Association mapping, comparative genomics and constructing linkage groups 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. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. 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 (50 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 (1295 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers.

限制性酶切位点关联DNA（Restriction-site associated DNA, RAD）测序是一种可对大量个体基因组实现靶向测序的高效新兴技术。该方法在非模式生物的遗传分析领域具备广泛应用潜力，可用于基因型-表型关联定位、系统地理学研究、群体遗传学分析，以及通过连锁作图完成基因组组装锚定。本研究利用RAD测序技术，为一种无既往基因组数据的生物从头构建遗传连锁图谱。我们从对杀虫剂多杀菌素（spinosad）抗性发生分离的小菜蛾（Plutella xylostella）回交群体的基因组DNA中构建RAD文库。在单个Illumina GAIIx测序泳道上完成了24个个体的测序，产出50碱基长度的双端测序读段（paired end reads）。借助鳞翅目雌性个体同源染色体无交叉互换的特性，我们将3177个母源遗传的RAD等位基因（allele）分配至31条染色体，从而实现了多杀菌素抗性位点以及W/Z性染色体的定位。将每个RAD等位基因对应的双端测序读段组装为重叠群（contig），并通过BLAST比对家蚕（Bombyx mori，n=28）的基因组，共发现28个同源匹配区域，同时存在3个预期的融合/断裂事件，以此解释二者之间的染色体数目差异。最终构建得到一张总长度为1295厘摩（cM）的全基因组连锁图谱，包含2878个来自回交父本的分离型RAD等位基因，可提供具备染色体及位置特异性的RAD测序标记。