Improved Methodology for Large Fragment Plant Resistance-Gene Capture

Plant resistance genes (R-genes) are the genetic basis of many disease resistances. Pathogen evolution rates typically outpace the rate at which resistant plant varieties can be generated. An alternative to traditional breeding strategies is finding novel resistances in wild relatives followed by marker assisted selection, or transgenic technology (especially as resistance is normally a dominant trait). Resistance gene enrichment sequencing (Ren-seq) is an approach to efficiently characterise a plants R-gene repertoire by targeted sequence enrichment. R-genes often appear in gene clusters with recently duplicated sequences. Therefore Ren-seq on short read platforms leads to potential false annotations and collapsing of R-gene sequences. By sequencing larger molecules we can capture whole genes, including intronic and intergenic sequences that are typically more polymorphic and so enable the resolving of gene structures of closely related genes. Ideally Ren-seq would capture the promoter, coding sequence and terminator useful for transient transfection assays and stable transgenes. By optimising the original (Illumina sequencing based) Ren-seq protocol for PacBio sequencing we demonstrate that our strategy can be used to specifically sequence inserts up to 7 kb with a high quality (≥ 97 % sequence accuracy) and assemble chromosomal regions containing multiple R-genes. This makes our pipeline useful for long and accurate R-gene sequencing and other applications of the specific capture of long fragments e.g. gap filling, other genome finishing, or structural variation verification.