Robert Ekblom

Applications of next generation sequencing in molecular ecology of non-model organisms

As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can ‘go genomic’, it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Adaptation genomics: the next generation

Understanding the genetics of how organisms adapt to changing environments is a fundamental topic in modern evolutionary ecology. The field is currently progressing rapidly because of advances in genomics technologies, especially DNA sequencing. The aim of this review is to first briefly summarise how next generation sequencing (NGS) has transformed our ability to identify the genes underpinning adaptation. We then demonstrate how the application of these genomic tools to ecological model species means that we can start addressing some of the questions that have puzzled ecological geneticists for decades such as: How many genes are involved in adaptation? What types of genetic variation are responsible for adaptation? Does adaptation utilise pre-existing genetic variation or does it require new mutations to arise following an environmental change?

Genomics and the challenging translation into conservation practice

The global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners.

Spatial pattern of MHC class II variation in the great snipe (Gallinago media)

The genes of the major histocompatibility complex (MHC) code for proteins involved in antigen recognition and triggering of the adaptive immune response, and are therefore likely to be under selection from parasites. These selection regimes may vary in space and time. Here we report a strong geographical structure in MHC class II B genes of a migrating bird, the great snipe (Gallinago media). Genetic differentiation in the MHC between two ecologically distinct distributional regions (Scandinavian mountain populations vs. East European lowland populations) was still present after statistically controlling for the effect of selectively neutral variation (microsatellites) using partial Mantel tests. This suggests a role for selection in generating this spatial structure and that it represents local adaptation to different environments. Differentiation between populations within the two regions was negligible. Overall, we found a high number of MHC alleles (50, from 175 individuals). This, together with a tendency for a higher rate of nonsynonymous than synonymous substitutions in the peptide binding sites, and high Tajimas D in certain regions of the gene, suggests a history of balancing selection. MHC variation is often thought to be maintained by some form of balancing selection, but the nature of this selection remains unclear. Our results support the hypothesis that spatial variation in selection regimes contributes to the high polymorphism.

A field guide to whole‐genome sequencing, assembly and annotation

Genome sequencing projects were long confined to biomedical model organisms and required the concerted effort of large consortia. Rapid progress in high‐throughput sequencing technology and the simultaneous development of bioinformatic tools have democratized the field. It is now within reach for individual research groups in the eco‐evolutionary and conservation community to generate de novo draft genome sequences for any organism of choice. Because of the cost and considerable effort involved in such an endeavour, the important first step is to thoroughly consider whether a genome sequence is necessary for addressing the biological question at hand. Once this decision is taken, a genome project requires careful planning with respect to the organism involved and the intended quality of the genome draft. Here, we briefly review the state of the art within this field and provide a step‐by‐step introduction to the workflow involved in genome sequencing, assembly and annotation with particular reference to large and complex genomes. This tutorial is targeted at scientists with a background in conservation genetics, but more generally, provides useful practical guidance for researchers engaging in whole‐genome sequencing projects.

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

BackgroundDue to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.ResultsThe zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.ConclusionThe zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.

Major histocompatibility complex variation and mate choice in a lekking bird, the great snipe (Gallinago media)

Genes of the major histocompatibility complex (MHC) play a major part in the activation of the vertebrate immune system. In addition, they also appear to function as cues for mate choice. In mammals especially, several kinds of MHC‐dependent mate choice have been hypothesized and observed. These include choice of mates that share no or few alleles with the choosing individual, choice of mates with alleles that differ as much as possible from the choosing individual, choice of heterozygous mates, choice of certain genotypes and choice of rare alleles. We investigated these different aspects of mate choice in relation to MHC in a lekking bird species, the great snipe (Gallinago media). We found no evidence for MHC disassortative mating, no preference for males with many MHC alleles and no preference for rare alleles. However, we did find that some allelic lineages were more often found in males with mating success than in males without mating success. Females do not seem to use themselves as references for the MHC‐dependent mate choice, rather they seem to prefer males with certain allele types. We speculate that these alleles may be linked to resistance to common parasites.

High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species

BackgroundMicrosatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility.ResultsWe selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species.ConclusionsWhen combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available.

Digital gene expression analysis of the zebra finch genome

BackgroundIn order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC).ResultsAlmost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function.ConclusionsOur study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates.