Abstract
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.
Highlights
Discovering genes that control morphological, behavioural and physiological phenotypes is critical for understanding adaptive evolution, for plant and animal breeding and for tracking the evolutionary responses of natural populations, such as insecticide resistance in crop pests
We estimated that there should be,6500 Sbf1 Restriction-site associated DNA (RAD) loci in the P. xylostella genome, and generated raw Illumina reads to a depth of 65x per locus per individual based on this estimate
Each forward sequence contained a 5 base molecular identifying sequence (MID) that allowed reads to be assigned to a specific individual, followed by 6 bases of the SbfI restriction enzyme footprint (TGCAGG), leaving 40 locus-specific bases per read
Summary
Discovering genes that control morphological, behavioural and physiological phenotypes is critical for understanding adaptive evolution, for plant and animal breeding and for tracking the evolutionary responses of natural populations, such as insecticide resistance in crop pests. Linkage maps have been constructed for scores of organisms, generally to identify a genome region controlling a trait of interest, such as skeletal armour morphology in stickleback fish [1,2], wing patterns in butterflies [3,4] and morphological and physiological traits in sunflowers [5]. Amplified fragment length polymorphism (AFLP), RAPDs, microsatellites and single copy gene markers used to construct such maps are all problematic, either being expensive and difficult to develop, or anonymous and difficult to translate into useful sequence-based markers. Next-generation sequencing can greatly facilitate the process of genetic mapping, allowing rapid generation of dense genome linkage maps consisting of thousands of sequenced markers, such that useful sequences linked to a gene of interest can be identified in a single experiment
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