Planning a SMRT-RenSeq experiment: capture, sequence and clone disease resistance genes

Plant disease resistance genes of the NB-LRR/NLR (nucleotide-binding leucine rich repeat) protein class are characterized by a high sequence conservation and large abundance of several thousands in polyploid species. Plant reference genome sequences reveal NLR gene repertoires, and enable creation of a biotinylated RNA sequence capture library for exploration of NLR diversity in related taxa, termed R gene enrichment and sequencing (RenSeq). RenSeq on genomic DNA from parents and bulked resistant and susceptible genotypes, followed by short-read sequencing, assembly and comparison, enables genetic mapping of NLR genes. It also enables (re)annotation of NLR complements, and identification of transcribed NLRs (cDNA RenSeq).  The novel combination of RenSeq with SMRT (single molecule real-time; Pacific Biosciences) sequencing, enables for the first time de novo assembly of complete NLR genes. Captured flanking sequences include regulatory elements and allow assembly of complex multi-NLR loci from uncharacterized germplasm. The ability to rapidly assemble and test complete R gene candidates based on a rough genetic map position, significantly reduces time and cost compared to current R gene cloning strategies. We will present the technical aspects for a successful RenSeq and gene cloning experiment.