Identification And Function Analysis Of Flowering MicroRNAs In Common Wild Wice (Oryza Rufipogon Griff.)

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNA (sRNA) that negativelyregulated genes expression mainly by target gene mRNA sequence-specific cleavage or translationrepression in post-transcriptional level. Currently, there are661known rice miRNAs were annotatedand included in miRBase18.0. However, as the wild relatives and genetic germplasm of the cultivatedrice, the common wild rice miRNAs have not been known. In this study, we sequenced3small RNAlibraries constructed from the common wild rice by Solexa Illumina high-throughput sequencingtechnology, identified and verified miRNAs in different development stages. Combining targetsprediction and cleavage verification, we could analyze the function and expression of floweringmiRNAs, identify flowering-related miRNAs and the miRNAs that induced flowering variation in thecommon wild rice. Research results not only obtain lots of information about the common wild ricemiRNAs，but also lay the foundation for future research into the molecular mechanisms of floweringdiversity and the flowering process in the common wild rice. The detailed results are shown below:Data sets analysis of sRNA libraries for High-throughput sequencingWe used the common wild rice population collected from Gaozhou, Guangdong Province toconstructed3sRNA libraries, CWR-V1and CWR-V2were the vegetative stage libraries of the group ofonce flowering a year and twice flowering a year, respectively, CWR-F2was the flowering stage libraryof the group of twice flowering a year. A total number of20156098,21531511and20995942highquality clean reads were obtained from3libraries by Illumina High-throughput sequencing, respectively.The sRNA sequences perfectly matching the rice genome were analyzed by BLAST search,512knownrice miRNAs corresponding to214miRNA families were found. After secondary structure analysis ofsRNA by MIREAP,290new miRNAs were predicted and specific-existed in the common wild rice.Cloning identification and tissue expression pattern analysis of new miRNAs33new miRNAs were selected to verify by stem-loop RT-PCR and obtained the expected DNAsegments that contained miRNA sequences, the results of RT-PCR were consistent with IlluminaHigh-throughput sequencing. These new miRNAs have different expression in the common wild rice.Relative quantitative Real-Time PCR indicated that new miRNAs expressed with spatio temporalspecific, the expression most of new miRNAs in leaf was general higher than any other tis sues, such asroot, stem and booting panical.Targets prediction and verification of flowering miRNAAs query sequences, miRNAs were performed Blastn searches against the rice genome inpsRNATarget,454known rice miRNAs and226new oru-miRNAs were predicted to have total8791potential targets. After functional annotation, GO and KEGG pathway analys is of targets,262predictedmiRNA targets interacted with rice flowering genes RFT1, Hd1, OsGI and so on. These targetscorrespond to146known rice miRNAs and41new miRNAs and some flowering targets were verified to cleave by their corresponding miRNAs in the method of RLM-5’RACE.Expression pattern analysis of flowering miRNAsMost expression of flowering miRNAs has no significantly difference between two differentlibraries,95flowering miRNAs with significantly difference could preliminary divide into3parties,66flowering-related F-miRNAs,17E-miRNAs induced flowering variation and12N-miRNAs with noclear function. The expression of47flowering miRNAs corresponding to22miRNA families indifferent libraries was detected by using relative quantitative Real-Time PCR, Real-Time PCR revealedthe same expression patterns as the High-throughput sequencing data. This further illustrates differentexpression of these miRNAs in different development stages of the common wild rice.