High-throughput sequencing discovery of conserved and novel microRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

MicroRNAs (miRNAs) are a class of 21-24 nucleotide non-coding RNAs that down-regulate gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in a few model plant species such as Arabidopsis, rice and Populus, and partially investigated in other non-model plant species. However, only a few conserved miRNAs have been identified in Chinese cabbage, a common and economically important crop in Asia. To identify novel and conserved miRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) we constructed a small RNA library. Using high-throughput Solexa sequencing to identify microRNAs we found 11,210 unique sequences belonging to 321 conserved miRNA families and 228 novel miRNAs. We ran a Blast search with these sequences against the Chinese cabbage mRNA database and found 2,308 and 736 potential target genes for 221 conserved and 125 novel miRNAs, respectively. The BlastX search against the Arabidopsis genome and GO analysis suggested most of the targets were involved in plant growth, metabolism, development and stress response. This study provides the first large scale-cloning and characterization of Chinese cabbage miRNAs and their potential targets. These miRNAs add to the growing database of new miRNAs, prompt further study on Chinese cabbage miRNA regulation mechanisms, and help toward a greater understanding of the important roles of miRNAs in Chinese cabbage.

Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

“…The field studies did not involve endangered or protected species. Small RNA libraries of M. notabilis were constructed as described elsewhere with a few modifications [27]. Briefly, the total RNA of three M. notabilis tissues was extracted using RNAiso plus (D9108A, Takara, China) in accordance with the manufacturers' instruction.…”

Identification of Radish (Raphanus sativus L.) miRNAs and Their Target Genes to Explore miRNA-Mediated Regulatory Networks in Lead (Pb) Stress Responses by High-Throughput Sequencing and Degradome Analysis

“…Experimental approaches including genetic screening, direct cloning and sequencing, and microarrays have also been adopted to identify and characterize miRNAs. Because genetic screening and direct cloning sequencing were expensive, time-consuming and produced only a few miRNA genes, they were inefficient for miRNA gene discovery (Berezikov et al 2006;Wang et al 2012). Small RNA (sRNA) microarrays were dependent on predesigned probes, thus, allowing only for the identification of the expression profiles of known miRNAs, which is unsuitable for the discovery of new miRNAs (Mangrauthia et al 2013).…”

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

“…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]. …”