Ferramentas moleculares e estatsticas para estudos de estrutura de populaes

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Ferramentas moleculares e estatísticas para
estudos de estrutura de populações

• Guillermo Ortí
• University of Nebraska
• Lincoln, NE, USA

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Ferramentas, herramientas, or tools?

• Plan A pure, good academic English
• Plan B Español en el mejor estilo argentino
• Plano C Portunhol ruim, nem academico, mas com
  as melhores intençoes

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PDF versions of papers and powerpoints are (will
be) downloadable fromhttp//golab.unl.edu/teachi
ng
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What is population structure?

• Why is it important to study this?

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Population Structure (hist.)

• Ecological (Ecological Genetics, Ford 1964)
• Adjustments and adaptations of wild populations
  to their natural environment
• Genetic concepts, neodarwinian synthesis
  (Stebbins, 1950 Dobzhanski, 1970)
• Natural selection and the genetic basis for
  variation
• Documenting and measuring the magnitude of
  selection in natural systems (single population,
  phenotype under simple genetic control)

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In 1951, the problematic of population genetics
was the description and explanation of genetic
variation within and between populations. That
remains its problematic 40 years later R.C.
Lewontin 1991
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Population Structure (current)

• Population size (Ne)
• Subdivision of populations into local units
• Interaction among (sub)populations through
  migration and dispersal (gene flow)
• Interaction among local selection and genetic
  drift (to explain population differentiation)
• Neutral variation vs traits under selection
• Quantitative and qualitative feature of the
  phenotype (genetic basis, gene mapping, QTL,
  candidate gene)

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Why study Population Structure?

• Basic science the origin of species!
• Adaptive radiation, the evolution of ecological
  diversity (mechanistic appraisals of evolution)
• Effects of geomorphological/climatological
  factors
• Historical interactions among species
  (co-evolution)
• Applied research
• Conservation Biology (habitat fragmentation)
• Management (sustainability of harvested
  populations)
• Invasive species origin and fate
• GMO introduction and spread, risk assessment
• Molecular epidemiology, emergence of new disease
• Forensics

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Ferramentas moleculares

• What is a genetic marker?
• Hereditary information in biological
  macromolecules (proteins, nucleic acids) direct
  or indirect genotypic information from nucleic
  acid and protein sequences.
• What are the ideal attributes of molecular
  markers

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Ideal attributes of molecular markers

• Molecular data provide genetic information show
  no environmental or developmental influences
• Detect qualitative or quantitative (discrete)
  variation presence/absence, high or low
• Show simple codominant inheritance
• Detect changes in coding and non-coding regions
  (randomly distributed across the genome silent
  changes)
• Document evolutionary homologous changes

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TREE THINKING
Nearly all studies that utilize molecular
markers can be viewed as attempts to estimate
genetic relationships somewhere along a
hierarchical continuum of evolutionary
divergences ranging from recent to distant
(Avise 2004)

• Genetic identity versus non-identity (clones)
• Genetic parentage
• Extended kinship within local demes
• Genealogical affinities among geographic
  populations of a species
• Genetic divergence among species that separated
  recently
• Phylogenetic connections at intermediate and
  ancient branches of the Tree of Life

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Genetic relationships among individuals along a
hierarchical continuum
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• Trees within trees

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Gene trees within organismal trees
Lineage sorting
Gene duplication
Horizontal transfer
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Lineage sorting, genetic drift

• Lineage sorting will lead to reciprocal
  monophyly, but if alleles within a lineage
  persist for periods that are typically longer
  than the interval between successive speciation
  events, the paraphyletic condition may become
  fixed
• Depends on Pop size, neutrality

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Population?

• What is a population?
• Statistical definition
• universe of items under study
• Ecological definition
• Group of organisms of one taxon that occur in a
  particular area at a particular time
• Genetic definition
• All individuals connected by gene flow (the gene
  pool) ? genealogical implication!
• (How do we sample populations?)

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No single marker is good for all applications

• How do we compare the different techniques used
  to measure genetic diversity?
• Information content (mutation rate, level of
  polymorphism) enough variation?
• Multiplex ratio (number of loci represented by
  the marker) the more the better!

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Comparison of some markers
Other criteria Automation? Sequence info needed?
Start-up costs? Radioactivity needed?
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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)
1. Double digestion with restriction enzymes
EcoRI rare cutter, 6-cutter (5-GAATTC-3) MseI
frequent cutter, 4-cutter (5-TTAA-3) 2.
Ligation of double stranded adapters (110 ratio)
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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)

• What is the relative frequency of the restriction
  fragments obtained?
• MseI - MseI fragments gt 90 of all fragments
• EcoRI - MseI fragments will be about 2x the
  number of EcoRI sites in the genome
• A very small number will be EcoRI - EcoRI
  fragments

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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)

• Goal Preferential amplification of the EcoRI -
  MseI fragments
• Rationale for using two REs
• Freq cutter generates small fragm, easy to
  amplify and visualize
• Number of frag to be amplified is reduced by
  using a rare cutter
• Possible to labe one strand of the amplified
  dsDNA fragments
• Flexibility for tuning the number of frag to be
  amplified
• Large number of independent fingerprints can be
  generated by using combinations of a small
  number of primers

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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)
C
Step 3--Pre-amplification PCR primers designed
to match the sequence of the ligated adapters
use equal concentration of both primers
(unlabeled, but 1 bp at 3end)- only 20 cycles
in PCR. This is a selective preamplification
(1/16 of the original restriction fragments will
be amplified or 1/4 x 1/4)
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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)
C

• AFLP primers consist of 3 parts
• Core 5 part corresponding to the adapter
  sequence
• The restriction site sequence (provides
  specificity for each RE)
• The selective nucleotides (up to 3)
• If more than 3 selective nucleotides are used,
  there is loss of selectivity (C, CT, CTC,
  CTCA), so for complex genomes two selective PCR
  steps are necessary

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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)
Amplification AFLP primers 1 to 3 selective
bases are used to reduce the total number of
bands amplified to 50-100 bands on a 400 bp
sequencing gel. The EcoRI primer is labeled and
present in limiting condition (WHY?)
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AFLP Amplified Fragment Length Polymorphism (Vos
et al 1995)
MseI
EcoRI
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AFLP
65534
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AFLP gel
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Scoring AFLP gels

• Most are scored as diallelic markers
• Alleles detected as a band presence or absence
  ?they are DOMINANT
• Cannot distinguish between 1/1 and 1/0
  individuals
• Effect of indels?
• Result is a vector of 0s and 1s for each
  individual

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Wilding et al 2001 Differential gene exchange
between parapatric morphs of Littorina saxatilis
detected using AFLP markers. J Evol Biol 14
611-619

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Wilding et al 2001
Diallelic locus p 1 - q H-W expectation p2
2pq q2 for the equilibrium frequency of 1/1,
1/0, and 0/0 individuals
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Wilding et al 2001
From the estimated allelic frequency data for
each locus, other population parameters can be
estimated, in this case Fst -- a measure of
population subdivision (F-statistics, Wright
1951) The proportion of genetic variation
distributed among (as opposed to within)
subdivided populations FST (hT - hS)/hT
Where hS is the mean expected heterozygosity at
a locus (under H-W) within subpopulations, and hT
is the overall expected heterozygosity given
allele frequencies in the total population. Nei
(1977) and Nei and Chesser (1983) give a method
to calculate Fst.
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Wilding et al 2001
Two morphological forms of L. saxatilis in the
British marine coast (H and M) H thin-shelled,
wide aperture lives in the upper shore M
thicker-shelled, smaller aperture lower
shore Crab predation and physical habitat
differences may select for these
features Compare Fst distributions across loci,
between H and M populations with Fst
distributions in within-morph comparisons
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Wilding et al 2001
Hypothesis testing
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Wilding et al 2001
Results
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Wilding et al 2001
Results
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Wilding et al 2001
Results
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Wilding et al 2001
Results
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Wilding et al 2001
Results 15 loci detected by AFLP (from a total
of 306) seem to be either under selection or
(more likely) to be linked to loci that are (loci
that may affect shell thicknes and
shape). Analysis excluding those 15 loci give
very different results. Calculate Neis genetic
distances between H and M samples and build a NJ
tree (phenogram)
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Wilding et al 2001
All genes included
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Wilding et al 2001
15 genes excluded
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Other studies using AFLP
PNAS 101 (1)171-176, 2004

• Alpine grassland species, 177 individuals
  genotyped for 261 ALFP loci
• MCA multivariate component analysis to test for
  the influence of environmental variables, genetic
  distances, F-statistics

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Other studies using AFLP

• 24 individuals(3 species) genotyped for 434 ALFP
  loci
• Mean pairwise distances among individuals, NJ
  tree to estimate phylogeny
• Principal component analysis to test for clade
  differentiation
• Compared and reconciled this tree with
  phylogenies derived from mtDNA sequences