Comparative Transcriptomics Analysis Uncovers Alternative Splicing Events and Molecular Markers in Cabbage (Brassica Oleracea L.)

Alternative splicing (AS) events were identified and verified in cabbage by comparative transcriptome analysis. The corresponding markers were developed and the germplasm resources were identified. Alternative splicing (AS) is a central regulatory mechanism that greatly contributes to plant gene expression and transcriptome diversity. A large body of evidence has shown that AS complexity is relevant for plant development, evolution, complexity, and adaptation. Both insertion/deletion (InDel) and single nucleotide polymorphism (SNP) are typically co-dominant inheritance markers and have abundant polymorphisms. These have been widely used for marker-assisted selection, genetic mapping, and germplasm identification in plants. However, little is known about the molecular mechanisms underlying AS events and the development of markers including SNP and InDel from the cabbage transcriptome. In this study, three cabbage transcriptome datasets were collected and aligned to the cabbage reference genome to analyze AS events and marker development. 31,524 AS events were identified from three cabbage genotypes, accounting for 20.8% of the total cabbage genes. Alternative 3' splice site donor (A3SS) was the most frequent type of the four main AS events in cabbage. 70,475 InDels and 706,269 SNPs were identified with average frequencies of 1 InDel/6.9 kb and 1 SNP/0.7 kb, respectively. 71,942 potential SSRs were identified in 53,129 assembled unigenes with a density of 1 SSR/6.8 kb. The ratio of SNPs with synonymous/non-synonymous mutations was 1:0.65. 142 InDels and 36 SNPs were randomly selected and validated via Sanger sequencing and polymorphism was found among 66.2% of the InDels and 78.6% of the SNPs. Furthermore, 35 informative InDel markers were successfully used for genetic diversity analysis on 36 cabbage accessions. These results facilitate understanding of the molecular regulation mechanism underlying AS events in cabbage. They also provide molecular marker resource data for genetic mapping construction and germplasm identification, and facilitate the genetic improvement of cabbage via breeding.