Identification of microRNAs and their targets in inflorescences of an Ogura-type cytoplasmic male-sterile line and its maintainer fertile line of turnip (Brassica rapa ssp. rapifera) via high-throughput sequencing and degradome analysis.

Sue Lin,Youjian Yu,Da Sun,Hao Ji,Jian Xu,Libo Jin,Shiwen Su,Renyi Peng

doi:10.1371/journal.pone.0236829

Abstract

Cytoplasmic male sterility (CMS) is a widely used trait in angiosperms caused by perturbations in nucleus-mitochondrion interactions that suppress the production of functional pollen. MicroRNAs (miRNAs) are small non-coding RNAs that act as regulatory molecules of transcriptional or post-transcriptional gene silencing in plants. The discovery of miRNAs and their possible implications in CMS induction provides clues for the intricacies and complexity of this phenomenon. Previously, we characterized an Ogura-CMS line of turnip (Brassica rapa ssp. rapifera) that displays distinct impaired anther development with defective microspore production and premature tapetum degeneration. In the present study, high-throughput sequencing was employed for a genome-wide investigation of miRNAs. Six small RNA libraries of inflorescences collected from the Ogura-CMS line and its maintainer fertile (MF) line of turnip were constructed. A total of 120 pre-miRNAs corresponding to 89 mature miRNAs were identified, including 87 conversed miRNAs and 33 novel miRNAs. Among these miRNAs, the expression of 10 differentially expressed mature miRNAs originating from 12 pre-miRNAs was shown to have changed by more than two-fold between inflorescences of the Ogura-CMS line and inflorescences of the MF line, including 8 down- and 2 up-regulated miRNAs. The expression profiles of the differentially expressed miRNAs were confirmed by stem-loop quantitative real-time PCR. In addition, to identify the targets of the identified miRNAs, a degradome analysis was performed. A total of 22 targets of 25 miRNAs and 17 targets of 28 miRNAs were identified as being involved in the reproductive development for Ogura-CMS and MF lines of turnip, respectively. Negative correlations of expression patterns between partial miRNAs and their targets were detected. Some of these identified targets, such as squamosa promoter-binding-like transcription factor family proteins, auxin response factors and pentatricopeptide repeat-containing proteins, were previously reported to be involved in reproductive development in plants. Taken together, our results can help improve the understanding of miRNA-mediated regulatory pathways that might be involved in CMS occurrence in turnip.

Highlights

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is common across angiosperms and is caused by the interaction of a nuclear fertility restorer gene and a mitochondrial CMS gene, resulting in the inability to produce functional pollen [1]
To identify miRNAs related to Ogura-CMS in turnip, six independent small RNA libraries from inflorescences collected from the Ogura-CMS line and its maintainer fertile (MF) line were constructed
Using high-throughput sequencing coupled with degradome analysis, we identified a total of 22 targets for 25 miRNAs and 17 targets for 28 miRNAs involved in reproductive development for the Ogura-CMS line and its MF line of turnip, respectively (Table 5)

Summary

Introduction

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is common across angiosperms and is caused by the interaction of a nuclear fertility restorer gene and a mitochondrial CMS gene, resulting in the inability to produce functional pollen [1]. Mitochondrial retrograde regulation especially manifests with variation in the stages at which pollen/anther abortion occur and the inconsistency in microsporogenesis and tapetum development processes with specific mitochondrial genes in different nuclear backgrounds [21,22,23,24]. All these findings have directed attention in recent years to studies of nucleus-cytoplasm interactions at the whole-genome level

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Conclusion