Identification And Characterization Of MicroRNAs From Wild Barley By High-throughput Sequencing Combined With Degradome Sequencing

Wild barley,Hordeum spontaneum,the progenitor of cultivated barley,Hordeum vulagare,exhibits a tremendous level of genetic diversity for both abiotic and biotic stresses.As a result,wild barley becomes the primary gene pool of cultivated barley and holds the promise for barley improvement to meet the challenges of increasing food demand and changing global climate in the 21st century.McroRNAs(miRNAs)are small class of endogenous RNAs that regulates the geneexpression which is involved in various biological and metabolic processes and stress resisitance at the post-transcriptional level in both plants and animals.Many major crops have been traced for miRNAs in past few years,yet miRNAs of wild barley(Hordeum vulgare L.)are still questioned.The second generation sequcencing.degradome sequencing and qPCR were combined together to detect the miRNA profile of wild barley in this vivo study.The main results obtained are as follows:1)A mixed library was constructed for solexa sequencing.After blast with the identified miRNA of four species(brachypodium?wheat?barley?rice)in miRBase,216 conserved miRNAs belongs to 131 fimilies were detected,of which the major is 21nt in length and the 5'end generally starts with U.we also found the conservation of miRNA among the grass family.2)The Mireap software was usd to identify novel miRNAs,by the assessments of the secondary structure and free energy of pre-miRNA,we finally detected 29 novel miRNAs.3)After Go annotation and kegg pathway analysis,we found the target genes of miRNAs were involved in large amount of processes,such as biosynthesis,metabolic processes,cell differentiation,apoptosis,embryonic development,environmental stress and other processes,which indicats the relationship between miRNA and the growth and development of plats.4)By comparison with the miRNA profile of cultivated barley,we found both the variety and expression level were different among them,which suggest the possibility for improving the quality of cultivated barley.5)We finilly confirmed 34 target gens of 21 miRNA by degradation sequcing and the existence of 11 miRNA including 7 conserved and 4novel miRNA by qPCR.The existence of novel miRNA and the relative expression level were also confirmed by qPCRThrough this study,we obtained the miRNA expression profiles of wild barley.the combination of bioinformatics,the degradation sequcencing as well as qPCR sequencing confirmed the credibility of the results and validate the function of miRNA target genes,which was useful for the future research.