Comprehensive profiling of alternative splicing landscape during secondary dormancy in oilseed rape (Brassica napus L.)

Alternative splicing is a general mechanism that regulates gene expression at the post-transcriptional level, which increases the transcriptomic diversity. Oilseed rape (Brassica napus L.), one of the main oil crops worldwide, is prone to secondary dormancy. However, how alternative splicing landscape of oilseed rape seed changes in response to secondary dormancy is unknown. Here, we analyzed twelve RNA-seq libraries from varieties "Huaiyou-SSD-V1" and "Huaiyou-WSD-H2" which exhibited high (> 95%) and low (< 5%) secondary dormancy potential, respectively, and demonstrated that alternative splicing changes led to a significant increase with the diversity of the transcripts in response to secondary dormancy induction via polyethylene glycol 6000 (PEG6000) treatment. Among the four basic alternative splicing types, intron retention dominates, and exon skipping shows the rarest frequency. A total of 8% of expressed genes had two or more transcripts after PEG treatment. Further analysis revealed that global isoform expression percentage variations in alternative splicing in differently expressed genes (DEGs) is more than three times as much as those in non-DEGs, suggesting alternative splicing change is associated with the transcriptional activity change in response to secondary dormancy induction. Eventually, 342 differently spliced genes (DSGs) associated with secondary dormancy were identified, five of which were validated by RT-PCR. The number of the overlapped genes between DSGs and DEGs associated with secondary dormancy was much less than that of either DSGs or DEGs, suggesting that DSGs and DEGs may independently regulates secondary dormancy. Functional annotation analysis of DSGs revealed that spliceosome components are overrepresented among the DSGs, including small nuclear ribonucleoprotein particles (snRNPs), serine/arginine-rich (SR) proteins, and other splicing factors. Thus, it is proposed that the spliceosome components could be exploited to reduce secondary dormancy potential in oilseed rape.