MiRNA Prediction,Structural Andevolutionary Analysis In Brassica As Well As Related Molecular Marker Development

The micro-RNAs prediction by homologous alignment technique is an efficientapproach for the identification of novel miRNAs across different species. We adopted aseries of steps with high sensitivity and high strictness to predicate718BrassicamiRNAs (642in B. rapa,54in B. napus and22in B. oleracea). Through the analysis ofsome characterization of miRNAs and pre-miRNAs, including the length, the GCcontent, and the relationship between miRNAs and repetitive sequences (transposableelement and simple repeat sequences). We concluded (1) The GC content of maturemiRNAs was obviously higher than that in pre-miRNAs;(2)22.2%pre-miRNAs and13.9%mature miRNAs averagely originated from transposons in Brassica species,respectively;(3) miRNAs existed in the reverse-repeats-rich or tandem-repeats-richregions;(4)14.7%miRNAs could control transposons;(5) Each of450groups ofmiRNAs could regulate the same target site while each of420groups of target siteswere controlled by the same miRNA. These results suggested that the repetitivesequences could be an origin of miRNAs or target sites. The two modes: one miRNAvas multiple target sites, and multiple miRNA vas one target site, make the regulationpatterns of multiple-copy genes more efficient and swift than ever. These researcheswould be beneficial to understand the origin and evolution and interacting patterns ofmiRNAs and target genes in three Brassica species.DNA-based molecular marker plays a great role in gene mapping, geneticdiversity analysis, germplasm evaluation and molecular marker-assisted selection. Highquality and high-efficiency of molecular marker is critical to its wide application.Traditional molecular marker is difficult to simultaneously meet the requirements ofabundant polymorphism, good stability and high efficiency. Therefore it is necessary todevelop new-type markers which can absorb merits of traditional markers as much aspossible. Small RNA is one kind of endogenous non-coding RNA and widely exists invarious organisms. High conservation of miRNA and pre-miRNA provides a probabilityto develop novel marker related to miRNA. We predicated795miRNAs in Brassicaspecies through a series of rigorous screening steps, aligned pre-miRNA sequences anddesigned miRNA-related primers. These primers were tested in16varieties of sixBrassica species and the results showed that abundant polymorphism was present among these materials and polymorphism index content of miRNA-related markersranged from2to8with an average of3.8. Good transferability was also verified acrossspecies.45bands were sequenced and validated the truth of miRNA-related markers.These results show that the miRNA-related marker is a novel marker genotypingtechnique which covers the advantages of high efficiency, stability and goodtransferability and will become a potential molecular marker system.