Friday 20 October 2023
Lecture 17 – Adaptation Genomics: Simple Traits
Stickleback Pelvic Skeleton
The three-spined stickleback, Gasterosteus aculeatus, has been the subject of studies on the
molecular genetics of adaptation
Sticklebacks were originally marine (or at least estuarine – living in brackish water and
saltwater lakes) fish in the Northern Hemisphere. But they’ve invaded freshwater lakes more
than 20 times independently. When they do so, they have a stereotyped pattern of evolution
where they tend to lose the pelvic spines and girdle (which is a modified pelvic fin). This may
be because of the absence of predation or a lack of calcium to make bones (they also lose
armour plating). The genetic basis of pelvic bone loss has been studied
From an estuarine population, there is independent evolution of lake sticklebacks that
always show the same phenotypic outcome of loss of armour and loss of spines. Is it due to
the same molecular mechanisms?
To identify the pelvic girdle/spine loci, do a QTL analysis. Found the largest, but not only, QTL
is found on Chromosome 7. There’s an important transcription factor, homologous to Pitx1
(known to be important in skeletal formation in mice) in the area close to the QTL
Sequencing of Pitx shows no difference in the coding regions between marine and
freshwater morphs – it is not a coding mutation
But there is a difference in the expression pattern of Pitx1 between marine and freshwater
morphs – marine has Pitx1 switched on around the pelvic fins, not in estuarine ones
Get marine x freshwater hybrids to put the two Pitx1 alleles against a common F1
background. Compare the allele-specific expression of Pitx1 using QT PCR. The marine allele
has higher levels of expression, so the difference between the two must be due to a cis-
acting regulatory element
If the two alleles had the same expression, implies a mutation in regulatory proteins
in the area that binds to the DNA equivalently and regulate both at equal levels
To identify the causal mutation that is affecting the cis-acting regulatory element and
causing the difference in expression – but there are many variations in these regions which
can be spread out across the genome (due to looping of DNA during regulation)
Sequence the Pitx1 regulatory region of morphs. Freshwater strains are missing a 501bp
region (32 kb) upstream of the Pitx1 is missing. Perhaps this contains the causal regulatory
element
Take the saltwater sequence of the 2.5 kb region deleted in freshwater fish, and put it
behind a GFP reporter and transfect fish. Estuarine Pel-2.5kbSALR drives expression in the
pelvis, showing that there’s a pelvis-specific cis-acting regulatory element in the area – an
Lecture 17 – Adaptation Genomics: Simple Traits
Stickleback Pelvic Skeleton
The three-spined stickleback, Gasterosteus aculeatus, has been the subject of studies on the
molecular genetics of adaptation
Sticklebacks were originally marine (or at least estuarine – living in brackish water and
saltwater lakes) fish in the Northern Hemisphere. But they’ve invaded freshwater lakes more
than 20 times independently. When they do so, they have a stereotyped pattern of evolution
where they tend to lose the pelvic spines and girdle (which is a modified pelvic fin). This may
be because of the absence of predation or a lack of calcium to make bones (they also lose
armour plating). The genetic basis of pelvic bone loss has been studied
From an estuarine population, there is independent evolution of lake sticklebacks that
always show the same phenotypic outcome of loss of armour and loss of spines. Is it due to
the same molecular mechanisms?
To identify the pelvic girdle/spine loci, do a QTL analysis. Found the largest, but not only, QTL
is found on Chromosome 7. There’s an important transcription factor, homologous to Pitx1
(known to be important in skeletal formation in mice) in the area close to the QTL
Sequencing of Pitx shows no difference in the coding regions between marine and
freshwater morphs – it is not a coding mutation
But there is a difference in the expression pattern of Pitx1 between marine and freshwater
morphs – marine has Pitx1 switched on around the pelvic fins, not in estuarine ones
Get marine x freshwater hybrids to put the two Pitx1 alleles against a common F1
background. Compare the allele-specific expression of Pitx1 using QT PCR. The marine allele
has higher levels of expression, so the difference between the two must be due to a cis-
acting regulatory element
If the two alleles had the same expression, implies a mutation in regulatory proteins
in the area that binds to the DNA equivalently and regulate both at equal levels
To identify the causal mutation that is affecting the cis-acting regulatory element and
causing the difference in expression – but there are many variations in these regions which
can be spread out across the genome (due to looping of DNA during regulation)
Sequence the Pitx1 regulatory region of morphs. Freshwater strains are missing a 501bp
region (32 kb) upstream of the Pitx1 is missing. Perhaps this contains the causal regulatory
element
Take the saltwater sequence of the 2.5 kb region deleted in freshwater fish, and put it
behind a GFP reporter and transfect fish. Estuarine Pel-2.5kbSALR drives expression in the
pelvis, showing that there’s a pelvis-specific cis-acting regulatory element in the area – an
