Characterization, Comparison And Evolutionary Analyses Of Transposable Elements In Two Brassica Diploid Species And Their Allotetraploid Relative

Two diploid species Brassica rapa and B. oleracea, and their tetroploid relative B. napus are important vegetables and oilseed crops in China, and have been recently used as model system to study genome evolution. Recent progress on sequencing the genomes of these species has provided an unprecedant opportunity to study transposon-mediated genome evolution. Transposons or transposable elements (TEs) are ubiquitous and most abundant DNA components in higher eukaryotes. In addition to polyploidization, rapid amplification of TEs is largely responsible for genome expansion. It has also been found that TEs are important factors for maintenance of chromatin structure and regulation of gene expression. Therefore, TEs are recognized as sensors of genome evolution. Furthermore, active TEs can be used to create TE insertion mutants, as a powerful functional genomics tool to study gene functions through forward or reverse genetics approaches.It is documented that B. rapa and B. oleracea diverged from a common ancestor approximately4million years ago, and formed two genomes of approximately485Mb and630Mbin size through long-term independent evolution. However, the difference regarding the composition, structure and organization of these two genomes remained largely unknown before this study. Here, we performed comparative analysis of these three Brassica genomes using both assembled and unassembled genome sequences with an emphasis on TE-mediated genome evolution, and obtained the following results:1. Asymmetric evolution of B. rapa and B. oleracea genomesBy a combination of structural analysis and homology comparison, a comprehensive Brassica TE database was constructed, which contains4602TEs in B. rapa and13382TEs in B. oleracea with clearly defined boundaries. Our data demonstrated that96.8%and93.3%of the retrotransposons and DNA transposons were inserted after the split of the two genomes. These TEs explained at least60%of genome size difference between the two species. Ralative to B. rapa, B. oleracea showed considerably high degree of genomic expansion in gene-rich syntenic regions.Analysis of orthologous long-terminal repeat (LTR)-retrotransposons (LTR-RTs) revealed more rapid nucleotide substitution in B. rapa than in B. oleracea. Consistently, analysis of a set of triplicated genes in the two genomes revealed significant lower rate of synonymous substitution and higher rate of non-synonymous substitution in B. rapa than in B. oleracea. These observations suggest that genes in the B. rapa genome have undergone stronger purifying selection than their orthologs in the syntenic regions of the B. oleracea genome. The asymatric evolution between the two genomes may be associated with the distinct genomic features, such as gene densities and local rates of genetic recombination between the two genomes.Differential scales of TE amplification between the two species were primarily responsible for genome size difference. On the other hand, elimination of large portions of LTR-retrotransposons by unequal recombination between two LTRs of individual elements also contributed to genome size variation. Additionally, our data suggest that B. rapa and B. oleracea diverged from a common ancestor approximately4.05million years ago. A large proportion of TE insertions were shared by the A genomes in B. rapa and B. napus and by the C genomes in B. oleracea and B. napus, but substantial TE insertion polymorphisms were observed between the same genomes in the tetraploid and corresponding diploids. Our data showed that the genomic differentiation between A genome in B. rapa and the A genome in B. napus, caused by insertions of retrotranposons and DNA TEs, were significantly higher than that between the C genome in B. oleracea and the C genome in B. napus.Recent burst of the Bocl family belonging to the CACTA superfamily was observed in B. oleracea, but such a burst was not found in B. rapa. In addition, three AT-rich introns were acquired in transposase of the Boc1elements in B. oleracea after it split from B. rapa.2. Transposable elements and regulation of gene expressionTo demonstrate the TE effects on gene regulation and functions, we used the whole set of Arabidopsis genes as references, and identified337genes containing TE insertions in the two Brassica genomes. Of these genes harboring TEs,68.5%were not annotated previously or structurally wrongly annotated. Using RNA sequencing data from the two Brassica diploids, we evaluated the levels of TE expressions. Our data showed that overall TEs in either genome expressed at very low levels, and the expression levels of different categories of TEs varied among different tissues. These variations may be associated with the changes of methylation status of TE DNA across tissues. Of the LTR-RTs identified in the B. rapa and B. oleracea genomes,62and148were found to have transcripts that readout into their adjacent sequences, indicating the interplay between TEs and surrounding genomic enviroments.3. Centromere evolution of B. rapa and B. oleracea genomesThe chromosome structure and organization of the.B. rapa and B. oleracea genomes are associated with centromere evolution. Using a bioinformatics approach, we identified a Gypsy LTR-RT family (dubbed Family190), which are largely enriched in the centromere satellite repeats in B. rapa, B. olecarea, and B. napus. The enrichment of this family in centromeric regions of the studied genomes was verified by fluorescence in situ hybridization. In addition, we identified several LTR-RT families showing different levels of enrichments between the A and C genomes, suggesting rapid and dynamic evolution of RT sequences in centromeric or pericentromeric regions.