Discovery and Profiling of microRNAs at the Critical Period of Sex Differentiation in Xanthoceras sorbifolium Bunge

Abstract

Research Highlights: The critical period of sex differentiation in Xanthoceras sorbifolium was investigated. Multiple microRNAs (miRNAs) were identified to influence female and male flower development, with some complementary functions. Background and Objectives: Xanthoceras sorbifolium Bunge is widely cultivated owing to its multipurpose usefulness. However, as a monoecious plant, the low female–male flowers ratio and consequent low seed yield are the main bottlenecks for industrial-scale development of seed utilization. MiRNAs play crucial regulatory roles in flower development and sex differentiation; therefore, we evaluated the roles of miRNAs in the critical period of sex differentiation in X. sorbifolium. Materials and Methods: Four small RNA libraries for female and male flower buds of the critical period of sex differentiation were constructed from paraffin-embedded sections. The miRNAs were characterized by high-throughput sequencing, and differentially expressed miRNAs were validated by reverse transcription-quantitative polymerase chain reaction. Results: There were obvious differences in male and female pistil and stamen flower buds, with elongated inflorescence and clear separation of flower buds marking the critical period of sex differentiation. A total of 1619 conserved miRNAs (belonging to 34 families) and 219 novel miRNAs were identified. Among these, 162 conserved and 14 novel miRNAs exhibited significant differential expression in the four libraries, and 1677 putative target genes of 112 differentially expressed miRNAs were predicted. These target genes were involved in diverse developmental and metabolic processes, including 17 miRNAs directly associated with flower and gametophyte development, mainly associated with carbohydrate metabolism and glycan biosynthesis and metabolism pathways. Some miRNA functions were confirmed, and others were found to be complemented. Conclusions: Multiple miRNAs closely related to sex differentiation in X. sorbifolium were identified. The theoretical framework presented herein might guide sex ratio regulation to enhance seed yield.