SNP Discovery In Polyploid Crops By Transcriptome Sequencing Without Reference Sequences

Single nucleotide polymorphisms (SNPs) are the most common genetic variation in the genome and widely used as genetic markers in linkage map and molecular marker-assisted breeding. New high-throughput sequencing platforms have been widely applied for SNP calling in crops. However, SNP discovery in polyploid crops is still challenged because of their large and complex genomes. In this study, we describe a new method utilizing RNA-Seq to generate transcriptomic90bp reads, combining with virtual enzyme cutting, alignment and other bioinformatic tools to identify SNPs, for polyploid crops without reference sequences. The method was employed to genotype SNPs in two example DH genetic populations of two polyploid crops (wheat and tobacco). About1Gb RNA-Seq data was generated from two wheat parents (Yanzhanl and Neixiangl88) and two tobacco parents (HHDJY and Hicks Broad Leaf) of the two populations, respectively, and further used to call their SNPs. The EST and GSS sequences by traditional sequencing from wheat cultivar Chinese Spring and tobacco cultivar Hicks Broad Leaf were used as the reference sequences to verify our SNP results, respectively. Based on our approaches, we identified5489SNPs including685simple SNPs and4804hemi SNPs between the two wheat parents and136simple SNPs and1714hemi SNPs from the tobacco parents. At last219/1816and118/923simple/hemi SNPs were validated in wheat and tobacco, respectively. Our method therefore provides a new framework for SNP genotyping for polyploid crops of which genomes usually are complex and genomic sequences are not available yet.