Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics.

Decreasing costs of next-generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well-known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site-frequency-spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species-specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches ­ genotype calling, genotype­haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2­8X). Genotype­haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8­20X). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2­4X). Studies without species-specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype­haplotype calling, is recommended.

Mining the pig genome to investigate the domestication process.

Pig domestication began around 9000 YBP in the Fertile Crescent and Far East, involving marked morphological and genetic changes that occurred in a relatively short window of time. Identifying the alleles that drove the behavioural and physiological transformation of wild boars into pigs through artificial selection constitutes a formidable challenge that can only be faced from an interdisciplinary perspective. Indeed, although basic facts regarding the demography of pig domestication and dispersal have been uncovered, the biological substrate of these processes remains enigmatic. Considerable hope has been placed on new approaches, based on next-generation sequencing, which allow whole-genome variation to be analyzed at the population level. In this review, we provide an outline of the current knowledge on pig domestication by considering both archaeological and genetic data. Moreover, we discuss several potential scenarios of genome evolution under the complex mixture of demography and selection forces at play during domestication. Finally, we highlight several technical and methodological approaches that may represent significant advances in resolving the conundrum of livestock domestication.

Evolutionary study of a potential selection target region in the pig.

Domestication, modern breeding and artificial selection have shaped dramatically the genomic variability of domestic animals. In livestock, the so-called FAT1 quantitative trait locus (QTL) in porcine chromosome 4 was the first QTL uncovered although, to date, its precise molecular nature has remained elusive. Here, we characterize the nucleotide variability of 13 fragments of ∼500 bp equally spaced in a 2 Mb region in the vicinity of the FAT1 region in a wide-diversity panel of 32 pigs. Asian and European animals, including local Mediterranean and international pig breeds, were sequenced. Patterns of genetic variability were very complex and varied largely across loci and populations; they did not reveal overall a clear signal of a selective sweep in any breed, although FABP4 fragment showed a significantly higher diversity. We used an approximate Bayesian computation approach to infer the evolutionary history of this SSC4 region. Notably, we found that European pig populations have a much lower effective size than their Asian counterparts: in the order of hundreds vs hundreds of thousands. We show also an important part of extant European variability is actually due to introgression of Asian germplasm into Europe. This study shows how a potential loss in diversity caused by bottlenecks and possible selective sweeps associated with domestication and artificial selection can be counterbalanced by migration, making it much more difficult the identification of selection footprints based on naive demographic assumptions. Given the small fragment analyzed here, it remains to be studied how these conclusions apply to the rest of the genome.

Multilocus analysis of variation using a large empirical data set: phenylpropanoid pathway genes in Arabidopsis thaliana.

Detecting the signature of adaptation on nucleotide variation is often difficult in species that like Arabidopsis thaliana might have a complex demographic history. Recent re-sequencing surveys in this species provided genome-wide information that would mainly reflect its demographic history. We have used a large empirical data set (LED) as well as multilocus coalescent simulations to analyse sequence variation at loci involved in the phenylpropanoid pathway of this species. We surveyed and examined DNA sequence variation at nine of these loci (about 19.7 kb) in 23 accessions of A. thaliana and one accession of its closely related species Arabidopsis lyrata. Nucleotide variation was lower at nonsynonymous sites than at silent sites in all loci, indicating generalized functional constraint at the protein level. No association between variation and position in the metabolic pathway was detected. When the data were contrasted against the standard neutral model, significant deviations for silent variation were detected with Tajima's D, Fu's F(S) and Fay and Wu's H multilocus test statistics. These deviations were in the same direction than in previous large-scale multilocus analyses, suggesting a genome-wide effect. When the nine-locus data set was contrasted against the large empirical data set, the level (Watterson's theta) and pattern of variation (Tajima's D) detected in these loci did not deviate either at the single-locus or multilocus level from the corresponding empirical distributions. These results would support an important role of the demographic history of A. thaliana in shaping nucleotide variation at the nine studied phenylpropanoid loci. The potential and limitations of the empirical distribution approach are discussed.

Highly structured nucleotide variation within and among Arabidopsis lyrata populations at the FAH1 and DFR gene regions.

Nucleotide variation at the FAH1 and DFR gene regions was surveyed in four populations of Arabidopsis lyrata (two European A. l. petraea and two North American A. l. lyrata populations). In contrast to previous results, levels of variation were not consistently lower in A. l. lyrata than in A. l. petraea, and similar degrees of genetic differentiation were detected between and within subspecies. These observations and the significant genetic differentiation detected among populations suggest population substructure and no real subdivision between subspecies. For each gene studied, genotypic data were obtained, which allowed comparing nucleotide diversity within individuals (between sequences from the same individual) and within populations (between sequences from the same population). The generally lower level of variation within than among individuals detected in each population yielded a significant deviation from panmixia within populations. In three of the four populations studied, two highly divergent alleles were detected within populations at the highly variable DFR locus. This pattern and the significant excess of derived variants detected in most populations suggest that most variation segregating within populations results from rare migration events between relatively small and isolated populations exhibiting reduced panmixia.