• Formalin usually not good
• Ancient DNA methods
• Recover DNA from old and degraded material
• Scales or bones
• No methods needing high quality DNA
• Sample sizes
• Endangered species
• Sampling logistics
• Remote areas
• Seldom found
• Giant squid
• Difficult material
• Limited success rate
• Museum specimens

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Availability of samples

• Big issue can determine success of a study
• Small samples can sometimes be compensated by
  more information
• Single locus markers not useful if N5
• Higher variability (more alleles) larger sample
  sizes
• Microsatellites gt allozymes
• Sequencing only few individuals
• Individual based analysis
• Phylogenetic trees
• Long sequences
• Multi-locus markers
• Have to make sure thatdata can be analyzed

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The wide range of molecular applications
Marker

• Individuals
• Genomics
• Relatedness
• Dispersal
• Populations
• Population structure
• Mixed Fishery Analyses
• Phylogeography
• Species
• Hybridization
• Phylogeny
• Taxonomy
• Communities
• Species diversity
• Phylogenetic diversity
• Community function

mtDNA, cpDNA
allozymes
RAPD, AFLP
RNA
VNTRs
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Key Concepts

• Using and choosing molecular markers
• Very important step
• Marker criteria
• Mutation rates
• Selection
• Mode of inheritance
• Logistic criteria
• Primer availability
• Sample availability
• Facilities