An efficient CRISPR-Cas9 enrichment sequencing strategy applied to Prunus salicina MYB10 genes

We improved and demonstrate the use of the CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina), which has a length from 90 kb to 271 kb in the P. salicina genomes available. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4.6% and a depth per sample of 11.9x. From the alignment, 3,261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed the detection, at the haplotype level, of the variability in this region.