Exploration And Verification Of Stripe Rust-responsive MiRNA In Wild Emmer Wheat

Stripe rust caused by Puccinia striiformis f.sp.tritici(Pst)is an important fungal disease of wheat.The use of disease resistance genes in wheat varieties is the most efficient method for disease management.The expression of diseae resistance is regulated by several factors.MicroRNA(miRNA)is a kind of non-coding single-stranded small RNA widely existed in plants.MicroRNAs(miRNA)play essential roles in plant adaptation to stress through regulating target genes negatively at the post-transcriptional levels.Wild emmer,the tetraploid progenitor of cultivated wheat,haobors rich resisance to stripe rust.However,little is known about miRNA and their functions on Pst stress in wild emmer.we performed deep sequencing technology to profile the stripe rust responsive miRNA and their targets in wild emmer.We used transient expression system in tobacco to analysis the potential role of tae-miR9773 in regulating target gene Yr15.1.A total of 54661879 raw reads with length of 18-24 nt were obtained from the small RNA libraries of the Pst-inoculated and mock-inoculated groups.783 stripe rust responsive miRNA including 306 known miRNA and 477 unknown miRNA were identified.2.Among the 306 known miRNA,272 were plant conserved miRNA.208 out of 272 miRNA had sequences differences to the previously reported miRNA.We found 34 conserved miRNA were wheat specific and 22 miRNA showed sequences differences to the known wheat miRNA.3.The 477 unknown miRNA were compared with all the pre-miRNA in the PNRD database,and 404 new miRNA belonging to 135 miRNA families were identified.Of which,29 miRNA families(e.g.miR1136,miR6197,miR9777,miR5084 and miR9783),were only detected in wheat.4.87%(682/783)of miRNA could be mapped on the wild emmer wheat chromosomes.The chromosome 6B had the largest number of miRNA(95),accounting for 14%.Based on the precursor sequences of miRNA(pre-miRNA)in the wild emmer reference genome,we found that 124 pre-miRNA were in cluster distributed and can be classified into 36 clusters.The number of pre-miRNA in each cluster were ranged from 2 to 8.The expression levels of pre-miRNA in cluster were similar.5.Of 783 miRNA identified in wild emmer,168 miRNA were differentially expressed(FDR< 0.05)and 143 miRNA were expressed between Pst inoculated and control treatments.53 and 65 miRNA were only expressed in the Pst inoculated and control treatments,respectively.6.Target Finder was used to predict the target genes of the 168 miRNA.A total of 4982 target genes were identified,including genes related to disease resistance(NBS,RPM1,RPS2,CML,EFR and MAPK).7.We have predicted 16 differential expressed miRNA(e.g.miR9773,miR6300,and miR5079)that may guide the cleavage of Yr15 transcripts.RT-PCR analysis showed that the expression levels of miR9773 showed a divergent pattern compared with Yr15,whereas the cleavage function of miR9773 to Yr15 transcripts was not verified in co-transformation of Yr15 and miR9773 in tobacco leaves.