Double-digest Restriction Site-associated DNA Sequencing Research Articles

Identification of Insertion and Deletion (InDel) Markers for Chickpea (Cicer arietinum L.) Based on Double-Digest Restriction Site-Associated DNA Sequencing.

Enhancing the marker repository and the development of breeder-friendly markers in chickpeas is important in relation to chickpea genomics-assisted breeding applications. Insertion-deletion (InDel) markers are widely distributed across genomes and easily observed with specifically designed primers, leading to less time, cost, and labor requirements. In light of this, the present study focused on the identification and development of InDel markers through the use of double-digest restriction site-associated DNA sequencing (ddRADSeq) data from 20 chickpea accessions. Bioinformatic analysis identified 20,700 InDel sites, including 15,031 (72.61%) deletions and 5669 (27.39%) insertions, among the chickpea accessions. The InDel markers ranged from 1 to 25 bp in length, while single-nucleotide-length InDel markers were found to represent the majority of the InDel sites and account for 79% of the total InDel markers. However, we focused on InDel markers wherein the length was greater than a single nucleotide to avoid any read or alignment errors. Among all of the InDel markers, 96.1% were less than 10 bp, 3.6% were between 10 and 20 bp, and 0.3% were more than 20 bp in length. We examined the InDel markers that were 10 bp and longer for the development of InDel markers based on a consideration of the genomic distribution and low-cost genotyping with agarose gels. A total of 29 InDel regions were selected, and primers were successfully designed to evaluate their efficiency. Annotation analysis of the InDel markers revealed them to be found with the highest frequency in the intergenic regions (82.76%), followed by the introns (6.90%), coding sequences (6.90%), and exons (3.45%). Genetic diversity analysis demonstrated that the polymorphic information content of the markers varied from 0.09 to 0.37, with an average of 0.20. Taken together, these results showed the efficiency of InDel marker development for chickpea genetic and genomic studies using the ddRADSeq method. The identified markers might prove valuable for chickpea breeders.

Adaptive radiation of the Callicarpa genus in the Bonin Islands revealed through double-digest restriction site-associated DNA sequencing analysis.

The Bonin Islands, comprised of the Mukojima, Chichijima, and Hahajima Islands, are known for their isolated and distinctive habitats, hosting a diverse array of endemic flora and fauna. In these islands, adaptive radiation has played a remarkable role in speciation, particularly evident in the Callicarpa genus that is represented by three species: Callicarpa parvifolia and C. glabra exclusive to the Chichijima Islands, and Callicarpa subpubescens, distributed across the entire Bonin Islands. Notably, C. subpubescens exhibits multiple ecotypes, differing in leaf hair density, flowering time, and tree size. In this study, we aimed to investigate species and ecotype diversification patterns, estimate divergence times, and explore cryptic species within Callicarpa in the Bonin Islands using phenotypic and genetic data (double-digest restriction site-associated DNA sequencing). Genetic analysis revealed that C. parvifolia and C. glabra both formed single, distinct genetic groups. Conversely, C. subpubescens consisted of six genetic groups corresponding to different ecotypes and regions, and a hybrid group resulting from the hybridization between two of these genetic groups. Population demography analysis focusing on six Chichijima and Hahajima Islands-based species/ecotypes indicated that all species and ecotypes except one ecotype diverged simultaneously around 73-77 kya. The star-shaped neighbor-net tree also suggests the simultaneous divergence of species and ecotypes. The species and ecotypes that simultaneously diverged adapted to dry environments and understory forests, suggesting that aridification may have contributed to this process of adaptive radiation. Moreover, leaf morphology, flowering time, and genetic analyses suggested the presence of two cryptic species and one hybrid species within C. subpubescens.

Reduced representation sequencing reveals weak genetic differentiation between Canadian and European Larus hyperboreus (Glaucous Gull)

Abstract Climate change poses a significant threat to Arctic ecosystems. Evaluation of genetic diversity within and differentiation among populations is needed to effectively conserve Arctic species and ensure genetic variation is appropriately managed. This research examined population genetic structure in Larus hyperboreus (Glaucous Gull), a circumpolar Arctic species that is declining in parts of its range. Population genetic information is needed to help delineate management units, including information on genetic differences among related species, among subspecies (currently distinguished by subtle morphometric and plumage differences), and among colonies in North America. We conducted double-digest restriction site-associated DNA sequencing for 62 L. hyperboreus, 18 L. argentatus smithsonianus (American Herring Gull), 6 L. a. argentatus (European Herring Gull), and 15 L. glaucescens (Glaucous-winged Gull) sampled across the Canadian and European Arctic. Interspecific analyses using 2,145 loci were unable to distinguish members of this species complex. Despite the geographic distance among sampling locations, molecular assignments and principal coordinates analyses based on 621 loci uncovered only weak population genetic differentiation among sampled European and Canadian colonies of L. hyperboreus. As L. hyperboreus occupying the eastern Canadian Arctic appear to be acting as a single panmictic population, conservation plans that protect Arctic habitat may help slow or reverse population declines. Proactive conservation strategies will benefit both L. hyperboreus and associated coastal Arctic ecosystems.

Translocations, rising populations, and phylogeographic consequences: genomic implications for conservation of introduced Aoudad (Ammotragus lervia) in the southwestern United States

Abstract Over the last century, Texas has been inundated with exotic ungulates, with as many as 67 species represented by captive and free-ranging populations. One of the most successful species to have established free-ranging populations is the Aoudad (Ammotragus lervia). Herein, we expand on previous genetic studies in Aoudad by including new genomic profiles based on double-digest restriction site-associated DNA sequencing methods with the intent of better understanding the effects of >70 years of human-mediated translocations, population expansion, and gene flow among populations. Using a range of 4,338 to 5,529 single-nucleotide polymorphisms obtained from 73 individuals that potentially represented A. l. blainei, A. l. lervia, and A. l. sahariensis, we measured genetic patterns of diversity and connectivity between source populations from California and New Mexico and resultant aoudad populations in Texas. Principal component analyses identified phylogeographically structured populations across Texas. Further, we identified a signature of isolation by distance among Texas populations. To assess geographic regions that facilitated or blocked gene flow between populations, we used estimation of effective migration surfaces, which indicated 3 barriers to movement between populations as well as connectivity among populations in close proximity. Comparison of mtDNA haplogroups and nuclear genotypes among the 3 examined subspecies were discordant, indicating that the current subspecific taxonomic classification needs refinement. Given the vulnerable status of Aoudad in their native range of northern Africa and the exponential population growth and potential competition with native ungulates in Texas, we propose that efforts to manage non-native Aoudad should strive to maintain and preserve unique genetic units while minimizing competition and other detriments to native species in Texas. Conservation efforts are especially important given the vulnerable status of Aoudad within their native range of northern Africa.

Population genomics of four co-distributed frog species in a barrier island system

Abstract In nature, small populations are often of concern because of limited genetic diversity, which underlies adaptive potential in the face of environmental change. Assessing patterns of genetic variation within co-distributed species sampled across varied landscapes can therefore illuminate their capacity to persist over time. We sequenced new genome-wide sequence data (double-digest restriction site-associated DNA sequencing) for four frog species (Anaxyrus terrestris, Hyla cinerea, Hyla squirella, and Rana sphenocephala) sampled from two barrier islands and the adjacent mainland of northern Florida. We calculated genomic diversity metrics and analysed spatial patterns of genomic variation for each species. We found higher genomic diversity within mainland individuals compared to island individuals for all species, suggesting a consistent effect of small island area on diversity across species. Three species (all but A. terrestris) showed significant signatures of isolation by distance, and some clustering analyses indicated separation of island and mainland individuals within species. We identified subtle differences in the strength of these patterns among species, with the strongest genetic differentiation observed in R. sphenocephala. Finally, we found evidence of recent migration between island and mainland populations for all species, which likely explains the limited genetic structure observed and contributes to the persistence of these small populations.

Genomic signatures of hybridization between Ixodes ricinus and Ixodes persulcatus in natural populations.

Identifying hybridization between common pathogen vectors is essential due to the major public health implications through risks associated with hybrid's enhanced pathogen transmission potential. The hard-ticks Ixodes ricinus and Ixodes persulcatus are the two most common vectors of tick-borne pathogens that affect human and animal health in Europe. Ixodes ricinus is a known native species in Finland with a well-known distribution, whereas I. persulcatus has expanded in range and abundance over the past 60 years, and currently it appears the most common tick species in certain areas in Finland. Here we used double-digest restriction site-associated DNA (ddRAD) sequencing on 186 ticks (morphologically identified as 92 I. ricinus, and 94 I. persulcatus) collected across Finland to investigate whether RAD generated single nucleotide polymorphisms (SNPs) can discriminate tick species and identify potential hybridization events. Two different clustering methods were used to assign specific species based on how they clustered and identified hybrids among them. We were able to discriminate between the two tick species and identified 11 putative hybrids with admixed genomic proportions ranging from approximately 24 to 76 percent. Four of these hybrids were morphologically identified as I. ricinus while the remaining seven were identified as I. persulcatus. Our results thus indicate that RAD SNPs are robust in identifying both species of the ticks as well as putative hybrids. These results further suggest ongoing hybridization between I. ricinus and I. persulcatus in their natural populations in Finland. The unique ability of RAD markers to discriminate between tick species and hybrids adds a useful aspect to tick evolutionary studies. Our findings align with previous studies and suggest a shared evolutionary history between the species, with instances of individuals possessing a considerable proportion of the other species' genome. This study is a significant step in understanding the formation of hybridization zones due to range expansion potentially associated with climate change.

Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Phylogenetic insights into Central European Chorthippus and Pseudochorthippus (Orthoptera: Acrididae) species using ddRADseq data

The evolution of several orthopteran groups, especially within the grasshopper family Acrididae, remains poorly understood. This is particularly true for the subfamily Gomphocerinae, which comprises cryptic sympatric and syntopic species. Previous mitochondrial studies have highlighted major discrepancies between taxonomic and phylogenetic hypotheses, thereby emphasizing the necessity of genome-wide approaches.In this study, we employ double-digest restriction site-associated DNA sequencing (ddRADseq) to reconstruct the evolution of Central European Chorthippus and Pseudochorthippus species, especially C.smardai, P.tatrae and the C.biguttulus group.Our phylogenomic analyses recovered deep discordance with mitochondrial DNA barcoding, emphasizing its unreliability in Gomphocerinae grasshoppers. Specifically, our data robustly distinguished the C.biguttulus group and confirmed the distinctiveness of C.eisentrauti, also shedding light on its presence in the Berchtesgaden Alps. Moreover, our results support the reclassification of C.smardai to the genus Pseudochorthippus and of P.tatrae to the genus Chorthippus.Our study demonstrates the efficiency of high-throughput genomic methods such as RADseq without prior optimization to elucidate the complex evolution of grasshopper radiations with direct taxonomic implications. While RADseq has predominantly been utilized for population genomics and within-genus phylogenomics, its application extends to resolve relationships between deeply-diverged clades representative of distinct genera.

DdRAD Sequencing and DNA Barcoding.

Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including single-nucleotide polymorphisms (SNPs). These SNPs can be utilized to gain evolutionary insights into patterns observed in DNA barcodes, to infer population structure and phylogenies, to detect gene flow and introgression, and to perform species delimitation analyses. The protocol includes chemically shearing genomic DNA with restriction enzymes, unique tagging, size selection, and amplification of the resulting DNA fragments. Here we provide a detailed description of each step of the protocol, as well as information on essential equipment and common issues encountered during laboratory work.

Rapid seasonal changes in phenotypes in a wild Drosophila population

Seasonal environmental change is one of the most rapid and striking environmental variables. Although relatively rapid adaptation to environmental changes over several years or several decades has been described in many taxa, rapid responses to seasonal environments are delicate, and therefore, the detection of the evolutionary responses requires sensitive methods. In this study, we examined seasonal changes in phenotypes related to thermal tolerance and morphological traits of Drosophila lutescens collected at the spring and autumn periods from a single location. We first demonstrated that flies in the two seasonal periods were almost genetically identical using double-digest restriction site-associated DNA sequencing and analysis. Using an experimental design to eliminate the effect of possible confounding factors that influence phenotypes (i.e., maternal effects and the environmental conditions in which each phenotype was analyzed), we showed that the heat tolerance of D. lutescens was significantly higher in the autumn population than in the spring population. Furthermore, cold tolerance was slightly higher in the spring population than in the autumn one. Although wing length and thorax length did not change significantly between seasons, the ratio of wing length to thorax length changed significantly between them. These results suggest that seasonal environmental heterogeneity induces rapid phenotypic changes within a year. Finally, we discuss the possibility of rapid evolutionary responses to seasonal changes.

Optimizing ddRAD sequencing for population genomic studies with ddgRADer.

Double-digest Restriction-site Associated DNA sequencing (ddRADseq) is widely used to generate genomic data for non-model organisms in evolutionary and ecological studies. Along with affordable paired-end sequencing, this method makes population genomic analyses more accessible. However, multiple factors should be considered when designing a ddRADseq experiment, which can be challenging for new users. The generated data often suffer from substantial read overlaps and adaptor contamination, severely reducing sequencing efficiency and affecting data quality. Here, we analyse diverse datasets from the literature and carry out controlled experiments to understand the effects of enzyme choice and size selection on sequencing efficiency. The empirical data reveal that size selection is imprecise and has limited efficacy. In certain scenarios, a substantial proportion of short fragments pass below the lower size-selection cut-off resulting in low sequencing efficiency. However, enzyme choice can considerably mitigate inadvertent inclusion of these shorter fragments. A simple model based on these experiments is implemented to predict the number of genomic fragments generated after digestion and size selection, number of SNPs genotyped, number of samples that can be multiplexed and the expected sequencing efficiency. We developed ddgRADer - http://ddgrader.haifa.ac.il/ - a user-friendly webtool and incorporated these calculations to aid in ddRADseq experimental design while optimizing sequencing efficiency. This tool can also be used for single enzyme protocols such as Genotyping-by-Sequencing. Given user-defined study goals, ddgRADer recommends enzyme pairs and allows users to compare and choose enzymes and size-selection criteria. ddgRADer improves the accessibility and ease of designing ddRADseq experiments and increases the probability of success of the first population genomic study conducted in labs with no prior experience in genomics.

Genomic signatures of ecological divergence between savanna and forest populations of a Neotropical tree.

In eastern Neotropical South America, the Cerrado, a large savanna vegetation, and the Atlantic Forest harbour high biodiversity levels, and their habitats are rather different from each other. The biomes have intrinsic evolutionary relationships, with high lineage exchange that can be attributed, in part, to a large contact zone between them. The genomic study of ecotypes, i.e. populations adapted to divergent habitats, can be a model to study the genomic signatures of ecological divergence. Here, we investigated two ecotypes of the tree Plathymenia reticulata, one from the Cerrado and the other from the Atlantic Forest, which have a hybrid zone in the ecotonal zone of Atlantic Forest-Cerrado. The ecotypes were sampled in the two biomes and their ecotone. The evolutionary history of the divergence of the species was analysed with double-digest restriction site-associated DNA sequencing. The genetic structure and the genotypic composition of the hybrid zone were determined. Genotype-association analyses were performed, and the loci under putative selection and their functions were investigated. High divergence between the two ecotypes was found, and only early-generation hybrids were found in the hybrid zone, suggesting a partial reproductive barrier. Ancient introgression between the Cerrado and Atlantic Forest was not detected. The soil and climate were associated with genetic divergence in Plathymenia ecotypes and outlier loci were found to be associated with the stress response, with stomatal and root development and with reproduction. The high genomic, ecological and morphophysiological divergence between ecotypes, coupled with partial reproductive isolation, indicate that the ecotypes represent two species and should be managed as different evolutionary lineages. We advise that the forest species should be re-evaluated and restated as vulnerable. Our results provide insights into the genomic mechanisms underlying the diversification of species across savanna and forest habitats and the evolutionary forces acting in the species diversification in the Neotropics.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targetedGENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Construction of a high-density genetic linkage map and QTL mapping for bioenergy-related traits in sweet sorghum [Sorghum bicolor (L.) Moench

Sorghum is an important but arguably undervalued cereal crop, grown in large areas in Asia and Africa due to its natural resilience to drought and heat. There is growing demand for sweet sorghum as a source of bioethanol as well as food and feed. The improvement of bioenergy-related traits directly affects bioethanol production from sweet sorghum; therefore, understanding the genetic basis of these traits would enable new cultivars to be developed for bioenergy production. In order to reveal the genetic architecture behind bioenergy-related traits, we generated an F2 population from a cross between sweet sorghum cv. 'Erdurmus' and grain sorghum cv. 'Ogretmenoglu'. This was used to construct a genetic map from SNPs discovered by double-digest restriction-site associated DNA sequencing (ddRAD-seq). F3 lines derived from each F2 individual were phenotyped for bioenergy-related traits in two different locations and their genotypes were analyzed with the SNPs to identify QTL regions. On chromosomes 1, 7, and 9, three major plant height (PH) QTLs (qPH1.1, qPH7.1, and qPH9.1) were identified, with phenotypic variation explained (PVE) ranging from 10.8 to 34.8%. One major QTL (qPJ6.1) on chromosome 6 was associated with the plant juice trait (PJ) and explained 35.2% of its phenotypic variation. For fresh biomass weight (FBW), four major QTLs (qFBW1.1, qFBW6.1, qFBW7.1, and qFBW9.1) were determined on chromosomes 1, 6, 7, and 9, which explained 12.3, 14.5, 10.6, and 11.9% of the phenotypic variation, respectively. Moreover, two minor QTLs (qBX3.1 and qBX7.1) of Brix (BX) were mapped on chromosomes 3 and 7, explaining 8.6 and 9.7% of the phenotypic variation, respectively. The QTLs in two clusters (qPH7.1/qBX7.1 and qPH7.1/qFBW7.1) overlapped for PH, FBW and BX. The QTL, qFBW6.1, has not been previously reported. In addition, eight SNPs were converted into cleaved amplified polymorphic sequences (CAPS) markers, which can be easily detected by agarose gel electrophoresis. These QTLs and molecular markers can be used for pyramiding and marker-assisted selection studies in sorghum, to develop advanced lines that include desirable bioenergy-related traits.

A community genomics approach to natural hybridization.

Hybridization is a complicated, oft-misunderstood process. Once deemed unnatural and uncommon, hybridization is now recognized as ubiquitous among species. But hybridization rates within and among communities are poorly understood despite the relevance to ecology, evolution and conservation. To clarify, we examined hybridization across 75 freshwater fish communities within the Ozarks of the North American Interior Highlands (USA) by single nucleotide polymorphism (SNP) genotyping 33 species (N = 2865 individuals; double-digest restriction site-associated DNA sequencing (ddRAD)). We found evidence of hybridization (70 putative hybrids; 2.4% of individuals) among 18 species-pairs involving 73% (24/33) of study species, with the majority being concentrated within one family (Leuciscidae/minnows; 15 species; 66 hybrids). Interspecific genetic exchange-or introgression-was evident from 24 backcrossed individuals (10/18 species-pairs). Hybrids occurred within 42 of 75 communities (56%). Four selected environmental variables (species richness, protected area extent, precipitation (May and annually)) exhibited 73-78% accuracy in predicting hybrid occurrence via random forest classification. Our community-level assessment identified hybridization as spatially widespread and environmentally dependent (albeit predominantly within one diverse, omnipresent family). Our approach provides a more holistic survey of natural hybridization by testing a wide range of species-pairs, thus contrasting with more conventional evaluations.

The Genetic Diversity of Mink (Neovison vison) Populations in China.

The American mink (Neovison vison) is a semiaquatic species of Mustelid native to North America that is now widespread in China. However, the knowledge of genetic diversity of mink in China is still limited. In this study, we investigated the genetic diversity and identified significant single nucleotide polymorphisms (SNPs) in mink populations of five different color types in three different mink farms in China. Using double-digest restriction site-associated DNA sequencing, we identified a total of 1.3 million SNPs. After filtering the SNPs, phylogenetic tree, Fst, principal component, and population structure analyses were performed. The results demonstrated that red mink and black mink grouped, with separate clustering of all other color types. The population divergence index (Fst) study confirmed that different mink populations were distinct (K = 4). Two populations with different coat colors were subjected to the selection signature analysis, and 2300 genes were found to have a clear selection signature. The genes with a selection signature were subjected to Gene Ontology (GO) categorization and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the results revealed that the genes with a selection signature were enriched in the melanogenesis pathway. These study's findings have set the stage for improved breeding and conservation of genetic resources in real-world practical mink farming.

Genetic Assessment of Fertile F1 Hybrids between Two Hercules Beetles, Dynastes maya Hardy and D. grantii Horn (Scarabaeidae).

Although hybridization may complicate taxonomic practices, it can be common between animal species. Animal hybridization not only can help with generating phenotypic and species diversity in nature, but also with understanding the genetic and genomic basis of phenotypic evolution in the laboratory. We assessed the genetic composition of captive bred F1 hybrids between two Hercules beetle species using mitochondrial CO1 and nuclear loci from a double-digest restriction-site associated DNA sequencing (ddRADseq) library. We showed that the F1 hybrids were genetically clustered with samples from the maternal species, D. grantii, based on CO1 data. Nuclear genome data, on the other hand, clearly showed that the F1 individuals were genetically intermediate between D. maya, the paternal species, and D. grantii, based on a principal component analysis. Our results also revealed that sampling design may have a major impact on the inferred genetic structure and hybrid individuals using ddRADseq data sets. We discuss the importance and potential from studying the genomics of this hybrid progeny in terms of understanding the origin and maintenance of both intraspecific and interspecific phenotypic divergence and convergence.

Spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), exhibits large-scale spatial genetic structure across Canada but not fruit host–associated genetic structure

Abstract Spotted-wing drosophila, Drosophila suzukii, is a global pest of soft fruits that is capable of reproducing on a wide range of cultivated and wild plant species. In Canada, D. suzukii was first reported in British Columbia in 2009 and is now widespread across the country. Understanding the genetic structure of D. suzukii populations could be important for pest management if there are phenotypic differences between genetically distinct populations. For example, insect pest populations could respond differently to directional selection imposed by insecticides, differ in their host plant preferences, and vary in their susceptibility to biological control agents. Here, we used double-digest restriction site–associated DNA sequencing to examine large- and fine-scale patterns of the genetic structure of D. suzukii reared from fruit hosts in Canada. We found that this species has a large-scale spatial genetic structure; the flies collected formed two distinct genetic clusters, one of which was distinct to western Canada and the other to eastern Canada. At the local scale, D. suzukii populations showed no evidence of host-associated structuring in British Columbia, suggesting that pest management tactics may be best applied at the landscape level. Our results highlight the need to investigate phenotypic differences between western and eastern D. suzukii populations in Canada.

Detecting a novel locus associated with pellicle peelability in Japanese chestnut populations preselected for a major peelability gene

• The distribution of phenotypic values of peelability in Japanese chestnut populations preselected for a major peelability gene was biased toward maximum values. • Use of rank-based inverse normal transformation and categorization of peelability phenotypes was applied for GWAS to further identify QTLs associated with peelability. • A single QTL for peelability was identified at CCR1.0F_56177061 on chromosome F. Peelability is one of the most important traits in Japanese chestnut ( Castanea crenata Sieb. et Zucc.) breeding. Previous studies revealed that the peelability of Japanese chestnut is controlled by a major recessive peeling gene (MRPG). This locus has been used effectively for marker-assisted selection in chestnut breeding programs, but there are also likely to be minor loci that would further improve the efficiency of breeding for peelability. We conducted a genome-wide association study (GWAS) based on a variational approximation version of BayesB using 27 cultivars and MRPG-preselected populations consisting of 520 F 1 seedlings from 22 families. Using double-digest restriction site-associated DNA sequencing (ddRAD-Seq), we obtained 9164 tag single-nucleotide polymorphisms. Because the phenotypic values of peelability were distributed very asymmetrically, with a peak at the high end due to the preselection for MRPG, we converted the original data set to both inverse normal transformed values and categorical scores, and we analyzed both sets of converted data by GWAS. In both analyses, a quantitative trait locus (QTL) for peelability at CCR1.0F_56177061 was identified with posterior probability values of 0.68 and 1.00, respectively. The average values of genotypes at CCR1.0F_56177061 for cultivars and seedlings in this study were 60.4% (peelability rate) for CC, 63.7% for CT, and 86.5% for TT. This analysis reveals a significant association between CCR1.0F_56177061 genotype and peelability, indicating that this newly identified QTL will be useful for improving peelability in chestnut breeding programs.

Forest cover and geographical distance influence fine-scale genetic structure of leaf-toed geckos in the tropical dry forests of western Mexico

Abstract The biodiversity within tropical dry forests (TDFs) is astounding and yet poorly catalogued due to inadequate sampling and the presence of cryptic species. In the Mexican TDF, endemic species are common, and the landscape has been continually altered by geological and anthropogenic changes. To understand how landscape and environmental variables have shaped the population structure of endemic species, we studied the recently described species of leaf-toed gecko, Phyllodactylus benedettii, in coastal western Mexico. Using double-digest restriction site-associated DNA sequencing data, we first explore population structure and estimate the number of ancestral populations. The results indicate a high degree of genetic structure with little admixture, and patterns corresponding to both latitudinal and altitudinal gradients. We find that genetic structure cannot be explained purely by geographical distance, and that ecological corridors may facilitate dispersal and gene flow. We then model the spatial distribution of P. benedettii in the TDF through time and find that the coastline has been climatically suitable for the species since the Last Glacial Maximum. Landscape genetic analyses suggest that the combination of isolation by distance (IBD) and isolation by resistance (IBR; forest cover) has influenced the spatial genetic structure of the species. Overall, our genomic data demonstrate fine-scale population structure in TDF habitat, a complex colonization history, and spatial patterns consistent with both IBD and other ecological factors. These results further highlight the Mexican TDF as a diversity hotspot and suggest that continued anthropogenic changes are likely to affect native fauna.