Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis

Abstract

BackgroundMicroRNAs (miRNAs) are endogenous regulators of a broad range of physiological processes and act by either degrading mRNA or blocking its translation. Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases, but little is known about its miRNAs and their targets. To date, only 46 miRNAs have been identified in B. napus.ResultsForty-one conserved and 62 brassica-specific candidate B. napus miRNAs, including 20 miRNA* sequences, were identified using Solexa sequencing technology. Furthermore, 33 non-redundant mRNA targets of conserved brassica miRNAs and 19 new non-redundant mRNA targets of novel brassica-specific miRNAs were identified by genome-scale sequencing of mRNA degradome.ConclusionsThis study describes large scale cloning and characterization of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.

Tight regulation of the interaction between Brassica napus and Sclerotinia sclerotiorum at the microRNA level

“…E xpression data of AGO2 -targeted miRNAs and AGO2 gene in response to S. sclerotiorum inoculation as well as functional study of PTGS suppressors suggest the potential involvement of AGO2 in resistance to S. sclerotiorum more than previous report on simple tissue sRNA libraries generated through deep sequencing (Xu et al 2012a). Finally, we identified totally 280 B. napus miRNAs, which include 227 new members, variants and canonical members of known miRNA families and more importantly 53 novel B. napus miRNAs/candidates (Supplementary Table S2), which far exceeded the number in miRBase 21.0.…”

“…Finally, we identified totally 280 B. napus miRNAs, which include 227 new members, variants and canonical members of known miRNA families and more importantly 53 novel B. napus miRNAs/candidates (Supplementary Table S2), which far exceeded the number in miRBase 21.0. Significantly, among the 53 new B. napus miRNAs/candidates, 42 are reported for the first time in this study, only 11 were reported previously (Xu et al 2012a;Shen et al 2014Shen et al , 2015. This might be because we use both the normal leaves of B. napus and those inoculated with S. sclerotiorum as the source samples to generate sRNA library so that the library includes both constitutively expressed and S. sclerotioruminduced miRNAs.…”

“…However, this number is only 92 for B. napus, although it is polyploidy with a much larger and more complex genome than these plants. This indicates that although an increasing number of B. napus miRNAs have been annotated (Buhtz et al 2008;Korbes et al 2012;Xu et al 2012a; wild-type except line #21, which showed retarded growth and more severely serrated leaves (Fig. 4b).…”

Applications and challenges of next-generation sequencing in Brassica species

“…Degradome sequencing technology acts directly on the 3′-end of mRNA fragments with a poly-A tail, which are complementary with an miRNA, to figure out the target gene. Recently, Xu et al (2012a) performed whole-mRNA degradome sequencing of B. napus and found 33 conserved and 19 new mRNA targets, providing for the first platform for miRNA regulation functional research in Brassica. This exploited the mixed mRNA samples from different tissues, and abundant targets were detected.…”

MicroRNAs play an important role in the regulation of strawberry fruit senescence in low temperature

“…org/) were identified, but function of most miRNA is still unknown. To totally understand their biological functions in diverse biological processes, the recent development of degradome sequencing provided a new strategy for validating the splicing targets on a whole genome scale, which revolutionized the traditional computational target prediction and was successfully employed on identifying miRNA targets in plants ( Addo-Quaye et al, 2008;Xu et al, 2012Xu et al, , 2013.…”