Identification And Characterization Of A MITE-DTM-Brr4 In Brassica Rapa

Transposable elements(TEs)are mobile units of DNA.Eukaryotic organisms have dynamic genomes,with transposable elements(TEs)as a major contributing factor.Transposon can be divided into autonomous and non-autonomous transposon,according to the ability of encoding transposase.Although the large autonomous TEs can significantly shape genomic structures during evolution,genomes often harbor more miniature nonautonomous TEs that can infest genomic niches where large TEs are rare.In spite of their cut-and-paste transposition mechanisms that do not inherently favor copy number increase,miniature inverted-repeat transposable elements(MITEs)are abundant in eukaryotic genomes and exist in high copy numbers.Which may play important roles in gene regulation and genome evolution.However,few studies have investigated the molecular mechanisms.In this study,we found an insertion/deletion polymorphism site in the anthocyanin synthesis gene promoter region between light-induced anthocyanin synthesis mutant and wild type of Brassica rapa subsp.rapa 'Tsuda'.At the site we cloned a transposon named DTM-Brr4,which has been found by Bioinformatics method.However,its role in turnip about peculiar traits formation,origin,evolution,distribution and transposition ability as well as function have not been determined.In this study,first we used flanking PCR verification in the backcross population and related gene expression pattern to detect whether the transposon was associated with the target phenotype of turnip.And then analysis DTM-Brr4 sequence features,and copy number,phyletic evolution and genomic distribution in turnips and other Brassicaceae plants through bioinformatics method.Moreover,Analysis DTM-Brr4 transposable activity and other transposition features in 'Tsuda' through resequencing data and small RNA sequencing date as well as DNA methylation detection techniques.Results are as follows:1.We identified a 559bp insertion sequence polymorphism in the anthocyanin biosynthetic gene(ANS)promoter of light-sensitive and non-sensitive 'Tsuda'.Preliminary analysis of the sequence showed that it was a transposon DTM-Brr4,and its insertion or deletion is not associated with the wild and mutant phenotype of the mutant backcross population.Moreover upon DTM-Brr4 insertion in the promoter ANS expression was down regulated,but other anthocyanin biosynthesis-related genes upstream of the ANS,such as PAP,TT8,CHS,DFR were also down regulated in the transcriptional level.2.Bioinformatics analysis showed that DTM-Brr4 has structural characteristics as miniature inverted repeat transposon(MITE),which can form a stable secondary structure,its long(about 200-300bp)terminal inverted repeats and 9bp target site repeat sequence classified it into Mutator transposon superfamily.In Brassicaceae plants,we found that Arabidopsis contained no DTM-Brr4 transposable elements,and plants such as Brassica rapa and B.oleracea contained 134 and 13 DTM-Brr4 copies respectively in their genome,but their heterologous diploid plant Brassica nupas contained only 41 DTM-Brr4 copies.In addition,these copies are evenly distributed over turnip chromosomes of the genome.3.Though re-sequencing data analysis,we obtained that the total DTM-Brr4 copy number did not change in 14 different individuals genome of turnip,but there are differences in their transposition sites.In the sample group,there were several common sites(or transposition stable sites)and numerous specific sites(or transposition unstable sites);Flanking sequence analysis of the transposition stable sites showed that there was no conserved motif for DTM-Brr4 transposition.DNA methylation experiments showed DTM-Brr4,both in stable or unstable sites,was heavily methylated in the internal sequence,and the main methylation pattern was the CHH methylation.And we found 113 small RNA in 21~24nt length that perfectly match to the terminal end sequence of the DTM-Brr4,in the turnip small RNA sequencing database.In conclusion,We demonstrated that DTM-Brr4 was a specific MITE of Brassica,belonging to Mutator family,and it may not be associated with a target phenotype in turnip.We proposed that DTM-Brr4 was originated from Brassica and only actively amplified in Brassica rapa after divergence from Arabidopsis.It was an active MITE and randomly inserted to other places in the genome when it moved.It got high level of DNA methylation modification within its body region.DTM-Brr4 had the potential to produce small RNAs or microRNAs,which may be related to the host's silencing mechanism to the MITE transposon(and)or the MITE transposon's regulation to its host gene.This investigation focused on the systematic characterization of DTM-Brr4 and its ability to transpose to provide a better understanding of the roles played by MITEs in the plant genome and to develop tools for genetic and genomic studies.