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1.
    
Advances in high‐throughput sequencing (HTS) technologies coupled with increased interdisciplinary collaboration are rapidly expanding capacity in the scope and scale of wildlife genetic studies. While existing HTS methods can be directly applied to address some evolutionary and ecological questions, certain research goals necessitate tailoring methods to specific study organisms, such as high‐throughput genotyping of the same loci that are comparable over large spatial and temporal scales. These needs are particularly common for studies of highly mobile species of conservation concern like marine turtles, where life history traits, limited financial resources and other constraints require affordable, adaptable methods for HTS genotyping to meet a variety of study goals. Here, we present a versatile marine turtle HTS targeted enrichment platform adapted from the recently developed Rapture (RAD‐Capture) method specifically designed to meet these research needs. Our results demonstrate consistent enrichment of targeted regions throughout the genome and discovery of candidate variants in all species examined for use in various conservation genetics applications. Accurate species identification confirmed the ability of our platform to genotype over 1,000 multiplexed samples and identified areas for future methodological improvement such as optimization for low initial concentration samples. Finally, analyses within green turtles supported the ability of this platform to identify informative SNPs for stock structure, population assignment and other applications over a broad geographic range of interest to management. This platform provides an additional tool for marine turtle genetic studies and broadens capacity for future large‐scale initiatives such as collaborative global marine turtle genetic databases.  相似文献   

2.
    
Understanding the genetics of biological diversification across micro‐ and macro‐evolutionary time scales is a vibrant field of research for molecular ecologists as rapid advances in sequencing technologies promise to overcome former limitations. In palms, an emblematic, economically and ecologically important plant family with high diversity in the tropics, studies of diversification at the population and species levels are still hampered by a lack of genomic markers suitable for the genotyping of large numbers of recently diverged taxa. To fill this gap, we used a whole genome sequencing approach to develop target sequencing for molecular markers in 4,184 genome regions, including 4,051 genes and 133 non‐genic putatively neutral regions. These markers were chosen to cover a wide range of evolutionary rates allowing future studies at the family, genus, species and population levels. Special emphasis was given to the avoidance of copy number variation during marker selection. In addition, a set of 149 well‐known sequence regions previously used as phylogenetic markers by the palm biological research community were included in the target regions, to open the possibility to combine and jointly analyse already available data sets with genomic data to be produced with this new toolkit. The bait set was effective for species belonging to all three palm sub‐families tested (Arecoideae, Ceroxyloideae and Coryphoideae), with high mapping rates, specificity and efficiency. The number of high‐quality single nucleotide polymorphisms (SNPs) detected at both the sub‐family and population levels facilitates efficient analyses of genomic diversity across micro‐ and macro‐evolutionary time scales.  相似文献   

3.
    
The use of high‐throughput, low‐density sequencing approaches has dramatically increased in recent years in studies of eco‐evolutionary processes in wild populations and domestication in commercial aquaculture. Most of these studies focus on identifying panels of SNP loci for a single downstream application, whereas there have been few studies examining the trade‐offs for selecting panels of markers for use in multiple applications. Here, we detail the use of a bioinformatic workflow for the development of a dual‐purpose SNP panel for parentage and population assignment, which included identifying putative SNP loci, filtering for the most informative loci for the two tasks, designing effective multiplex PCR primers, optimizing the SNP panel for performance, and performing quality control steps for downstream applications. We applied this workflow to two adjacent Alaskan Sockeye Salmon populations and identified a GTseq panel of 142 SNP loci for parentage and 35 SNP loci for population assignment. Only 50–75 panel loci were necessary for >95% accurate parentage, whereas population assignment success, with all 172 panel loci, ranged from 93.9% to 96.2%. Finally, we discuss the trade‐offs and complexities of the decision‐making process that drives SNP panel development, optimization, and testing.  相似文献   

4.
    
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5.
    
The advent of high‐throughput sequencing (HTS) has made genomic‐level analyses feasible for nonmodel organisms. A critical step of many HTS pipelines involves aligning reads to a reference genome to identify variants. Despite recent initiatives, only a fraction of species has publically available reference genomes. Therefore, a common practice is to align reads to the genome of an organism related to the target species; however, this could affect read alignment and bias genotyping. In this study, I conducted an experiment using empirical RADseq datasets generated for two species of salmonids (Actinopterygii; Teleostei; Salmonidae) to address these questions. There are currently reference genomes for six salmonids of varying phylogenetic distance. I aligned the RADseq data to all six genomes and identified variants with several different genotypers, which were then fed into population genetic analyses. Increasing phylogenetic distance between target species and reference genome reduced the proportion of reads that successfully aligned and mapping quality. Reference genome also influenced the number of SNPs that were generated and depth at those SNPs, although the affect varied by genotyper. Inferences of population structure were mixed: increasing reference genome divergence reduced estimates of differentiation but similar patterns of population relationships were found across scenarios. These findings reveal how the choice of reference genome can influence the output of bioinformatic pipelines. It also emphasizes the need to identify best practices and guidelines for the burgeoning field of biodiversity genomics.  相似文献   

6.
    
Combining high‐throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short‐read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long‐read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom‐made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage‐specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long‐read capture as a versatile approach for comparative genomic studies by generation of a cross‐species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.  相似文献   

7.
    
Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.  相似文献   

8.
    
Analyzing genetic variation through time and space is important to identify key evolutionary and ecological processes in populations. However, using contemporary genetic data to infer the dynamics of genetic diversity may be at risk of a bias, as inferences are performed from a set of extant populations, setting aside unavailable, rare, or now extinct lineages. Here, we took advantage of new developments in next‐generation sequencing to analyze the spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus, a steppic Southwestern‐Palearctic species. We applied a recently developed hybridization capture (hyRAD) protocol that allows retrieving orthologous sequences even from degraded DNA characteristic of museum specimens. We identified single nucleotide polymorphisms in 68 historical and 51 modern samples in order to (i) unravel the spatial genetic structure across part of the species distribution and (ii) assess the loss of genetic diversity over the past century in Swiss populations. Our results revealed (i) the presence of three potential glacial refugia spread across the European continent and converging spatially in the Alpine area. In addition, and despite a limited population sample size, our results indicate (ii) a loss of allelic richness in contemporary Swiss populations compared to historical populations, whereas levels of expected heterozygosities were not significantly different. This observation is compatible with an increase in the bottleneck magnitude experienced by central European populations of O. decorus following human‐mediated land‐use change impacting steppic habitats. Our results confirm that application of hyRAD to museum samples produces valuable information to study genetic processes across time and space.  相似文献   

9.
    
Understanding how risk factors affect populations across their annual cycle is a major challenge for conserving migratory birds. For example, disease outbreaks may happen on the breeding grounds, the wintering grounds, or during migration and are expected to accelerate under climate change. The ability to identify the geographic origins of impacted individuals, especially outside of breeding areas, might make it possible to predict demographic trends and inform conservation decision-making. However, such an effort is made more challenging by the degraded state of carcasses and resulting low quality of DNA available. Here, we describe a rapid and low-cost approach for identifying the origins of birds sampled across their annual cycle that is robust even when DNA quality is poor. We illustrate the approach in the common loon (Gavia immer), an iconic migratory aquatic bird that is under increasing threat on both its breeding and wintering areas. Using 300 samples collected from across the breeding range, we develop a panel of 158 single-nucleotide polymorphisms (SNP) loci with divergent allele frequencies across six genetic subpopulations. We use this SNP panel to identify the breeding grounds for 142 live nonbreeding individuals and carcasses. For example, genetic assignment of loons sampled during botulism outbreaks in parts of the Great Lakes provides evidence for the significant role the lakes play as migratory stopover areas for loons that breed across wide swaths of Canada, and highlights the vulnerability of a large segment of the breeding population to botulism outbreaks that are occurring in the Great Lakes with increasing frequency. Our results illustrate that the use of SNP panels to identify breeding origins of carcasses collected during the nonbreeding season can improve our understanding of the population-specific impacts of mortality from disease and anthropogenic stressors, ultimately allowing more effective management.  相似文献   

10.
    
Minimally invasive samples are often the best option for collecting genetic material from species of conservation concern, but they perform poorly in many genomic sequencing methods due to their tendency to yield low DNA quality and quantity. Genotyping-in-thousands by sequencing (GT-seq) is a powerful amplicon sequencing method that can genotype large numbers of variable-quality samples at a standardized set of single nucleotide polymorphism (SNP) loci. Here, we develop, optimize, and validate a GT-seq panel for the federally threatened northern Idaho ground squirrel (Urocitellus brunneus) to provide a standardized approach for future genetic monitoring and assessment of recovery goals using minimally invasive samples. The optimized panel consists of 224 neutral and 81 putatively adaptive SNPs. DNA collected from buccal swabs from 2016 to 2020 had 73% genotyping success, while samples collected from hair from 2002 to 2006 had little to no DNA remaining and did not genotype successfully. We evaluated our GT-seq panel by measuring genotype discordance rates compared to RADseq and whole-genome sequencing. GT-seq and other sequencing methods had similar population diversity and FST estimates, but GT-seq consistently called more heterozygotes than expected, resulting in negative FIS values at the population level. Genetic ancestry assignment was consistent when estimated with different sequencing methods and numbers of loci. Our GT-seq panel is an effective and efficient genotyping tool that will aid in the monitoring and recovery of this threatened species, and our results provide insights for applying GT-seq for minimally invasive DNA sampling techniques in other rare animals.  相似文献   

11.
    
Colour patterns in animals have long offered an opportunity to observe adaptive traits in natural populations. Colour plays myriad roles in interactions within and among species, from reproductive signalling to predator avoidance, leading to multiple targets of natural and sexual selection and opportunities for diversification. Understanding the genetic and developmental underpinnings of variation in colour promises a fuller understanding of these evolutionary processes, but the path to unravelling these connections can be arduous. The advent of genomic techniques suitable for nonmodel organisms is now beginning to light the way. Two new studies in this issue of Molecular Ecology use genomic sequencing of laboratory crosses to map colour traits in cichlid fishes, a remarkably diverse group in which coloration has played a major role in diversification. They illustrate how genomic approaches, specifically RAD sequencing, can rapidly identify both simple and more complex genetic variation underlying ecologically important traits. In the first, Henning et al. ( 2014 ) detect a single locus that appears to control in a Mendelian fashion the presence of horizontal stripes, a trait that has evolved in numerous cichlid lineages. In the second, Albertson et al. ( 2014 ) identify several genes and epistatic interactions affecting multiple colour traits, as well as a novel metric describing integration across colour traits. Albertson et al. ( 2014 ) go further, by quantifying differential expression of parental alleles at a candidate locus and by relating differentiation among natural populations at mapped loci to trait divergence. Herein lies the promise of ecological genomics – efficiently integrating genetic mapping of phenotypes with population genomic data to both identify functional genes and unravel their evolutionary history. These studies offer guidance on how genomic techniques can be tailored to a research question or study system, and they also add to the growing body of empirical examples addressing basic questions about how ecologically important traits evolve in natural populations.  相似文献   

12.
    
Single nucleotide polymorphisms (SNPs) are replacing microsatellites for population genetic analyses, but it is not apparent how many SNPs are needed or how well SNPs correlate with microsatellites. We used data from the gopher tortoise, Gopherus polyphemus—a species with small populations, to compare SNPs and microsatellites to estimate population genetic parameters. Specifically, we compared one SNP data set (16 tortoises from four populations sequenced at 17 901 SNPs) to two microsatellite data sets, a full data set of 101 tortoises and a partial data set of 16 tortoises previously genotyped at 10 microsatellites. For the full microsatellite data set, observed heterozygosity, expected heterozygosity and FST were correlated between SNPs and microsatellites; however, allelic richness was not. The same was true for the partial microsatellite data set, except that allelic richness, but not observed heterozygosity, was correlated. The number of clusters estimated by structure differed for each data set (SNPs = 2; partial microsatellite = 3; full microsatellite = 4). Principle component analyses (PCA) showed four clusters for all data sets. More than 800 SNPs were needed to correlate with allelic richness, observed heterozygosity and expected heterozygosity, but only 100 were needed for FST. The number of SNPs typically obtained from next‐generation sequencing (NGS) far exceeds the number needed to correlate with microsatellite parameter estimates. Our study illustrates that diversity, FST and PCA results from microsatellites can mirror those obtained with SNPs. These results may be generally applicable to small populations, a defining feature of endangered and threatened species, because theory predicts that genetic drift will tend to outweigh selection in small populations.  相似文献   

13.
    
Species occupying habitats subjected to frequent natural and/or anthropogenic changes are a challenge for conservation management. We studied one such species, Viola uliginosa, an endangered perennial wetland species typically inhabiting sporadically flooded meadows alongside rivers/lakes. In order to estimate genomic diversity, population structure, and history, we sampled five sites in Finland, three in Estonia, and one each in Slovenia, Belarus, and Poland using genomic SNP data with double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq). We found monophyletic populations, high levels of inbreeding (mean population FSNP = 0.407–0.945), low effective population sizes (Ne = 0.8–50.9), indications of past demographic expansion, and rare long‐distance dispersal. Our results are important in implementing conservation strategies for V. uliginosa, which should include founding of seed banks, ex situ cultivations, and reintroductions with individuals of proper origin, combined with continuous population monitoring and habitat management.  相似文献   

14.
    
Restriction‐site‐associated DNA tag (RAD‐tag) sequencing has become a popular approach to generate thousands of SNPs used to address diverse questions in population genomics. Comparatively, the suitability of RAD‐tag genotyping to address evolutionary questions across divergent species has been the subject of only a few recent studies. Here, we evaluate the applicability of this approach to conduct genome‐wide scans for polymorphisms across two cetacean species belonging to distinct families: the short‐beaked common dolphin (Delphinus delphis; n = 5 individuals) and the harbour porpoise (Phocoena phocoena; n = 1 individual). Additionally, we explore the effects of varying two parameters in the Stacks analysis pipeline on the number of loci and level of divergence obtained. We observed a 34% drop in the total number of loci that were present in all individuals when analysing individuals from the distinct families compared with analyses restricted to intraspecific comparisons (i.e. within D. delphis). Despite relatively stringent quality filters, 3595 polymorphic loci were retrieved from our interfamilial comparison. Cetaceans have undergone rapid diversification, and the estimated divergence time between the two families is relatively recent (14–19 Ma). Thus, our results showed that, for this level of divergence, a large number of orthologous loci can still be genotyped using this approach, which is on par with two recent in silico studies. Our findings constitute one of the first empirical investigations using RAD‐tag sequencing at this level of divergence and highlights the great potential of this approach in comparative studies and to address evolutionary questions.  相似文献   

15.
    
Fusion genes formed by chromosomal rearrangements are common drivers of cancer. Recent innovations in the field of next-generation sequencing (NGS) have seen a dynamic shift from traditional fusion detection approaches, such as visual characterization by fluorescence, to more precise multiplexed methods. There are many different NGS-based approaches to fusion gene detection and deciding on the most appropriate method can be difficult. Beyond the experimental approach, consideration needs to be given to factors such as the ease of implementation, processing time, associated costs, and the level of expertise required for data analysis. Here, the different NGS-based methods for fusion gene detection, the basic principles underlying the techniques, and the benefits and limitations of each approach are reviewed. This article concludes with a discussion of how NGS will impact fusion gene detection in a clinical context and from where the next innovations are evolving.  相似文献   

16.
    
Sequencing them all. That is the ambitious goal of the recently launched Earth BioGenome project (Proceedings of the National Academy of Sciences of the United States of America, 115, 4325–4333), which aims to produce reference genomes for all eukaryotic species within the next decade. In this perspective, we discuss the opportunities of this project with a plant focus, but highlight also potential limitations. This includes the question of how to best capture all plant diversity, as the green taxon is one of the most complex clades in the tree of life, with over 300 000 species. For this, we highlight four key points: (i) the unique biological insights that could be gained from studying plants, (ii) their apparent underrepresentation in sequencing efforts given the number of threatened species, (iii) the necessity of phylogenomic methods that are aware of differences in genome complexity and quality, and (iv) the accounting for within‐species genetic diversity and the historical aspect of conservation genetics.  相似文献   

17.
    
《Evolutionary Applications》2018,11(8):1322-1341
Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double‐digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.  相似文献   

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19.
    
Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.  相似文献   

20.
    
Effective resource management depends on our ability to partition diversity into biologically meaningful units. Recent evolutionary divergence, however, can often lead to ambiguity in morphological and genetic differentiation, complicating the delineation of valid conservation units. Such is the case with the “coregonine problem,” where recent postglacial radiations of coregonines into lacustrine habitats resulted in the evolution of numerous species flocks, often with ambiguous taxonomy. The application of genomics methods is beginning to shed light on this problem and the evolutionary mechanisms underlying divergence in these ecologically and economically important fishes. Here, we used restriction site‐associated DNA (RAD) sequencing to examine genetic diversity and differentiation among sympatric forms in the Coregonus artedi complex in the Apostle Islands of Lake Superior, the largest lake in the Laurentian Great Lakes. Using 29,068 SNPs, we were able to clearly distinguish among the three most common forms for the first time, as well as identify putative hybrids and potentially misidentified specimens. Population assignment rates for these forms using our RAD data were 93%–100% with the only mis‐assignments arising from putative hybrids, an improvement from 62% to 77% using microsatellites. Estimates of pairwise differentiation (FST: 0.045–0.056) were large given the detection of hybrids, suggesting that reduced fitness of hybrid individuals may be a potential mechanism for the maintenance of differentiation. We also used a newly built C. artedi linkage map to look for islands of genetic divergence among forms and found widespread differentiation across the genome, a pattern indicative of long‐term drift, suggesting that these forms have been reproductively isolated for a substantial amount of time. The results of this study provide valuable information that can be applied to develop well‐informed management strategies and stress the importance of re‐evaluating conservation units with genomic tools to ensure they accurately reflect species diversity.  相似文献   

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