Characterization of microRNAs involved in asymbiotic germination of Bletilla striata (Orchidaceae) seeds

Abstract

Orchids are distributed worldwide, and some species have considerable economic value. Orchid seeds are minute in size, simple in structure, and deficient in nutrient reserves. Asymbiotic seed germination is an important propagation strategy for orchids. MicroRNAs (miRNAs) play an essential role in seed germination. However, few studies have examined miRNAs involved in seed germination in orchids. Here, we conducted comparative small RNA sequencing at five stages to characterize the miRNAs involved in asymbiotic seed germination in Bletilla striata. A total of 253 known and 125 novel miRNAs were identified. Of them, 71 known and 29 novel miRNAs showed distinct expression among the five stages. Quantitative PCR revealed negative correlations of expression between differentially expressed miRNAs (DE miRNAs) and their targets. Function annotation and enrichment analyses of the targets of DE miRNAs between adjacent stages indicate that miRNA-target regulations are involved in many important processes during germination, such as signaling, biosynthesis, and transport of plant hormones. Twenty-two miRNAs were inferred to participate in plant hormone-related processes. The contents of abscisic acid, gibberellin A3, indole-3-acetic acid, jasmonic acid, trans zeatin riboside, and N6-(Δ2-isopentenyl) adenine varied significantly among the five stages. Nine tested plant hormone-related miRNAs and their targets exhibited significant correlations with at least one plant hormone. 5′-RLM-RACE validated that a transcript encoding auxin response factor was cleaved by Bst-miR160e as predicted. For the first time, we characterized miRNAs associated with the asymbiotic seed germination of an orchid species, which will help understand the miRNA-mediated regulatory mechanism of seed germination in orchids.