Linkage Mapping and Comparative Genomics Using Next-Generation RAD Sequencing of a Non-Model Organism

Restriction-site associated DNA (RAD) sequencing is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis of non-model organisms including genotype-phenotype association mapping, phylogeography, population genetics and scaffolding genome assemblies through linkage mapping. We constructed a RAD library using genomic DNA from a Plutella xylostella (diamondback moth) backcross that segregated for resistance to the insecticide spinosad. Sequencing of 24 individuals was performed on a single Illumina GAIIx lane (51 base paired-end reads). Taking advantage of the lack of crossing over in homologous chromosomes in female Lepidoptera, 3,177 maternally inherited RAD alleles were assigned to the 31 chromosomes, enabling identification of the spinosad resistance and W/Z sex chromosomes. Paired-end reads for each RAD allele were assembled into contigs and compared to the genome of Bombyx mori (n = 28) using BLAST, revealing 28 homologous matches plus 3 expected fusion/breakage events which account for the difference in chromosome number. A genome-wide linkage map (1292 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. Comparative analysis of P. xyloxtella linkage groups with B. mori chromosomes shows for the first time, genetic synteny appears common beyond the Macrolepidoptera. RAD sequencing is a powerful system capable of rapidly generating chromosome specific data for non-model organisms

Mis-Spliced Transcripts of Nicotinic Acetylcholine Receptor α6 Are Associated with Field Evolved Spinosad Resistance in Plutella xylostella (L.)

The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR) and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxα6), at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxα6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxα6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species