Identification and characterisation of tobacco microRNA transcriptome using high‐throughput sequencing

Abstract

MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in numerous biological processes in plants. In this study, we investigate miRNAs in Honghua Dajinyuan, an agronomically important species of tobacco in China. Here, we report a comprehensive analysis of miRNA expression profiles in the leaf, stem and root using a high-throughput sequencing approach. A total of 165 miRNAs, representing 55 conserved families, and 50 novel miRNAs, representing 19 families, were identified in three libraries. In addition, 12 miRNAs were randomly selected from a differentially expressed conserved miRNA family in three libraries with expression alterations and subjected to qRT-PCR validation. Of these, the expression level of nta-miR167d is highly enriched in the leaf tissue. In addition, the expression level of nta-miR319a is prominently enriched in the stem, while nta-miR160c is highly enriched in the root. Moreover, the target prediction showed that most of the targets coded for transcription factors that are involved in cellular and metabolic processes. GO analysis showed that most of the targets were involved in organelle function, served binding functions, and take part in cellular and metabolic processes. This study helps shed new light on understanding the role of miRNAs in different parts of the tobacco plant and adds a significant number of novel miRNAs to the tobacco miRNA transcriptome.

Identification and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection

“…Among the 49 novel miRNAs, only 17 had at least 0.01 TPM in both libraries, with the highest abundance, 130.31 TPM, for hop-miR41. The low abundance of novel miRNAs compared with those of conserved miRNAs in our data observed in this study was consistent with previous reports [23–30]. Normalized expression profiles of the novel miRNAs in HP and CIP libraries were compared to identify CBCVd-responsive novel miRNAs (Table 3).…”

“…The U content in the identified hop pre-miRNAs, in particular, was higher than the C and G contents, which were below 20% average (Additional file 3: Table S3 and Additional file 4: Figure S1). The MFE (minimum free energy), AMFE (adjusted MFE) and MFEI (MFE index) of the identified hop pre-miRNAs (Additional file 3: Table S3 and Additional file 4: Figure S1) were consistent with previous findings [30] and indicated high-confidence prediction of miRNAs in hop.…”

“…Their response to CBCVd-infection was also investigated, delivering valuable information for gaining in-depth knowledge of the function and regulatory mechanisms of miRNAs in the CBCVd-defence response. Comparison of sequencing data of the two libraries showed that sRNAs of 21-nt and 24-nt formed two major classes, consistent with the distribution patterns of sRNAs in other plant species [29, 30]. However, their distributions were significantly different between two analysed libraries.…”

“…Furthermore, the characteristic features of pre-miRNAs (such as length distribution, MFE) and miRNAs (such as size distribution, nucleotide composition of miRNAs and prevalence of 5′-uridine) were similar to those observed in other plants [28–30]. MFEI is considered as an another valuable criterion to distinguish miRNAs from other types of coding and non-coding RNAs.…”

“…The approach has been successfully used to discover novel miRNAs (which are generally expressed at a lower level), due to its ability to generate millions of reads randomly and independently with a determined length, which is implausible to identify by sequencing a low number of clones (sRNA library sequencing) or in situ hybridization-based methods (sRNA Northern blot and miRNA array analyses) [22]. Over the past years, high-throughput sequencing technologies have been implemented to investigate miRNAs across different plant species such as maize [23], potato [24], peanut [25], barley [26], soybean [27], Chinese cabbage [28], mulberry [29] and tobacco [30]. …”