ABSTRACT: BACKGROUND:Cold tolerance is important for plants' geographical distribution and survival in extreme seasonal variations of climate. However, Populus simonii × P. nigra shows wide adaptability and strong cold resistance. Transcriptional and post-transcriptional regulation of cold-responsive genes is crucial for cold tolerance in plants. To understand the roles of regulatory RNAs under cold induction in Populus simonii × P. nigra, we constructed cDNA and small RNA libraries from leaf buds treated or not with -4 °C for 8 h for analysis. RESULTS:Through high-throughput sequencing and differential expression analysis, 61 miRNAs and 1229 DEGs were identified under cold induction condition in Populus simonii × P. nigra. The result showed that miR167a, miR1450, miR319a, miR395b, miR393a-5p, miR408-5p, and miR168a-5p were downregulated, whereas transcription level of miR172a increased under the cold treatment. Thirty-one phased-siRNA were also obtained (reads ? 4) and some of them proceeded from TAS3 loci. Analysis of the differentially expressed genes (DEGs) showed that transcription factor genes such as Cluster-15451.2 (putative MYB), Cluster-16493.29872 (putative bZIP), Cluster-16493.29175 (putative SBP), and Cluster-1378.1 (putative ARF) were differentially expressed in cold treated and untreated plantlets of Populus simonii × P. nigra. Integrated analysis of miRNAs and transcriptome showed miR319, miR159, miR167, miR395, miR390, and miR172 and their target genes, including MYB, SBP, bZIP, ARF, LHW, and ATL, were predicted to be involved in ARF pathway, SPL pathway, DnaJ related photosystem II, and LRR receptor kinase, and many of them are known to resist chilling injury. Particularly, a sophisticated regulatory model including miRNAs, phasiRNAs, and targets of them was set up. CONCLUSIONS:Integrated analysis of miRNAs and transcriptome uncovered the complicated regulation of the tolerance of cold in Populus simonii × P. nigra. MiRNAs, phasiRNAs, and gene-encoded transcription factors were characterized at a whole genome level and their expression patterns were proved to be complementary. This work lays a foundation for further research of the pathway of sRNAs and regulatory factors involved in cold tolerance.
