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Abstract/Summary
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Nasonia, a genus of four closely related parasitoid insect species, is a model system for genetic research. Their haplodiploid genetics (haploid males and diploid females) and interfertile species are advantageous for the genetic analysis of complex traits and the genetic basis of species differences. A fine-scale genomic map is an important tool for advancing genetic studies in this system. We developed and used a hybrid genotyping microarray to generate a high-resolution genetic map that covers 79% of the sequenced genome of Nasonia vitripennis. The microarray is based on differential hybridization of species-specific oligos between N. vitripennis and Nasonia giraulti at more than 20,000 markers spanning the Nasonia genome. The map places 729 scaffolds onto the five linkage groups of Nasonia, including locating many smaller scaffolds that would be difficult to map by other means. The microarray was used to characterize 26 segmental introgression lines containing chromosomal regions from one species in the genetic background of another. These segmental introgression lines have been used for rapid screening and mapping of quantitative trait loci involved in species differences. Finally, the microarray is extended to bulk-segregant analysis and genotyping of other Nasonia species combinations. These resources should further expand the usefulness of Nasonia for studies of the genetic basis and architecture of complex traits and speciation. The parasitic wasp genus Nasonia (Insecta: Hymenoptera) is a model system in genetics, particularly evolutionary genetics, the genetics of complex traits, developmental genetics, and host−parasite interactions (Beukeboom and Desplan 2003; Werren and Loehlin 2009). Nasonia consists of four closely related species, Nasonia vitripennis, Nasonia giraulti, Nasonia longicornis, and the recently discovered Nasonia oneida, which are all cross-fertile once cured of their endosymbiotic Wolbachia (Breeuwer and Werren 1995; Raychoudhury et al. 2010). The presence of interfertile species is advantageous for evolutionary genetic research because it allows movement of genetic regions between species for the identification, mapping, and cloning of quantitative trait loci (QTL) involved in species differences. Several additional features make Nasonia an excellent genetic model, including short (2-wk) generation time, ease of laboratory rearing, systemic RNA interference, and haplodiploid sex determination (females are diploid, whereas males are haploid and derived from unfertilized eggs, facilitating mutation screening) (Lynch and Desplan 2006; Werren and Loehlin 2009; Werren et al. 2009). The N. vitripennis−N. giraulti species pair has been used widely to identify QTL involved in a diverse array of phenotypes, including wing size (Gadau et al. 2002; Loehlin et al. 2010a,b; Loehlin and Werren 2012), cuticular hydrocarbons (Niehuis et al. 2011), hybrid incompatibilities (Breeuwer and Werren 1995; Gadau et al. 1999; Gibson et al. 2010; Koevoets et al. 2012; Niehuis et al. 2008; Werren et al. 2010), and host preference (Desjardins et al. 2010). The recent sequencing of the genomes of three Nasonia species (Werren et al. 2010) provides a key resource for advancing this new model system. Using the genome sequence data in conjunction with haploid males from hybrid crosses between N. vitripennis and N. giraulti, a genetic map of the Nasonia genome was generated (Niehuis et al. 2010; Werren et al. 2010). This approach was facilitated by the high level of single-nucleotide polymorphisms (SNPs) between species (average coding sequence difference between N. vitripennis and N. giraulti is 1%). The mapping population consisted of haploid male embryos from F1 hybrid females. The advantage of hybrid haploid males is that for any locus, a hybrid male carries only the allele for one of the parental species. With the use of this strategy, 265 scaffolds (covering 64% of the assembled genome) were mapped onto the five chromosomes of Nasonia (Niehuis et al. 2010). The combination of a genome sequence and genetic map has allowed investigators studying Nasonia to do forward genetics efficiently, i.e., quickly proceed from detection of QTL to positional cloning of QTL and identification of the genetic architecture that underlie phenotypes of interest (Loehlin et al. 2010b; Loehlin and Werren 2012). To further advance Nasonia as a genetic system, a high-resolution genetic map is needed that places more scaffolds onto the linkage map and has a finer scale of resolution. In addition, the availability of a cost-effective tool for high-throughput genotyping and bulk-segregant analysis could help advance studies geared toward investigating the genetic basis of complex adaptive phenotypes, genetic incompatibilities, and species differences. Here, we developed a comparative hybridization genotyping (CGH) microarray that uses clusters of SNPs and insertion-deletions (indels) in a high-density genotyping microarray to differentiate N. vitripennis from N. giraulti sequence at a large number of markers spanning the Nasonia genome.
Additional resources being developed for Nasonia include a set of segmental introgression lines (SILs), which contain specific genomic regions from one species (typically N. giraulti) in the genetic background of another (typically N. vitripennis) (Werren and Loehlin 2009). These are produced by performing an interspecific cross followed by repeated backcrossing of hybrid females to males of one species. To “hold onto” the introgressed genetic region during the backcrossing process, either visible mutant markers, phenotypic species-differences (such as wing size), or molecular markers are used. Eventually (usually greater than eight generations depending on the size of the introgressed region) the introgression lines are made homozygous for the “foreign” target region within the other species’ genetic background. SILs already have been used for efficient mapping of genes affecting phenotypic differences between Nasonia species (Loehlin et al. 2010b; Loehlin and Werren 2012). To further develop Nasonia SIL resources for QTL mapping, here we used the microarray to genotype 26 SILs. We also tested the applicability of the microarray to bulk segregant analysis, another useful tool for efficiently identifying genomic regions associated with phenotypes of interest (Michelmore et al. 1991). In bulk segregant analysis, a population is divided into two subsamples based on phenotype, and then allele proportions are estimated for each subsample. It is an alternative to genotyping a large population at the level of individuals, and is an economical way to quickly and efficiently identify genomic regions associated with traits of interest. For example, this approach was used in combination with genotyping to identify regions associated with nuclear-cytoplasmic incompatibility in hybrids of N. vitripennis and N. giraulti (Werren et al. 2010). Here we broadly applied the methodology to mapping phenotypes in Nasonia by using the CGH microarray. The CGH array will be of even greater utility if it can be applied to hybrid crosses involving other Nasonia species, such as N. longicornis and N. oneida, as a number of research groups are currently investigating these species (L. Beukeboom, personal communication; Koevoets 2012). Although the array was not designed for this purpose, the close phylogenetic relationship of both N. longicornis and N. oneida to N. giraulti suggest that their DNA would be more likely to hybridize to the N. giraulti oligo than the N. vitripennis oligo (Raychoudhury et al. 2010). We therefore tested how many markers on the microarray can accurately distinguish between N. vitripennis−N. longicornis and N. vitripennis−N. oneida sequences.
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