Neutral and adaptive

molecular variation
18 February 2020
Reading: Rowe, Sweet & Beebee
Chapter 8

Blokkebær
Bog bilberry
(Vaccinium uliginosum)

Photo: Truls Moum

 Adaptive molecular variation

Using genomics to study ecologically

important traits that are under the
influence of natural selection
Ecologically important traits

Fitness associated traits, e.g.

- reproductive performance
- Life history traits

https://en.wikipedia.org/wiki/Frog
 Adaptive variation
Alternative approaches to studying adaptive variation

Phenotypic traits associated with fitness

https://u.osu.edu/buckmdblog/ The genomic landscape of species divergence in Ficedula flycatchers

Ellegren et al. Nature 2012
 Adaptive variation
Alternative approaches to studying adaptive variation

Phenotypic traits associated with fitness

Non-neutral genetic markers

… identify markers that are influenced by natural
selection, either directly or through linkage to
other genes (“hitch-hiking”)
 Adaptive variation

Little yellow croaker (Larimichthys polyactis)

The frequencies of
4 alleles of the
microsatellite A study of geographic variation in
locus H16 varies the little yellow croaker using 28
geographically microsatellite markers and mtDNA

Figure 1. Map of sampling

localities and allele
frequencies at locus H16.

Wang L, Liu S, Zhuang Z, Guo L, Meng Z, et al. (2013) Population Genetic Studies Revealed Local Adaptation in a High Gene-Flow Marine Fish, the
Small Yellow Croaker (Larimichthys polyactis). PLoS ONE 8(12): e83493.
 Adaptive variation

Little yellow croaker (Larimichthys polyactis)

Outlier test
Fst neutral: 0.008
Fst overall: 0.004
Fst H16: 0.092

Wang L, Liu S, Zhuang Z, Guo L, Meng Z, et al. (2013) Population Genetic Studies Revealed Local Adaptation in a High Gene-Flow Marine Fish, the
Small Yellow Croaker (Larimichthys polyactis). PLoS ONE 8(12): e83493.
 Adaptive variation
Microsatellite H16 maps
to the proteincoding
gene BAHCC1 Little yellow croaker (Larimichthys polyactis)

The BAHCC1 gene is

involved in
development and This suggests that selection is
growth acting on the BAHCC1 gene of the
species, with different alleles being
selected for in the north and south
of the distribution area

Figure 1. Map of sampling

localities and allele
frequencies at locus H16.

Wang L, Liu S, Zhuang Z, Guo L, Meng Z, et al. (2013) Population Genetic Studies Revealed Local Adaptation in a High Gene-Flow Marine Fish, the
Small Yellow Croaker (Larimichthys polyactis). PLoS ONE 8(12): e83493.
 Adaptive variation
Alternative approaches to studying adaptive variation

Phenotypic traits associated with fitness

Non-neutral genetic markers

Genetic mechanisms of adaptation

 Molecular ecology

- the application of molecular genetic methods to ecological problems

Genetic markers

population genetics

the neutrality assumption

 The neutrality assumption

Are markers really neutral?

Anonymous nuclear markers (ANMs)

Allozymes, NPCL – nuclear protein coding loci

DNA markers, the likelihood of being

influenced by selection
 The neutrality assumption

Are markers really neutral?

Anonymous nuclear markers (ANMs)

Allozymes, NPCL – nuclear protein coding loci

DNA markers, the likelihood of being

influenced by selection

Linkage (hitch-hiking) A and B are closely linked. A/B and C are

less closely linked. A recombination event
is more likely to happen between A/B and
C, than between A and B.
 Deviation from neutrality in DNA markers
Mitochondrial DNA
Supposedly neutral marker, but:

• Disease causing mutations are selected against, i.e. negative selection

• Bias in codon usage

• Empirical evidence – e.g. regional variation in humans

• Experimental evidence

• (Complete) linkage - selective sweeps, suggesting positive selection

Bazin et al. 2006: Population size does not influence mitochondrial

genetic diversity in animals. Science, 312, 570-572.
 Adaptive variation – the problem

identification of (a few) genes involved in adaptive variation

Further complication:
combined activity of several genes

Quantitative trait loci (QTL):

growth rate, adult size etc
 Adaptive variation – the problem

identification of (a few) genes involved in adaptive variation

Basis of fitness association can be:

• Allelic differences in properties of the gene product of interest

• Differential gene expression

 Genetic variation in cod (Gadus morhua)
Spatial variation in Atlantic cod:
- Is it genetically determined?
- Is it due to isolation (i.e. genetic drift) or differential selection?

Atlantic cod (Gadus morhua)

 Genetic variation in cod (Gadus morhua)
Large distribution area

Atlantic cod (Gadus morhua)

http://en.academic.ru/dic.nsf/enwiki/26785
 Genetic variation in cod (Gadus morhua)
Large distribution area

Geographic variation in protein coding genes (e.g. Pantophysin I,

haemoglobin)

Wide range of physical and biotic parameters:

Temperature, salinity, light, food supply, pathogens, predators...

Atlantic cod (Gadus morhua)

 Selection revealed

Comparison of several markers:

one (or a few) marker(s) behaves differently from the rest

Geographic variation in protein coding genes (e.g. Pantophysin I,

haemoglobin)

Pan IA
Pan IB
arctic cod vs coastal cod

and

northern vs southern populations

 Selection revealed

Comparison of several markers:

one (or a few) marker(s) behaves differently from the rest

environmental correlates

northern cooler

E.g. latitudinal clines

 Selection revealed

Comparison of several markers:

one (or a few) marker(s) behaves differently from the rest

environmental correlates

identifying the molecular basis for selection

Pan I locus in Atlantic cod

dN/dS (ratio of nonsynonymous/synonymous

substitutions)

Positive selection was detected in the two intravesicular

loops of the integral membrane protein
 Selection revealed

Comparison of several markers:

one (or a few) marker(s) behaves differently from the rest

environmental correlates

identifying the molecular basis for selection

optimum enzyme performance (temperature, salinity etc)

Structure-function comparison of haemoglobin of extant elephants to that

of the extinct mammoth (p. 254 Rowe, Sweet & Beebee)
 Selection revealed

Comparison of several markers:

one (or a few) marker(s) behaves differently from the rest

environmental correlates

identifying the molecular basis for selection

optimum enzyme performance (temperature, salinity etc)

revealing how selection operates in natural populations

 Heterozygosity and fitness associations
The assumptions:

1. Heterozygosity at marker loci reflects heterozygosity at loci associated with fitness traits

2. Heterozygosity at functional loci is associated with high fitness

There is no consensus among studies that heterozygosity and fitness is actually correlated!

Significant negative correlation

Based on 27 allozyme loci between heterozygosity and
developmental abnormalities in
toads (Bufo bufo)

Fig. 8.6 R&S

 Adaptive variation – the problem

Basis of fitness association:

• Allelic differences in properties of the gene product of interest

• Differential gene expression

How to detect alterations in gene expression?
Gene expression and microarrays
Generating a cDNA library
Generating a cDNA library
 Microarrays
Generating a cDNA library

mRNAs are polyadenylated

poly-T fishing

mRNA

HIV reverse transcriptase

cDNA
 Microarrays
Generating a cDNA library
 Microarrays
Generating a cDNA library

Expressed Sequence Tag (EST)

Single sequencing run from one end

of a cDNA

Piece of sequence information from

an expressed gene that is often
sufficient to identify the gene
 Microarrays

Gene expression studies from 1995

Gene expression within individuals

Comparing genomes between species

Generate genotypes for population studies (“SNP array”)

Microarrays
 Microarrays
probes
Sequences 20 – 100 nucleotides (synthesized oligos)

1. Known genes and targeted sequences (a few

model organisms with sequenced genome or
cDNA libraries)

2. Anonymous probes derived from cDNA library

 Microarrays
Verification with qPCR
Potential for experimental error

Differences in gene expression should be verified

using qPCR

Alternatively, analyses using RNA-seq!

 Microarrays
Variation within individuals
Arabidopsis thaliana
Gene expression changes
related to attacks from 5
herbivores/pathogens:

• Aphids
• Thrips
• Caterpillar
• 2 microbial pathogens Probes for 23 750 A. thaliana genes

151 – 2181 genes showed changes in gene expression

De Vos et al. 2005
 Microarrays
Variation between species

Compare gene sequences or

gene expression patterns to
baseline divergence

Gene sequences – phenotypic

differences

Gene expression patterns –

same genes regulated
differentially
 Microarrays and NGS

Microarrays can to a large

extent be replaced by NGS

microarrays

high throughput sequencing

 Variation within species

Phenotypic adaptations to freshwater and seawater in three spine sticklebacks (Gasterosteus aculeatus)
Variation within species

Marine populations

Freshwater populations
Variation within species

Expression of Pitx1 (Pituitary

homeobox transcription factor 1)

Chan et al. Science 2010

 Variation within species
A genetic switch in sticklebacks

Recurrent mutations (deletions) of a tissue specific enhancer of the Pitx1 (Pituitary

homeobox transcription factor 1) gene at chromosome 7 involved in pelvic reduction

Major morphological changes can arise by single mutational leaps in natural populations!
 Variation within species
A genetic switch in sticklebacks

Approx. 80% of freshwater adaptations

are located in regulatory DNA
sequences, only 20% in coding DNA
 Linkage mapping

Linkage group – linkage disequilibrium (LD)

http://ibbiologyhelp.com/Genetics/dihybridcrosslinkage.html
 Linkage mapping

Development of microsatellite markers from an enriched genomic

library for genetic analysis of melon (Cucumis melo L.)

Ritschel et al., BMC Plant Biology (2004)

Linkage mapping

Microsatellites:
genome wide coverage
Linkage mapping

E.g. 13 microsatellite markers at chromosome 8

Linkage mapping

Pedigree
Autosomal dominant inheritance
 Linkage mapping

Several candidate genes at 1q21-q23

POU genes encode a family of DNA-binding transcription factors

The POU2F1 gene was reported to be expressed in the cochlea during rat
embryogenesis, consistent with its contribution to inner ear development.
 Quantitative trait analysis

Quantitative trait loci (QTL)

Specific regions of DNA that are responsible for the

expression of a quantitative phenotype, such as size,
weight, reproductive performance, and so on

Quantitative trait analysis requires a large number of

markers to provide a good coverage of the genome

Markers should be codominant, i.e. microsatellites or SNPs

 Quantitative trait analysis
The number of loci involved in a particular trait is often large
Growth rate – at least 650 genes have some phenotypic effect on growth
in mice

Heritability
Genetic and environmental factors contributing to phenotypic variation

Quantitative trait analysis permits determination of heritability and

estimating the number of loci that contribute to the trait under study

Linkage analyses using pedigree information:

1. Breeding experiments
2. Natural populations, requiring pedigree information from molecular
ecology

More relevant to plant species than animals

 The redpoll complex (genus Acanthis)

Understanding the relationships between phenotypic

variation, adaptation, and speciation

Redpolls

A species complex with boreal and arctic,

circumpolar distributions

Common redpoll (Acanthis flammea) Arctic redpoll (Acanthis hornemanni)

 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

Arctic redpoll (Acanthis hornemanni)

A. h. hornemanni (Greenland Arctic
redpoll)
A. h. exilipes (hoary redpoll)

Common redpoll (Acanthis flammea)

A. f. flammea (mealy redpoll)
A. f. islandica (Icelandic redpoll)
A. f. rostrata (Greenland redpoll)

Lesser redpoll (Acanthis cabaret)

 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

~ 20 000 anonymous SNPs throughout genome

~ 200 000 SNPs in transcriptome

Gene expression data

Environmental data

Mason & Taylor 2015

 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

Mason & Taylor 2015 used:

~ 20 000 anonymous SNPs throughout genome

~ 200 000 SNPs in transcriptome

Gene expression data

Environmental data
 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

Mason & Taylor 2015 used:

~ 20 000 anonymous SNPs throughout genome

~ 200 000 SNPs in transcriptome

Gene expression data

Environmental data

Genomes were undifferentiated – they all belong to

a common gene pool
 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

Genomes of species were undifferentiated

But, there was a relationship between gene

expression, phenotype and environment

Thus, phenotypic differences seem to be driven by

ecological, local selection on a few regulatory genes

Mason & Taylor 2015

 The redpoll complex (genus Acanthis)
Phenotypically diverse with unsettled taxonomy:
One to five species/subspecies described, but with no genetic
differentiation using traditional genotyping

Conclusion:
The redpoll complex consists of a single,
locally adapted polymorphic species!

Mason & Taylor 2015

 Reverse genetics
Classical genetics:
establish the genetic basis of a phenotype

”Reverse genetics”:
Starting with a particular DNA sequence,
trying to establish the phenotype associated
Tobacco (Nicotinia tabacum)
with it

Gene inactivation
• Knockout
• RNA interference (siRNA; miRNA)
Knockout mice
Homology searching

The ultimate goal is to establish a link between the genome and the phenotype