Identification And Expression Analysis Of MicroRNA In Populus Infected With Stem Canker Disease

Stem canker disease causes stem canker on poplars. In north-central China, lots of Populus species easily infected the disease, resulting in significant production losses. MicroRNAs (miRNAs) are small RNAs, generally of20-24nt, that down-regulate target gene expression during development, differentiation, growth and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity and mechanical stresses have been performed, however, there are few reports about profiling of the miRNA identification and expression patterns during pathogen stress. We obtained almost38million raw reads through Solexa sequencing of two libraries from plants inoculated and uninoculated with canker disease pathogen. The majority of total sRNA reads were24nt for both libraries, followed by21nt. Sequence analyses identified74conserved miRNA sequences belonging to37miRNA families from154loci in the Populus genome and27novel miRNA sequences from35loci, including their complementary miRNA*strands, To investigate whether these35novel miRNAs were conserved across plant species, we used them as query sequences to perform Blastn searches against the other plant genome databases in Phytozome v7.0. No perfect matches were found except that pbe-SR2, pbe-SR6and pbe-SR14existed in Ricinus comunis, suggesting that these newly identified miRNAs were not broadly conserved within angiosperms. Intriguingly, the miRNA*of three conserved miRNAs (pbe-miR396b*, pbe-miR396c*and pbe-miR408*) were more abundant than their corresponding miRNAs. These miRNA*s might have functions related to pathogen stress. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of33miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and63target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. The target genes encoding transcription factors, metabolism proteins and stress resistance proteins exhibited important roles in the gene network when subjected to pathogen stress. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of tree defense responses during pathogen infection.