Unravelling The MicroRNA-mediated Gene Regulation In Developing Pongamia Seeds By High-throughput Small RNA Profiling

Pongamia(Millettia pinnata)is a leguminous plant whose seeds have high yield of non-edible oils.In recent years,as a biodiesel feedstock tree species,it have attracted more and more research attention.Breeding Pongamia for favorable traits will rely on a comprehensive understanding of molecular mechanism regulating oil accumulation during its seed development.To date,only limited genomic or transcript sequences are available for Pongamia,while gene expression patterns in developing seeds is still lacking in this species.Regulation of gene expression mainly functions at transcriptional and post-transcriptional levels.MicroRNA(miRNA)is a kind of endogenous non-coding small RNA involved in post-transcriptional gene regulation.In this study,profiling of small RNAs by deep sequencing technology was performed in three developmental stages of Pongamia seeds to understand the characteristics of expressional changes and their putative correlation with seed development.A total of about 283 million raw reads were obtained from nine small RNA libraries.After sequence analysis,241 conserved miRNAs belonging to 54 families and 143 novel miRNAs were identified in Pongamia.Among the identified miRNAs,we found 119miRNAs(DEmiRs)that were significantly differentially expressed between different stages of seed development.At the same time,we obtained 1,327 and 107 target genes by bioinformatic prediction and degradome sequencing analysis,respectively.Functional annotation of the predicted target genes indicated that the target genes of DEmiRs were significantly enriched in certain metabolic pathways such as non-homologous end joining,base excision repair,and photosynthesis.Finally,we performed qPCR analyses for 15 randomly selected DEmiRs and their target genes.The results proved a good reliability of the sequencing results.This study will lay a solid foundation for further study on specific miRNAs with roles in Pongamia seed development and their potential applications in plant breeding.