Linkage mapping and comparative genomics using next-generation RAD sequencing of a non-model organism

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.

Figure 1. Assembling RAD alleles and creating RAD contigs from RAD paired-end reads.

Inset. Each forward sequence read contains a 5 base molecular identifier (MID), followed by 6 bp of SbfI restriction enzyme footprint. After identifying candidate RAD alleles, RAD contigs were assembled from the paired-end reads. Scatter plot. The size distribution of RAD assembled paired-end contigs. The X-axis is contig length in base pairs and Y-axis the number of RAD contigs with each length. The mean read length is 333 bases. One RAD allele can have multiple RAD paired-end contigs, as significant variation in the paired-end sequence can result in assembly of more than one contig, or low coverage may prevent complete assembly. Read lengths of 57 bases (containing 542 contigs) and 61 bases (containing 596 contigs) plus 200 contigs containing stretches of Ns were deemed to be assembly artefacts and omitted.

Figure 2. Linkage map of the Plutella xylostella (n = 31) genome.

This was inferred from 2,878 RAD alleles collapsed into 285 discrete RAD markers. Each linkage group contains between 10 and 158 RAD alleles (labelled RADs) and the total map length is 1,292 cM. Each RAD marker is labelled with three numbers (i_ii_iii) corresponding to (i) the RAD marker (1–285), (ii) the chromosome number (1–31) and (iii) the number of RAD alleles at that marker. Linkage groups 1–28 are homologous to the B. mori (n = 28) chromosome numbering system, and LG29, LG30 and LG31 represent fusions to chromosomes 11, 23 and 24 respectively in B. mori. Dashed lines represent manual linkages inferred from 3, 4 or 5 genotype differences that were otherwise left ungrouped due to small sample size. As 20 progeny were used to construct the map, distances were approximated as 5 cM (1/20) per 1 crossing-over (c/o) event. On chromosomes 14, 16 and 22, markers formed two distinct groups and may be separated by regions of high recombination rates or chromosomal assignment error. In total, 11 of the 285 RAD markers could not be confidently assigned to their predicted chromosome. Linkage group 28 contained only four RAD markers at a single locus. Six additional markers were identified for this chromosome using JoinMap 3.0, from the remaining paternal markers not assigned to linkage groups.