quantitative trait locus (qtl) mapping
DESCRIPTION
How can we identify the genes and molecules responsible for phenotypic differences within and between species?. Quantitative genetics: direct mapping of genes in crosses between species or divergent strains within species Unbiased and does not require prior knowledge - PowerPoint PPT PresentationTRANSCRIPT
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How can we identify the genes and molecules responsible for phenotypic differences within and between species?
Quantitative genetics: direct mapping of genes in crosses between species or divergent strains within species
Unbiased and does not require prior knowledgeOffers conclusive proof of a gene's involvementSpecies must be able to hybridizeYou have to develop genetic markers and mapsHard to go from map position to single gene
Comparative developmental biology: using our knowledge of developmental biology and molecular genetics to test evolutionary hypotheses
Most direct route from phenotype to moleculesDoes not require species to be crossable or closely relatedRequires good knowledge of developmentSpecial tools and techniques must often be developedHard to go from correlation to functional proof
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Quantitative trait locus (QTL) mapping
Most traits in nature are controlled by multiple genes and therefore vary in a quantitative fashion
To map the genes responsible for this variation, we need:
1. Identify pure-breeding lines (or species) that differ in the trait of interest
2. Develop a large number of molecular markers throughout the genome
3. Cross these lines, then cross F1 offspring to each other or to one of the parental strains
4. Genotype F2 progeny for each marker5. Determine which markers are linked to the trait, and how
strongly6. Infer QTL positions based on this information
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1. Identify pure-breeding lines (or species) that differ in the trait of interest2. Develop a large number of molecular markers throughout the genome3. Cross these lines, then cross F1 offspring to each other or to one of the parental strains4. Genotype F2 progeny for each marker5. Determine which markers are linked to the trait, and how strongly6. Infer QTL positions based on this information
What does it take to map QTLs with high resolution?
Highest resolution is required for identification and cloning
Mapping procedure:
Factors affecting power and precision:
Lines should differ as much as possibleLines (species) should be able to hybridize
Marker density should be very highNumber of progeny should be very large
For polygenic traits, very sophisticated math & software are required
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Mapping agriculturally important traits in tomatoes
!
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Mapping agriculturally important traits in tomatoes
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What can QTL mapping tell us?
Easy (relatively):How many genes contribute to phenotypic differences?What are the contributions of individual genes?
Key question: are evolutionary changes due to many genes of small effect, or to few genes of large effect?100 genes that contribute 1% each, or 4 genes that contribute 25% each?
Very hard:What are these genes??? (TFs, enzymes, etc.)What are their normal developmental/biochemical functions?Why do changes in these genes cause phenotypic differences?What are these changes at the molecular level? (coding or non-
coding, how many mutations per gene, etc.)
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How many genes are responsible for phenotypic differences between species?
These are sibling species!
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How many genes are responsible for phenotypic differences between species?
These are sibling species!
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How strong are phenotypic effects of individual genes?
In practice, we are only likely to find these
Allen Orr's theoretical prediction
Conclusion: genes of large effect do play an important role in phenotypic evolution, but our technical limitations may lead us to overestimate this role
Note the log scale
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Why is it so hard to identify QTLs?
Factors affecting power and precision:
Lines should differ as much as possibleLines (species) should be able to hybridizeMarker density should be very high (expensive)Number of progeny should be very large (expensive)
Often incompatible
There have been hundreds of QTL mapping studies in a variety of organisms
Hundreds or thousands of QTLs have been detected and roughly mapped
But, only a handful of genes (< 20 total?) have ever been identified at the molecular level
So what can we do about this?
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So what can we do about this?
Identification of QTLs can be made much easier if we already know something about the developmental and biochemical basis of the trait
OK, so we know the responsible gene is somewhere in the red interval. Now, how do we identify it?
1. Map it with ever-increasing resolution until we reach single-gene density (positional cloning - long and expensive but certain)
2. Test specific genes that are located in that interval and that might be responsible for the trait based on their molecular function (candidate gene approach - fast and cheap but uncertain).
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Combining QTL and developmental-genetic approaches
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Pelvic spine reduction in sticklebacks
Spined (marine) Spineless (freshwater)
This transition has occurred many times in stickleback evolution during independent transitions from marine to freshwater habitats
Many changes have occurred in the last 10,000 years (after glaciation)
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QTL mapping of pelvic spine reduction
Candidate genes known to be involved in appendage development in all vertebrates
Note that the mapping is very crude
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Pitx1 expression in the pelvic spine region is lost in freshwater sticklebacks
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Combining experimental and comparative information
We don't know the mutant phenotype of Pitx1 in sticklebacks
But, we know what its phenotype is in mice, and it is consistent with its expression pattern in sticklebacks
The combination of these two lines of evidence suggests that changes in Pitx1 contribute to phenotypic differences
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What happened to Pitx1?
Pitx1 is expressed in multiple tissues
Its expression is controlled by separate tissue-specific regulatory sequences
The coding sequence of Pitx1 is unchanged
But it is no longer expressed in the pelvic girdle in spineless fish
Its expression in other tissues is unchanged
Hypothesis: Mutations in the tissue-specific regulatory sequence are the cause of phenotypic divergence
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The tb1 gene underlies a major difference in growth pattern between corn and teosinte
Wild-type teosinte
Wild-type maize
Maize tb1 mutant
Last 10,000 years!
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The difference in branching architecture between maize and teosinte maps near the tb1 locus
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tb1 is expressed at a higher level in maize than in teosinte
Maize and teosinte alleles were introgressed into the same genetic background
tb1 expression in axillary meristems and stamens of the ear primordia is required to suppress branching
Hypothesis: increased expression of tb1 in maize is responsible for the architectural differences between maize and teosinte
(Northern blots, normalized by ubiquitin)
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Selection has been limited to the non-coding region
Directional selection has removed most variation in maize
No evidence of selection
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Different species of Danio (zebrafish)
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Parallels between mutant phenotypes and natural diversity
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How the zebrafish got its stripes
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Pigment patterns in interspecific Danio hybrids
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An evolutionary complementation test
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Are candidate genes responsible for interspecific differences?
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Are candidate genes responsible for interspecific differences?
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ovo/shavenbaby controls denticle pattern in the larval epidermis
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Divergence of svb expression is responsible for differences between D. melanogaster and D. sechellia
D. melanogaster D. sechellia
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D. sechellia D.mel svb x D.sec
D.mel x D.sec D.mel svb
Genetic analysis and mapping of divergent phenotype
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svb expression correlates with morphological differences
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svb expression and function in the virilis species group
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A very simple trait: Excretory system in C. elegans
excretory duct cell lin-48 expression
wild type lin-48 mutant
longer duct
ces-2 mutant
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Genetic control of excretory duct development in C. elegans
wild-type
wild-type
C. elegans mutants
C. elegans coding sequence controlled by C. elegans
regulatory sequence
C. elegans coding sequence controlled by C. briggsae
regulatory sequence
Length of ED
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Derived excretory system morphology in C. elegans
derived morphology!
Length of EDspecies strain
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Derived excretory system function in C. elegans
Salt resistance
Derived function
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Divergence of lin-48 cis-regulatory regions
known regulator of lin-48
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Evolution of lin-48 regulation: alleles from different species introduced into C. elegans
The function of regulatory sequences of lin-48 has diverged!
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Functional tests: Necessity and Sufficiency
Is C. elegans lin-48 necessary for elegans-specific morphology?
Yes
Is C. elegans lin-48 sufficient for elegans-specific morphology?
Yes
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Functional sufficiency tests: Morphology
Conclusion: changes map to regulatory, not coding, sequences
C. briggsae coding sequence controlled by C. elegans regulatory sequence
C. briggsae coding sequence controlled by C. elegans regulatory sequence
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Functional sufficiency tests: Physiology
Necessary…
… but not sufficient
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From observation to testable hypothesis
Comparative analysis can suggest candidate genes that may be responsible for phenotypic changes
However, candidate gene hypotheses cannot be tested without functional
assays
These assays can take the form of either genetic crosses (where hybridization is possible) or transgenic tests
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Changes in development need not result in morphological changes
Changes in development need not result from genetic changes
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Dobzhansky - Muller incompatibilities and developmental drift
Reproductive isolation and speciation are caused by accumulation of genetic differences among independently evolving populations.
Accumulation of genetic differences does not necessarily result in phenotypic divergence
“Co-adaptation” of accumulated genetic changes is disrupted in hybrids, leading to inviability or sterility
Speciation is due to the evolution of developmental pathways
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Xiphophorus helleri
Xiphophorus maculatus x X. helleri
Developmental incompatibility in Xyphophorus hybrids
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Tu consists of two RTK genes, Xmrk1 and Xmrk2
Xmrk1 is found in all Xyphophorus species
Xmrk2 has evolved by exchange/fusion of Xmrk1 and D (donor) locus
Xmrk2 is present only in some species, including X. Maculatus
The two Xmrk genes are very similar in sequence, but Xmrk is controlled by a D-derived enhancer
Xmrk1 is expressed at a low level in all tissues; Xmrk2 is expressed at a high level only in hybrid melanomas
Xmrk2 overexpression in non-hybrid fish causes melanic tumors
In X. maculatus strains that do not induce melanomas, Xmrk2 is disrupted
r = cdk2 ???
Genetic basis of reproductive isolation in Xyphophorus
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Developmental defects in interspecific hybrids
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D. melanogaster C(1)RM/Y females x wild males
Bristle loss in hybrids between D. melanogaster and D. simulans
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The extent of bristle loss depends of parental phenotype
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neuralized-lacZ
Early neuralized expression is normal
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cut
Late loss of sensory organ markers
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Bristle loss in hybrids is sensitive to asc dosage
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Expression of many enhancers is altered in interspecific hybrids
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If it ain’t broke, fix it anyway:Cryptic divergence of developmental pathways
Developmental pathways can diverge rapidly among closely related species
This divergence may occur without any overt phenotypic consequences (“developmental drift”)
Cryptic divergence of developmental pathways is revealed by hybrid breakdown
Speciation is a consequence of developmental changes
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Polyphenic development in termites
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Pheidole castes
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Continuous and discontinuous environments
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Environmental control of development - and developmental adaptations to environment
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Genetic control of wing growth and patterning
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Photoperiod, temperature
Diet
Polyphenism in Pheidole morrisi
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Different gene expression in genetically identical individuals
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Different gene expression in genetically identical individuals
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Different gene expression in genetically identical individuals
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Polyphenism and the evolution of genetic networks