Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing

Abstract

BackgroundMicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored.ResultsPomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5’ end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock.ConclusionOur first and preliminary report of miRNAs will provide information on the synthesis of biochemical compounds of pomegranate for future research. The functions of the targets of the novel miRNAs need further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0807-3) contains supplementary material, which is available to authorized users.

Identification and Target Prediction of MicroRNAs in Ulmus pumila L. Seedling Roots under Salt Stress by High-Throughput Sequencing

“…Moreover, such techniques do not always detect non-conserved or species-specific miRNAs that may accumulate at lower levels [30]. High-throughput sequencing technologies provide a rapid and more efficient approach for identifying novel species-specific or low-abundance miRNAs and for profiling populations of small RNAs at different plant developmental stages and under various environmental conditions [6,38].…”

“…These sizes and proportions are consistent with typical small RNA distributions in angiosperms such as cotton [7], Lycium chinense Mill. [28], Punica granatum L. [38], and potato [39]. Forests 2016, 7, 318 6 of 14 Figure 1.…”

Small RNA and mRNA Sequencing Reveal the Roles of microRNAs Involved in Pomegranate Female Sterility

“…Several conserved and novel miRNAs in pomegranate have been identified in previous studies through high-throughput small RNA sequencing [28][29][30]. Compared with the miRNAs expressed in fruit [30], a total of 28 known miRNAs identified in this study were conserved in pistil and fruit, while 67 known miRNAs were newly identified in pistil.…”

“…To accurately evaluate the expression levels of miRNAs, stem-loop qRT-PCR was performed as described by previous studies [29,59] and 5.8S was used as the reference gene [29]. Total RNA was isolated from floral pistils at different development stages, using the approach mentioned above [46].…”

“…The study of small RNAs in pomegranate has been reported previously [28][29][30]. However, the involvement of small RNAs in pomegranate FMFs' ovule development cessation has not yet been determined.…”