2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador

Background: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment. Author summary: Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread

Special features of RAD Sequencing data:implications for genotyping

Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008; Andrews et al. 2016). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011; Gamble & Zarkower 2014). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998; Mossman & Waser 1999), the management and breeding of endangered species (Taberlet et al. 1993; Griffiths & Tiwari 1995; Robertson et al. 2006) and sexing embryonic material (Hacker et al. 1995; Smith et al. 1999). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010; Gamble & Zarkower 2014). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi (2016) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig. 1), Fowler & Buonaccorsi (2016) uncover shared sex‐specific markers and a conserved sex chromosome system

The Discovery of XY Sex Chromosomes in a \u3cem\u3eBoa\u3c/em\u3e and \u3cem\u3ePython\u3c/em\u3e

For over 50 years, biologists have accepted that all extant snakes share the same ZW sex chromosomes derived from a common ancestor [1, 2, 3], with different species exhibiting sex chromosomes at varying stages of differentiation. Accordingly, snakes have been a well-studied model for sex chromosome evolution in animals [1, 4]. A review of the literature, however, reveals no compelling support that boas and pythons possess ZW sex chromosomes [2, 5]. Furthermore, phylogenetic patterns of facultative parthenogenesis in snakes and a sex-linked color mutation in the ball python (Python regius) are best explained by boas and pythons possessing an XY sex chromosome system [6, 7]. Here we demonstrate that a boa (Boa imperator) and python (Python bivittatus) indeed possess XY sex chromosomes, based on the discovery of male-specific genetic markers in both species. We use these markers, along with transcriptomic and genomic data, to identify distinct sex chromosomes in boas and pythons, demonstrating that XY systems evolved independently in each lineage. This discovery highlights the dynamic evolution of vertebrate sex chromosomes and further enhances the value of snakes as a model for studying sex chromosome evolution

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques—Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species’ monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence

XX/XY Sex Chromosomes in the South American Dwarf Gecko (\u3cem\u3eGonatodes humeralis\u3c/em\u3e)

Sex-specific genetic markers identified using restriction site-associated DNA sequencing, or RADseq, permits the recognition of a species’ sex chromosome system in cases where standard cytogenetic methods fail. Thus, species with male-specific RAD markers have an XX/XY sex chromosome system (male heterogamety) while species with female-specific RAD markers have a ZZ/ZW sex chromosome (female heterogamety). Here, we use RADseq data from 5 male and 5 female South American dwarf geckos (Gonatodes humeralis) to identify an XX/XY sex chromosome system. This is the first confidently known sex chromosome system in a Gonatodes species. We used a low-coverage de novo G. humeralis genome assembly to design PCR primers to validate the male-specificity of a subset of the sex-specific RADseq markers and describe how even modest genome assemblies can facilitate the design of sex-specific PCR primers in species with diverse sex chromosome systems

Development of a Next-Generation NIL Library in Arabidopsis Thaliana for Dissecting Complex Traits

The identification of the loci and specific alleles underlying variation in quantitative traits is an important goal for evolutionary biologists and breeders. Despite major advancements in genomics technology, moving from QTL to causal alleles remains a major challenge in genetics research. Near-isogenic lines are the ideal raw material for QTL validation, refinement of QTL location and, ultimately, gene discovery. Results: In this study, a population of 75 Arabidopsis thaliana near-isogenic lines was developed from an existing recombinant inbred line (RIL) population derived from a cross between physiologically divergent accessions Kas-1 and Tsu-1. First, a novel algorithm was developed to utilize genome-wide marker data in selecting RILs fully isogenic to Kas-1 for a single chromosome. Seven such RILs were used in 2 generations of crossing to Tsu-1 to create BC1 seed. BC1 plants were genotyped with SSR markers so that lines could be selected that carried Kas-1 introgressions, resulting in a population carrying chromosomal introgressions spanning the genome. BC1 lines were genotyped with 48 genome-wide SSRs to identify lines with a targeted Kas-1 introgression and the fewest genomic introgressions elsewhere. 75 such lines were selected and genotyped at an additional 41 SNP loci and another 930 tags using 2b-RAD genotyping by sequencing. The final population carried an average of 1.35 homozygous and 2.49 heterozygous introgressions per line with average introgression sizes of 5.32 and 5.16 Mb, respectively. In a simple case study, we demonstrate the advantage of maintaining heterozygotes in our library whereby fine-mapping efforts are conducted simply by self-pollination. Crossovers in the heterozygous interval during this single selfing generation break the introgression into smaller, homozygous fragments (sub-NILs). Additionally, we utilize a homozygous NIL for validation of a QTL underlying stomatal conductance, a low heritability trait. Conclusions: The present results introduce a new and valuable resource to the Brassicaceae research community that enables rapid fine-mapping of candidate loci in parallel with QTL validation. These attributes along with dense marker coverage and genome-wide chromosomal introgressions make this population an ideal starting point for discovery of genes underlying important complex traits of agricultural and ecological significance.NSF DEB-1022196, DEB-0618302, DEB-0618347, IOS-09221457Integrative Biolog