Using genomic prediction to detect microevolutionary change of a quantitative trait.

Detecting microevolutionary responses to natural selection by observing temporal changes in individual breeding values is challenging. The collection of suitable datasets can take many years and disentangling the contributions of the environment and genetics to phenotypic change is not trivial. Furthermore, pedigree-based methods of obtaining individual breeding values have known biases. Here, we apply a genomic prediction approach to estimate breeding values of adult weight in a 35-year dataset of Soay sheep (Ovis aries). Comparisons are made with a traditional pedigree-based approach. During the study period, adult body weight decreased, but the underlying genetic component of body weight increased, at a rate that is unlikely to be attributable to genetic drift. Thus cryptic microevolution of greater adult body weight has probably occurred. Genomic and pedigree-based approaches gave largely consistent results. Thus, using genomic prediction to study microevolution in wild populations can remove the requirement for pedigree data, potentially opening up new study systems for similar research.

A high-density SNP chip for genotyping great tit (Parus major) populations and its application to studying the genetic architecture of exploration behaviour.

High-density SNP microarrays ("SNP chips") are a rapid, accurate and efficient method for genotyping several hundred thousand polymorphisms in large numbers of individuals. While SNP chips are routinely used in human genetics and in animal and plant breeding, they are less widely used in evolutionary and ecological research. In this article, we describe the development and application of a high-density Affymetrix Axiom chip with around 500,000 SNPs, designed to perform genomics studies of great tit (Parus major) populations. We demonstrate that the per-SNP genotype error rate is well below 1% and that the chip can also be used to identify structural or copy number variation. The chip is used to explore the genetic architecture of exploration behaviour (EB), a personality trait that has been widely studied in great tits and other species. No SNPs reached genomewide significance, including at DRD4, a candidate gene. However, EB is heritable and appears to have a polygenic architecture. Researchers developing similar SNP chips may note: (i) SNPs previously typed on alternative platforms are more likely to be converted to working assays; (ii) detecting SNPs by more than one pipeline, and in independent data sets, ensures a high proportion of working assays; (iii) allele frequency ascertainment bias is minimized by performing SNP discovery in individuals from multiple populations; and (iv) samples with the lowest call rates tend to also have the greatest genotyping error rates.

Multivariate selection and intersexual genetic constraints in a wild bird population.

When selection differs between the sexes for traits that are genetically correlated between the sexes, there is potential for the effect of selection in one sex to be altered by indirect selection in the other sex, a situation commonly referred to as intralocus sexual conflict (ISC). While potentially common, ISC has rarely been studied in wild populations. Here, we studied ISC over a set of morphological traits (wing length, tarsus length, bill depth and bill length) in a wild population of great tits (Parus major) from Wytham Woods, UK. Specifically, we quantified the microevolutionary impacts of ISC by combining intra- and intersex additive genetic (co)variances and sex-specific selection estimates in a multivariate framework. Large genetic correlations between homologous male and female traits combined with evidence for sex-specific multivariate survival selection suggested that ISC could play an appreciable role in the evolution of this population. Together, multivariate sex-specific selection and additive genetic (co)variance for the traits considered accounted for additive genetic variance in fitness that was uncorrelated between the sexes (cross-sex genetic correlation = -0.003, 95% CI = -0.83, 0.83). Gender load, defined as the reduction in a population's rate of adaptation due to sex-specific effects, was estimated at 50% (95% CI = 13%, 86%). This study provides novel insights into the evolution of sexual dimorphism in wild populations and illustrates how quantitative genetics and selection analyses can be combined in a multivariate framework to quantify the microevolutionary impacts of ISC.

The genetic architecture of sexually selected traits in two natural populations of Drosophila montana.

We investigated the genetic architecture of courtship song and cuticular hydrocarbon traits in two phygenetically distinct populations of Drosophila montana. To study natural variation in these two important traits, we analysed within-population crosses among individuals sampled from the wild. Hence, the genetic variation analysed should represent that available for natural and sexual selection to act upon. In contrast to previous between-population crosses in this species, no major quantitative trait loci (QTLs) were detected, perhaps because the between-population QTLs were due to fixed differences between the populations. Partitioning the trait variation to chromosomes suggested a broadly polygenic genetic architecture of within-population variation, although some chromosomes explained more variation in one population compared with the other. Studies of natural variation provide an important contrast to crosses between species or divergent lines, but our analysis highlights recent concerns that segregating variation within populations for important quantitative ecological traits may largely consist of small effect alleles, difficult to detect with studies of moderate power.

No evidence for MHC class I-based disassortative mating in a wild population of great tits.

Genes of the major histocompatibility complex (MHC) are regarded as a potentially important target of mate choice due to the fitness benefits that may be conferred to the offspring. According to the complementary genes hypothesis, females mate with MHC dissimilar males to enhance the immunocompetence of their offspring or to avoid inbreeding depression. Here, we investigate whether selection favours a preference for maximally dissimilar or optimally dissimilar MHC class I types, based on MHC genotypes, average amino acid distances and the functional properties of the antigen-binding sites (MHC supertypes); and whether MHC type dissimilarity predicts relatedness between mates in a wild great tit population. In particular, we explore the role that MHC class I plays in female mate choice decisions while controlling for relatedness and spatial population structure, and examine the reproductive fitness consequences of MHC compatibility between mates. We find no evidence for the hypotheses that females select mates on the basis of either maximal or optimal MHC class I dissimilarity. A weak correlation between MHC supertype sharing and relatedness suggests that MHC dissimilarity at functional variants may not provide an effective index of relatedness. Moreover, the reproductive success of pairs did not vary with MHC dissimilarity. Our results provide no support for the suggestion that selection favours, or that mate choice realizes, a preference for complimentary MHC types.

Replicated high-density genetic maps of two great tit populations reveal fine-scale genomic departures from sex-equal recombination rates.

Linking variation in quantitative traits to variation in the genome is an important, but challenging task in the study of life-history evolution. Linkage maps provide a valuable tool for the unravelling of such trait-gene associations. Moreover, they give insight into recombination landscapes and between-species karyotype evolution. Here we used genotype data, generated from a 10k single-nucleotide polymorphism (SNP) chip, of over 2000 individuals to produce high-density linkage maps of the great tit (Parus major), a passerine bird that serves as a model species for ecological and evolutionary questions. We created independent maps from two distinct populations: a captive F2-cross from The Netherlands (NL) and a wild population from the United Kingdom (UK). The two maps contained 6554 SNPs in 32 linkage groups, spanning 2010 cM and 1917 cM for the NL and UK populations, respectively, and were similar in size and marker order. Subtle levels of heterochiasmy within and between chromosomes were remarkably consistent between the populations, suggesting that the local departures from sex-equal recombination rates have evolved. This key and surprising result would have been impossible to detect if only one population was mapped. A comparison with zebra finch Taeniopygia guttata, chicken Gallus gallus and the green anole lizard Anolis carolinensis genomes provided further insight into the evolution of avian karyotypes.

Detecting genes for variation in parasite burden and immunological traits in a wild population: testing the candidate gene approach.

Identifying the genes underlying phenotypic variation in natural populations can provide novel insight into the evolutionary process. The candidate gene approach has been applied to studies of a number of traits in various species, in an attempt to elucidate their genetic basis. Here, we test the application of the candidate gene approach to identify the loci involved in variation in gastrointestinal parasite burden, a complex trait likely to be controlled by many loci, in a wild population of Soay sheep. A comprehensive literature review, Gene Ontology databases, and comparative genomics resources between cattle and sheep were used to generate a list of candidate genes. In a pilot study, these candidates, along with 50 random genes, were then sequenced in two pools of Soay sheep; one with low gastrointestinal nematode burden and the other high, using a NimbleGen sequence capture experiment. Further candidates were identified from single nucleotide polymorphisms (SNPs) that were highly differentiated between high- and low-resistance sheep breeds. A panel of 192 candidate and control SNPs were then typed in 960 individual Soay sheep to examine whether they individually explained variation in parasite burden, as measured as faecal egg count, as well as two immune measures (Teladorsagia circumcincta-specific antibodies and antinuclear antibodies). The cumulative effect of the candidate and control SNPs were estimated by fitting genetic relationship matrices (GRMs) as random effects in animal models of the three traits. No more significant SNPs were identified in the pilot sequencing experiment and association study than expected by chance. Furthermore, no significant difference was found between the proportions of candidate or control SNPs that were found to be significantly associated with parasite burden/immune measures. No significant effect of the candidate or control gene GRMs was found. There is thus little support for the candidate gene approach to the identification of loci explaining variation in parasitological and immunological traits in this population. However, a number of SNPs explained significant variation in multiple traits and significant correlations were found between the proportions of variance explained by individual SNPs across multiple traits. The significant SNPs identified in this study may still, therefore, merit further investigation.

Rates of assay success and genotyping error when single nucleotide polymorphism genotyping in non-model organisms: a case study in the Antarctic fur seal.

Although single nucleotide polymorphisms (SNPs) are increasingly being recognized as powerful molecular markers, their application to non-model organisms can bring significant challenges. Among these are imperfect conversion rates of assays designed from in silico resources and the enhanced potential for genotyping error relative to pre-validated, highly optimized human SNPs. To explore these issues, we used Illumina's GoldenGate assay to genotype 480 Antarctic fur seal (Arctocephalus gazella) individuals at 144 putative SNPs derived from a 454 transcriptome assembly. One hundred and thirty-five polymorphic SNPs (93.8%) were automatically validated by the program GenomeStudio, and the initial genotyping error rate, estimated from nine replicate samples, was 0.004 per reaction. However, an almost tenfold further reduction in the error rate was achieved by excluding 31 loci (21.5%) that exhibited unclear clustering patterns, manually editing clusters to allow rescoring of ambiguous or incorrect genotypes, and excluding 18 samples (3.8%) with unreliable genotypes. After stringent quality filtering, we also found a counter-intuitive negative relationship between in silico minor allele frequency and the conversion rate, suggesting that some of our assays may have been designed from paralogous loci. Nevertheless, we obtained over 45 000 individual SNP genotypes with a final error rate of 0.0005, indicating that the GoldenGate assay is eminently capable of generating large, high-quality data sets for non-model organisms. This has positive implications for future studies of the evolutionary, behavioural and conservation genetics of natural populations.

Inbreeding causes early death in a passerine bird.

Inbreeding typically reduces fitness. Related partners may fail to reproduce and any inbred offspring may die early or fail to reproduce themselves. Here we show that inbreeding causes early death in the zebra finch Taeniopygia guttata, and among inbred individuals of the same inbreeding coefficient (F), those that die early are more homozygous (estimated from single nucleotide polymorphisms) than those that survive to adulthood. Therefore, we identify two ways by which inbreeding depression may be underestimated in studies of inbreeding. First, a failure to study early life history could mean that the magnitude of inbreeding depression is routinely underestimated. Second, the observation that the most homozygous individuals of the same pedigree F were the least likely to survive to sexual maturity provides evidence that realized inbreeding, estimated from a high density of markers spread throughout the genome, explains variation in survival above and beyond what pedigree-based measures of inbreeding can explain.

Selection and microevolution of coat pattern are cryptic in a wild population of sheep.

Understanding the maintenance of genetic variation in natural populations is a core aim of evolutionary genetics. Insight can be gained by quantifying selection at the level of the genotype, as opposed to the phenotype. Here, we show that in a natural population of Soay sheep which is polymorphic for coat pattern, recessive genetic variants at the causal gene, agouti signalling protein (ASIP) are associated with reduced lifetime fitness. This was due primarily to a reduction in juvenile survival of uniformly coloured (self-type) sheep, which are homozygous recessive, and occurs despite significantly higher reproductive success in surviving self-type adults. Consistent with their relatively low fitness, we show that the frequency of self-type individuals has declined from 1985 to 2008. Remarkably though, the frequency of the underlying self-allele has increased, because the frequency of heterozygous individuals (who harbour the majority of all self-alleles) has increased. Indeed, the ratio of observed/expected heterozygous individuals has increased during the study, such that there is now a significant excess of heterozygotyes. By employing gene-dropping simulations, we show that microevolutionary trends in the frequency and excess of ASIP heterozygotes are too pronounced to be caused by genetic drift. Studying this polymorphism at the level of phenotype rather than underlying genotype would have failed to detect cryptic fitness differences. We would also have been unable to rule out genetic drift as an evolutionary force driving genetic change. This highlights the importance of resolving the underlying genetic basis of phenotypic variation in explaining evolutionary dynamics.

Natural and sexual selection in a wild insect population.

The understanding of natural and sexual selection requires both field and laboratory studies to exploit the advantages and avoid the disadvantages of each approach. However, studies have tended to be polarized among the types of organisms studied, with vertebrates studied in the field and invertebrates in the lab. We used video monitoring combined with DNA profiling of all of the members of a wild population of field crickets across two generations to capture the factors predicting the reproductive success of males and females. The factors that predict a male's success in gaining mates differ from those that predict how many offspring he has. We confirm the fundamental prediction that males vary more in their reproductive success than females, and we find that females as well as males leave more offspring when they mate with more partners.

The genetic basis of recessive self-colour pattern in a wild sheep population.

Bridging the genotype-phenotype gap for traits of ecological and evolutionary importance in natural populations can provide a novel insight into the origin and maintenance of variation. Here, we identify the gene and putative causal mutations underlying a recessive colour pattern phenotype ('self' or uniform colour) in a wild population of primitive Soay sheep. We targeted the agouti signalling protein (ASIP) gene, a positional candidate based on previous study that mapped the Coat pattern locus to a presumptive region on chromosome 13. We found evidence for three recessive mutations, including two functional changes in the coding sequence and a putative third cis-regulatory mutation that inactivates the promoter. These mutations define up to five haplotypes in Soays, which collectively explained the coat pattern in all but one member of a complex multi-generational pedigree containing 621 genotyped individuals. The functional mutations are in strong linkage disequilibrium in the study population, and are identical to those known to underlie the self phenotype in domestic sheep. This is indicative of a recent (and simultaneous) origin in Soay sheep, possibly as a consequence of past interbreeding with modern domestic breeds. This is only the second study in which ASIP has been linked to variation in pigmentation in a natural population. Knowledge of the genetic basis of self-colour pattern in Soay sheep, and the recognition that several mutations are segregating in the population, will aid future studies investigating the role of selection in the maintenance of the polymorphism.

Horn type and horn length genes map to the same chromosomal region in Soay sheep.

The evolution of male weaponry in animals is driven by sexual selection, which is predicted to reduce the genetic variability underlying such traits. Soay sheep have an inherited polymorphism for horn type in both sexes, with males presenting with either large, normal horns or small, deformed horns (scurs). In addition, there is additive genetic variation in horn length among males with normal horns. Given that scurred males cannot win conflicts with normal-horned males, it is unusual that genes conferring scurs should persist in the population. Identifying the genetic basis of these traits should help us in understanding their evolution. We developed microsatellite markers in a targeted region of the Soay sheep genome and refined the location of the Horns locus (Ho) to a approximately 7.4 cM interval on chromosome 10 (LOD=8.78). We then located quantitative trait loci spanning a 34 cM interval with a peak centred close to Ho, which explained the majority of the genetic variation for horn length and base circumference in normal-horned males (LOD=2.51 and LOD=1.04, respectively). Therefore, the genetic variation in both horn type and horn length is attributable to the same chromosomal region. Understanding the maintenance of horn type and length variation will require an investigation of selection on genotypes that (co)determine both traits.

Mitochondrial haplotype does not affect sperm velocity in the zebra finch Taeniopygia guttata.

Mitochondrial DNA (mtDNA) variation has been suggested as a possible cause of variation in male fertility because sperm activity is tightly coupled to mitochondrial oxidative phosphorylation and ATP production, both of which are sensitive to mtDNA mutations. Since male-specific phenotypes such as sperm have no fitness consequences for mitochondria due to maternal mitochondrial (and mtDNA) inheritance, mtDNA mutations that are deleterious in males but which have negligible or no fitness effect in females can persist in populations. How often such mutations arise and persist is virtually unknown. To test whether there were associations between mtDNA variation and sperm performance, we haplotyped 250 zebra finches Taeniopygia guttata from a large pedigreed-population and measured sperm velocity using computer-assisted sperm analysis. Using quantitative genetic 'animal' models, we found no effect of mtDNA haplotype on sperm velocity. Therefore, there is no evidence that in this system mitochondrial mutations have asymmetric fitness effects on males and females, leading to genetic variation in male fertility that is blind to natural selection.

A quantitative review of heterozygosity-fitness correlations in animal populations.

The ease of obtaining genotypic data from wild populations has renewed interest in the relationship between individual genetic diversity and fitness-related traits (heterozygosity-fitness correlations, or HFC). Here we present a comprehensive meta-analysis of HFC studies using powerful multivariate techniques which account for nonindependence of data. We compare these findings with those from univariate techniques, and test the influence of a range of factors hypothesized to influence the strength of HFCs. We found small but significantly positive effect sizes for life-history, morphological, and physiological traits; while theory predicts higher mean effect sizes for life-history traits, effect size did not differ consistently with trait type. Newly proposed measures of variation were no more powerful at detecting relationships than multilocus heterozygosity, and populations predicted to have elevated inbreeding variance did not exhibit higher mean effect sizes. Finally, we found evidence for publication bias, with studies reporting weak, nonsignificant effects being under-represented in the literature. In general, our review shows that HFC studies do not generally reveal patterns predicted by population genetic theory, and are of small effect (less than 1% of the variance in phenotypic characters explained). Future studies should use more genetic marker data and utilize sampling designs that shed more light on the biological mechanisms that may modulate the strength of association, for example by contrasting the strength of HFCs in mainland and island populations of the same species, investigating the role of environmental stress, or by considering how selection has shaped the traits under investigation.

Global patterns of gene expression in rice cultivars undergoing a susceptible or resistant interaction with the parasitic plant Striga hermonthica.

Striga hermonthica is a root hemiparasite of cereals that causes devastating loss of yield. Recently, a rice cultivar, Nipponbare, was discovered, which exhibits post-attachment resistance to this parasite and quantitative trait loci (QTL) associated with the resistance were identified. Changes in gene expression in susceptible (IAC 165) and resistant (Nipponbare) rice cultivars were profiled using rice whole-genome microarrays. In addition to a functional categorization of changes in gene expression, genes that were significantly up-regulated within resistance QTL were identified. The resistance reaction was characterized by up-regulation of defence genes, including pathogenesis-related proteins, pleiotropic drug resistance ABC transporters, genes involved in phenylpropanoid metabolism and WRKY transcription factors. These changes in gene expression resemble those associated with resistance to microbial pathogens. Three genes encoding proteins of unknown function, within a major resistance QTL on chromosome 12, were highly up-regulated and are excellent candidate resistance genes. The susceptible interaction was characterized by large-scale down-regulation of gene expression, particularly within the functional categories plant growth regulator signalling and metabolism, biogenesis of cellular components and cell division. Up-regulated genes included nutrient transporters, enzymes of amino acid metabolism and some abiotic stress genes.

A comparison of synteny and gene order on the homologue of chicken chromosome 7 between two passerine species and between passerines and chicken.

Wild populations of passerine birds have frequently been used in studies of ecology and evolution. However, the majority of species lack genetic linkage maps. The completion of a model avian genome sequence (the jungle fowl, Gallus gallus) has created an opportunity to advance genetic knowledge of other birds. Here we constructed genetic linkage maps of the homologue of chicken chromosome 7 for two passerine species, the zebra finch (Taeniopygia guttata) and the house sparrow (Passer domesticus). Linkage groups were constructed with a combination of SNP and microsatellite markers that were developed both in silico based on similarity to the chicken genome sequence, and de novo in the laboratory. Synteny was well conserved throughout the chromosome; however there were some gene rearrangements between chickens and passerines. This suggests that the transfer of map information from chicken to passerine and between different passerine species should not assume conserved gene order. The length of linkage maps of chromosome 7 were on average 60% that of chicken, consistent with the idea that passerines have a reduced recombination rate relative to chicken. Some evidence of heterochiasmy, i.e. a difference in the recombination rate between the sexes, was observed.

A linkage map of the zebra finch Taeniopygia guttata provides new insights into avian genome evolution.

Passeriformes are the largest order of birds and one of the most widely studied groups in evolutionary biology and ecology. Until recently genomic tools in passerines relied on chicken genomic resources. Here we report the construction and analysis of a whole-genome linkage map for the zebra finch (Taeniopygia guttata) using a 354-bird pedigree. The map contains 876 SNPs dispersed across 45 linkage groups and we found only a few instances of interchromosomal rearrangement between the zebra finch and the chicken genomes. Interestingly, there was a greater than expected degree of intrachromosomal rearrangements compared to the chicken, suggesting that gene order is not conserved within avian chromosomes. At 1068 cM the map is approximately only one quarter the length of the chicken linkage map, providing further evidence that the chicken has an unusually high recombination rate. Male and female linkage-map lengths were similar, suggesting no heterochiasmy in the zebra finch. This whole-genome map is the first for any passerine and a valuable tool for the zebra finch genome sequence project and for studies of quantitative trait loci.