iMSAT: a novel approach to the development of microsatellite loci using barcoded Illumina libraries

Background

Illumina sequencing with its high number of reads and low per base pair cost is an attractive technology for development of molecular resources for non-model organisms. While many software packages have been developed to identify short tandem repeats (STRs) from next-generation sequencing data, these methods do not inform the investigator as to whether or not candidate loci are polymorphic in their target populations.

Results

We provide a python program iMSAT that uses the polymorphism data obtained from mapping individual Illumina sequence reads onto a reference genome to identify polymorphic STRs. Using this approach, we identified 9,119 candidate polymorphic STRs for use with the parasitoid wasp Trioxys pallidus and 2,378 candidate polymorphic STRs for use with the aphid Chromaphis juglandicola. For both organisms we selected 20 candidate tri-nucleotide STRs for validation. Using fluorescent-labeled oligonucleotide primers, we genotyped 91 female T. pallidus collected in nine localities and 46 female C. juglandicola collected in 4 localities and found 15 of the examined markers to be polymorphic for T. pallidus and 12 of the examined markers to be polymorphic for C. juglandicola.

Conclusions

We present a novel approach that uses standard Illumina barcoding primers and a single Illumina HiSeq run to target polymorphic STR fragments to develop and test STR markers. We validate this approach using the parasitoid wasp T. pallidus and its aphid host C. juglandicola. This approach, which would also be compatible with 454 Sequencing, allowed us to quickly identify markers with known variability. Accordingly, our method constitutes a significant improvement over existing STR identification software packages.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-858) contains supplementary material, which is available to authorized users.     

Differential introgression from a sister species explains high FST outlier loci within a mussel species

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F_ST outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F_ST outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F_ST = 0.03), but seven loci (2%) were strong F_ST outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister‐hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi‐permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.     

Response of walnut aphid populations to increasing foliar nitrogen content

相似文献   

Disentangling the effects of geographic peripherality and habitat suitability on neutral and adaptive genetic variation in Swiss stone pine

It is generally accepted that the spatial distribution of neutral genetic diversity within a species’ native range mostly depends on effective population size, demographic history, and geographic position. However, it is unclear how genetic diversity at adaptive loci correlates with geographic peripherality or with habitat suitability within the ecological niche. Using exome‐wide genomic data and distribution maps of the Alpine range, we first tested whether geographic peripherality correlates with four measures of population genetic diversity at > 17,000 SNP loci in 24 Alpine populations (480 individuals) of Swiss stone pine (Pinus cembra) from Switzerland. To distinguish between neutral and adaptive SNP sets, we used four approaches (two gene diversity estimates, F_ST outlier test, and environmental association analysis) that search for signatures of selection. Second, we established ecological niche models for P. cembra in the study range and investigated how habitat suitability correlates with genetic diversity at neutral and adaptive loci. All estimates of neutral genetic diversity decreased with geographic peripherality, but were uncorrelated with habitat suitability. However, heterozygosity (H_e) at adaptive loci based on Tajima's D declined significantly with increasingly suitable conditions. No other diversity estimates at adaptive loci were correlated with habitat suitability. Our findings suggest that populations at the edge of a species' geographic distribution harbour limited neutral genetic diversity due to demographic properties. Moreover, we argue that populations from suitable habitats went through strong selection processes, are thus well adapted to local conditions, and therefore exhibit reduced genetic diversity at adaptive loci compared to populations at niche margins.     

Rapid evolution and the genomic consequences of selection against interspecific mating

While few species introduced into a new environment become invasive, those that do provide critical information on ecological mechanisms that determine invasions success and the evolutionary responses that follow invasion. Aedes albopictus (the Asian tiger mosquito) was introduced into the naturalized range of Aedes aegypti (the yellow fever mosquito) in the United States in the mid‐1980s, resulting in the displacement of A. aegypti in much of the south‐eastern United States. The rapid displacement was likely due to the superior competitive ability of A. albopictus as larvae and asymmetric mating interference competition, in which male A. albopictus mate with and sterilize A. aegypti females, a process called “satyrization.” The goal of this study was to examine the genomic responses of a resident species to an invasive species in which the mechanism of character displacement is understood. We used double‐digest restriction enzyme DNA sequencing (ddRADseq) to analyse outlier loci between selected and control lines of laboratory‐reared A. aegypti females from two populations (Tucson, AZ and Key West, Florida, USA), and individual females classified as either “resisted” or “mated with” A. albopictus males via mating trials of wild‐derived females from four populations in Florida. We found significant outlier loci in comparing selected and control lines and between mated and nonmated A. aegypti females in the laboratory and wild‐derived populations, respectively. We found overlap in specific outlier loci between different source populations that support consistent genomic signatures of selection within A. aegypti. Our results point to regions of the A. aegypti genome and potential candidate genes that may be involved in mating behaviour, and specifically in avoiding interspecific mating choices.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.     

Development and preliminary evaluation of a genomewide single nucleotide polymorphisms resource generated by RAD‐seq for the small yellow croaker (Larimichthys polyactis)

Recent advances in high‐throughput sequencing technologies have offered the possibility to generate genomewide sequence data to delineate previously unidentified genetic structure, obtain more accurate estimates of demographic parameters and to evaluate potential adaptive divergence. Here, we identified 27 556 single nucleotide polymorphisms for the small yellow croaker (Larimichthys polyactis) using restriction‐site‐associated DNA (RAD) sequencing of 24 individuals from two populations. Significant sources of genetic variation were identified, with an average nucleotide diversity (π) of 0.00105 ± 0.000425 across individuals, and long‐term effective population size was thus estimated to range between 26 172 and 261 716. According to the results, no differentiation between the two populations was detected based on the SNP data set of top quality score per contig or neutral loci. However, the two analysed populations were highly differentiated based on SNP data set of both top F_ST value per contig and the outlier SNPs. Moreover, local adaptation was highlighted by an F_ST‐based outlier tests implemented in LOSITAN and a total of 538 potentially locally selected SNPs were identified. blast2go annotation of contigs containing the outlier SNPs yielded hits for 37 (66%) of 56 significant blastx matches. Candidate genes for local adaptation constituted a wide array of biological functions, including cellular response to oxidative stress, actin filament binding, ion transmembrane transport and synapse assembly. The generated SNP resources in this study provided a valuable tool for future population genetics and genomics studies of L. polyactis.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

基于CLIMEX的2种核桃蚜虫潜在适生区分析

【目的】分析核桃黑斑蚜与核桃全斑蚜在全球范围内的潜在分布,比较气候变化对其分布的影响,为核桃有害生物综合管理策略的制定提供依据。【方法】基于实验室和野外试验,结合CLIMEX软件对核桃黑斑蚜与核桃全斑蚜在目前及未来气候条件下的潜在地理分布进行了模拟和系统评估。【结果】2种核桃蚜虫适生区十分相似,在世界范围内主要分布区集中在欧洲、北美洲、亚洲等区域,核桃黑斑蚜适生区范围大于核桃全斑蚜,但在我国,核桃黑斑蚜适生区范围小于核桃全斑蚜。气候变化将决定2种蚜虫分布的差异性,未来气候情景下,2种蚜虫适生区将发生变化,在欧洲、北美洲和亚洲适生区范围向高纬度延伸;在我国适生区范围逐渐减小。【结论】在世界范围内,核桃黑斑蚜与核桃全斑蚜适生区域主要分布在25°N-75°N内的亚洲、欧洲、北美洲的部分地区;在我国,其适生区域主要分布在东部季风区内的东北的南部、西北东南部、西南中部、华中北部以及华北地区。     

Natural Selection and Neutral Evolution Jointly Drive Population Divergence between Alpine and Lowland Ecotypes of the Allopolyploid Plant Anemone multifida (Ranunculaceae)

Population differentiation can be driven in large part by natural selection, but selectively neutral evolution can play a prominent role in shaping patters of population divergence. The decomposition of the evolutionary history of populations into the relative effects of natural selection and selectively neutral evolution enables an understanding of the causes of population divergence and adaptation. In this study, we examined heterogeneous genomic divergence between alpine and lowland ecotypes of the allopolyploid plant, Anemone multifida. Using peak height and dominant AFLP data, we quantified population differentiation at non-outlier (neutral) and outlier loci to determine the potential contribution of natural selection and selectively neutral evolution to population divergence. We found 13 candidate loci, corresponding to 2.7% of loci, with signatures of divergent natural selection between alpine and lowland populations and between alpine populations (Fst  = 0.074–0.445 at outlier loci), but neutral population differentiation was also evident between alpine populations (F_ST  = 0.041–0.095 at neutral loci). By examining population structure at both neutral and outlier loci, we determined that the combined effects of selection and neutral evolution are associated with the divergence of alpine populations, which may be linked to extreme abiotic conditions and isolation between alpine sites. The presence of outlier levels of genetic variation in structured populations underscores the importance of separately analyzing neutral and outlier loci to infer the relative role of divergent natural selection and neutral evolution in population divergence.     

Genetic isolation in an endemic African habitat specialist

The Chestnut‐banded Plover Charadrius pallidus is a Near‐Threatened shorebird species endemic to mainland Africa. We examined levels of genetic differentiation between its two morphologically and geographically distinct subspecies, C. p. pallidus in southern Africa (population size 11 000–16 000) and C. p. venustus in eastern Africa (population size 6500). In contrast to other plover species that maintain genetic connectivity over thousands of kilometres across continental Africa, we found profound genetic differences between remote sampling sites. Phylogenetic network analysis based on four nuclear and two mitochondrial gene regions, and population genetic structure analyses based on 11 microsatellite loci, indicated strong genetic divergence, with 2.36% mitochondrial sequence divergence between individuals sampled in Namibia (southern Africa) and those of Kenya and Tanzania (eastern Africa). This distinction between southern and eastern African populations was also supported by highly distinct genetic clusters based on microsatellite markers (global F_ST = 0.309,  = 0.510, D = 0.182). Behavioural factors that may promote genetic differentiation in this species include habitat specialization, monogamous mating behaviour and sedentariness. Reliance on an extremely small number of saline lakes for breeding and limited dispersal between populations are likely to promote reproductive and genetic isolation between eastern and southern Africa. We suggest that the two Chestnut‐banded Plover subspecies may warrant elevation to full species status. To assess this distinction fully, additional sample collection will be needed, with analysis of genetic and phenotypic traits from across the species’ entire breeding range.     

Positive selection of IL‐33 in adaptive immunity of domestic Chinese goats

The identification of the candidate genes that play key role in phenotypic variation in livestock populations can provide new information about evolution and positive selection. IL‐33 (71954) (Interleukin) gene is associated with the increased nematode resistance in small ruminants; however, the role of IL‐33 for the genetic control of different diseases in Chinese goat breeds is poorly described in scientific literature. Therefore, the current investigation was performed for the better understanding of the molecular evolution and the positive selection of single‐nucleotide polymorphism in IL‐33 gene. Fixation Index (F_ST)‐based method was used for the outlier loci determination and found that IL‐33 was present in outlier area with the provisional combined allocation of mean heterozygosity and F_ST. Positively selected IL‐33 gene was significantly, that is, p(Simul F_ST < sample F_ST = 0.98*) present in corresponding positive selection area. Hence, our study provided novel information about the nucleotide variations in IL‐33 gene and found to be nonsynonymous which may helpful for the genetic control of diseases by enhancing the immune system in local Chinese goat breeds as well as in other analyzed vertebrate species.     

Genetic divergence and isolation by thermal environment in geothermal populations of an aquatic invertebrate

Temperature is one of the most influential forces of natural selection impacting all biological levels. In the face of increasing global temperatures, studies over small geographic scales allowing investigations on the effects of gene flow are of great value for understanding thermal adaptation. Here, we investigated genetic population structure in the freshwater gastropod Radix balthica originating from contrasting thermal habitats in three areas of geothermal activity in Iceland. Snails from 32 sites were genotyped at 208 AFLP loci. Five AFLPs were identified as putatively under divergent selection in Lake Mývatn, a geothermal lake with an almost 20 °C difference in mean temperature across a distance of a few kilometres. In four of these loci, variation across all study populations was correlated with temperature. We found significant population structure in neutral markers both within and between the areas. Cluster analysis using neutral markers classified the sites mainly by geography, whereas analyses using markers under selection differentiated the sites based on temperature. Isolation by distance was stronger in the neutral than in the outlier loci. Pairwise differences based on outlier F_ST were significantly correlated with temperature at different spatial scales, even after correcting for geographic distance or neutral pairwise F_ST differences. In general, genetic variation decreased with increasing environmental temperature, possibly suggesting that natural selection had reduced the genetic diversity in the warm origin sites. Our results emphasize the influence of environmental temperature on the genetic structure of populations and suggest local thermal adaptation in these geothermal habitats.     

Genetic structure of wild populations of the endangered Desert Pupfish complex (Cyprinodontidae: Cyprinodon)

Data from 10 microsatellite DNA loci were used to describe the genetic structure of the two extant species (Cyprinodon macularius and C. eremus) of the endangered Desert Pupfish complex of southwestern United States and northwestern Mexico. Variation at microsatellite loci was significantly correlated (Mantel test) with that of previous mtDNA results, both for the complex and for the relatively wide-ranging C. macularius alone. Both species showed unusually high levels of microsatellite diversity for non-marine fish (H _e = 0.84–0.93; A_R = 11.9–17.0). There was evidence (R _ST > F _ST) that the two extant populations of C. eremus have been isolated sufficiently long for mutation to contribute significantly to genetic divergence, whereas divergence among the nine assayed populations of C. macularius could be attributed to genetic drift alone. Correspondingly, 10% of the diversity in C. eremus was attributable to differences between the two populations, whereas, for C. macularius, only 2.7% was attributable to among-population variation. Within C. macularius, a small (0.8%), but statistically significant, portion was attributable to differences between populations in the Salton Sea area and those on the lower Colorado River delta. The two populations of C. eremus and five groups of populations of C. macularius are recommended as management units for conservation genetics management of the two species.     

Diversity and genetic structure of the endangered cycad Dioon sonorense (Zamiaceae) from Sonora, Mexico: Evolutionary and conservation implications

The diversity and genetic structure of the cycad Dioon sonorense (De Luca, Sabato & Vázq. Torres) Chemnick, T.J. Greg. & S. Salas-Mor. were evaluated from the four known populations in tropical dry forests of Sonora, Mexico. The estimates of genetic diversity in the 19 loci analysed were: mean number of alleles per locus (A = 2.0), percentage of polymorphic loci (P = 81.6) and expected heterozygosity (H_E = 0.314). The subpopulations were found to be in Hardy–Weinberg equilibrium at the local level (F_is), even though we found a global excess of homozygotes (F_it = 0.13). Genetic variation attributed to differences among populations was 15%. Our results indicate that the geographical isolation caused by the historical effects of the Pleistocene among populations has probably generated clinal variation of the allelic frequencies at two loci, in relation to their latitudinal distribution. We compare our results with the long-lived southern hemisphere trees Fitzroya and Nothofagus in relationship to the Pleistocene glaciations. We also infer that the populations have become fragmented recently due to increasing pressure of habitat conversion, disturbance and illegal extraction of plants and seeds for the international horticultural trade. We therefore recommend three aspects: (i) propagation of this species for reintroduction, (ii) declare the localities of its northern distribution as a protected area, and (iii) an amendment of the current International Union for Conservation of Nature Red List categorisation for this species to ‘Critically Endangered’.     

Speciation genomics and a role for the Z chromosome in the early stages of divergence between Mexican ducks and mallards

Speciation is a continuous and dynamic process, and studying organisms during the early stages of this process can aid in identifying speciation mechanisms. The mallard (Anas platyrhynchos) and Mexican duck (A. [p.] diazi) are two recently diverged taxa with a history of hybridization and controversial taxonomy. To understand their evolutionary history, we conducted genomic scans to characterize patterns of genetic diversity and divergence across the mitochondrial DNA (mtDNA) control region, 3523 autosomal loci and 172 Z‐linked sex chromosome loci. Between the two taxa, Z‐linked loci (Φ_ST = 0.088) were 5.2 times more differentiated than autosomal DNA (Φ_ST = 0.017) but comparable to mtDNA (Φ_ST = 0.092). This elevated Z differentiation deviated from neutral expectations inferred from simulated data that incorporated demographic history and differences in effective population sizes between marker types. Furthermore, 3% of Z‐linked loci, compared to <0.1% of autosomal loci, were detected as outlier loci under divergent selection with elevated relative (Φ_ST) and absolute (d_XY) estimates of divergence. In contrast, the ratio of Z‐linked and autosomal differentiation among the seven Mexican duck sampling locations was close to 1:1 (Φ_ST = 0.018 for both markers). We conclude that between mallards and Mexican ducks, divergence at autosomal markers is largely neutral, whereas greater divergence on the Z chromosome (or some portions thereof) is likely the product of selection that has been important in speciation. Our results contribute to a growing body of literature indicating elevated divergence on the Z chromosome and its likely importance in avian speciation.