Download How many genes are responsible for phenotypic differences

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
Offers conclusive proof of a gene's involvement
Species must be able to hybridize
You have to develop genetic markers and maps
Hard 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 molecules
Does not require species to be crossable or closely related
Requires good knowledge of development
Special tools and techniques must often be developed
Hard to go from correlation to functional proof
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 marker
5. Determine which markers are linked to the trait, and how
strongly
6. 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:
1.
2.
3.
4.
5.
6.
Identify pure-breeding lines (or species) that differ in the trait of interest
Develop a large number of molecular markers throughout the genome
Cross these lines, then cross F1 offspring to each other or to one of the parental strains
Genotype F2 progeny for each marker
Determine which markers are linked to the trait, and how strongly
Infer QTL positions based on this information
Factors affecting power and precision:
Lines should differ as much as possible
Lines (species) should be able to hybridize
Marker density should be very high
Number of progeny should be very large
For polygenic traits, very sophisticated math & software are required
Mapping agriculturally important traits in tomatoes
!
Mapping agriculturally important traits in tomatoes
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 noncoding, how many mutations per gene, etc.)
How many genes are responsible for phenotypic
differences between species?
These are sibling species!
How many genes are responsible for phenotypic
differences between species?
These are sibling species!
How strong are phenotypic effects of individual genes?
Allen Orr's theoretical
prediction
Note the log scale
In practice, we are only likely to find these
Conclusion: genes of large effect do play an important role in phenotypic
evolution, but our technical limitations may lead us to overestimate this role
Why is it so hard to identify QTLs?
Factors affecting power and precision:
Lines should differ as much as possible
Lines (species) should be able to hybridize
Marker density should be very high
Number of progeny should be very large
Often
incompatible
(expensive)
(expensive)
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?
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).
Combining QTL and developmental-genetic approaches
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)
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
Pitx1 expression in the pelvic spine region is lost in
freshwater sticklebacks
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
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 tissuespecific regulatory sequence are the
cause of phenotypic divergence
The tb1 gene underlies a major difference in growth
pattern between corn and teosinte
Wild-type maize
Wild-type teosinte
Last 10,000 years!
Maize tb1 mutant
The difference in branching architecture between maize and
teosinte maps near the tb1 locus
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
(Northern blots, normalized by ubiquitin)
Hypothesis: increased
expression of tb1 in maize
is responsible for the
architectural differences
between maize and teosinte
Selection has been limited to the non-coding region
Directional selection has removed
most variation in maize
No evidence of selection
Different species of Danio (zebrafish)
Parallels between mutant phenotypes and natural diversity
How the zebrafish got its stripes
Pigment patterns in interspecific Danio hybrids
An evolutionary complementation test
Are candidate genes responsible for interspecific differences?
Are candidate genes responsible for interspecific differences?
ovo/shavenbaby controls denticle pattern in the larval epidermis
Divergence of svb expression is responsible for differences
between D. melanogaster and D. sechellia
D. melanogaster
D. sechellia
Genetic analysis and mapping of divergent phenotype
D. sechellia
D.mel x D.sec
D.mel svb x D.sec
D.mel svb
svb expression correlates with morphological differences
svb expression and function in the virilis species group
A very simple trait: Excretory system in C. elegans
excretory duct cell
wild type
lin-48 expression
lin-48 mutant
ces-2 mutant
Genetic control of excretory duct development in C. elegans
Length of ED
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
Derived excretory system morphology in C. elegans
species
strain
Length of ED
derived
morphology!
Derived excretory system function in C. elegans
Derived function
Salt
resistance
Divergence of lin-48 cis-regulatory regions
known regulator of lin-48
Evolution of lin-48 regulation: alleles from different species
introduced into C. elegans
The function of regulatory sequences of lin-48 has diverged!
Functional tests: Necessity and Sufficiency
Is C. elegans lin-48
necessary for elegansspecific morphology?
Yes
Is C. elegans lin-48
sufficient for elegansspecific morphology?
Yes
Functional sufficiency tests: Morphology
C. briggsae coding
sequence controlled by
C. elegans regulatory
sequence
C. briggsae coding
sequence controlled
by C. elegans regulatory
sequence
Conclusion: changes map to regulatory, not coding, sequences
Functional sufficiency tests: Physiology
Necessary…
… but not
sufficient
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
Changes in development need not result in morphological changes
Changes in development need not result from genetic changes
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
Speciation is due to the
hybrids, leading to inviability
evolution of developmental
or sterility
pathways
Developmental incompatibility in Xyphophorus hybrids
Xiphophorus helleri
Xiphophorus maculatus x X. helleri
Genetic basis of reproductive isolation in Xyphophorus
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
r = cdk2 ???
Xmrk2 overexpression in non-hybrid fish causes
melanic tumors
In X. maculatus strains that do not induce
melanomas, Xmrk2 is disrupted
Developmental defects in interspecific hybrids
Bristle loss in hybrids between D. melanogaster and D. simulans
D. melanogaster C(1)RM/Y females x wild males
The extent of bristle loss depends of parental phenotype
Early neuralized expression is normal
neuralized-lacZ
Late loss of sensory organ markers
cut
Bristle loss in hybrids is sensitive to asc dosage
Expression of many enhancers is altered in interspecific hybrids
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
Polyphenic development in termites
Pheidole castes
Continuous and discontinuous environments
Environmental control of development and developmental adaptations to environment
Genetic control of wing growth and patterning
Polyphenism in Pheidole morrisi
Photoperiod, temperature
Diet
Different gene expression in genetically identical individuals
Different gene expression in genetically identical individuals
Different gene expression in genetically identical individuals
Polyphenism and the evolution of genetic networks