Impacts Of Whole Genome Triplication On MIRNA And CNSs Evolution In Brassiceae Tribe

Brassiceae is an important tribe of Brassicaceae, which has many vegetables with great nutrient and economic values. Apart from the shared experience of their ancestral polyploidization events (y, β and a) in Brassicaceae, Brassiceae underwent an additional whole genome triplication (WGT) event about 10 million years ago. MIRNAs and CNSs are two categories of important sequences which can regulate genes’ expression. However, little was known on how WGT affects evolution of these regulatory sequences, which limited the application of these sequences on molecular breeding. In our study, we not only annotated the MIRNAs and CNSs in Brassiceae, but also systematically investigated the impacts of WGT on MIRNAs and CNSs in these triplicated genomes. The main results are listed as follows:(1) We first annotated MIRNAs of Brassica rapa by a comprehensive strategy, including of analyzing sequencing reads of small RNAs in B. rapa, integrating with previous B. rapa MIRNA datasets and surveying synteny with three relative species. By our efforts, a MIRNA dataset of B. rapa with 680 MIRNAs and 969 miRNAs was provided. A total of 97 non-redundant MIRNA loci in relative species had 178 orthologous MIRNA loci in B. rapa with different copy numbers. To be detail,40 MIRNA loci were singlets, with 33 doublets and 24 triplets. Following WGT, MIRNAs were over-retained than whole genome genes and their flanking genes. Besides, MIRNAs also showed biased distribution among three subgenomes as genes, that is to say, retention of MIRNAs on subgenome LF was significantly higher than those of the other two subgenomes MF1 and MF2. Furthermore, we found that multiple-copy MIRNAs are more conserved than single-copy MIRNAs, which indicated that the experienced purifying selection and functional importance of the multiple-copy MIRNAs during the WGT process. Additionally, we predicted 2,396 target genes of MIRNAs, which were involved with divergently biological process. This result reflected functional divergence of MIRNAs. Strikingly, relationships of miRNA-targets had great divergence with loss of binding sites following WGT, which unveiled the fast evolution and loss for the function of miRNAs on their target genes.(2) We determined a highly confident pre-Brassiceae WGT CNSs dataset by a series of steps, including of integration of reprehensive CNSs datasets in Arabidopsis thaliana, sequence alignment, synteny analysis and multiple species comparison. A total of 106,280 complete CNSs which belong to 19,326 genes and 10,095 CNSs which belong to 4,875 genes were annotated respectively by our efforts. Overlap comparison showed that CNSs highly covered the datasets of cis-regulatory elements and DHS. Besides, CNSs associated genes were significantly enriched in the GO term of "transcription regulation, DNA-dependent". Additionally, transcription factors genes have significant higher count of CNSs than whole genome genes, which showed the CNSs’ multiple functions and their important role in transcription regulation. Interestingly, CNSs abundance in Brassicaceae plants was strongly correlated with their divergence relationship. The proportion of shared CNSs decreased with the increase of diverged time between Brassicaceae species, which indicated the variation and loss of CNSs during evolution history. Following WGT, CNSs had stronger tendency of single-copy status than their associated genes, which indicated the functional divergence of multiple-copy genes. Furthermore, CNSs also showed biased retention as genes, with the dominance of subgenome LF over subgenomes MF1 and MF2. Retained bias of CNSs was due to biased retention of their associated genes. For multiple-copy genes, retention of CNSs form copies in dominant subgenomes or dominant expressing genes were significantly higher than those of the others. This result showed that dominance of CNSs counts were important reason of subgenome dominance. Finally, impacts of CNSs mainly concentrated on counts of CNSs, not on sequence variation of CNSs.Together, our in silico analysis not only offered data resource of MIRNAs and CNSs in Brassiceae, but also deciphering the impacts of WGT on their evolution, which further the understanding of functional divergence of multiple-copy genes at transcriptional and post-transcriptional levels in polyploidy genomes.