Functional identification of lncRNAs in sweet cherry (Prunus avium) pollen tubes via transcriptome analysis using single-molecule long-read sequencing

Yang Li,Jie Yu,Xiaoming Zhang,Xuwei Duan,Kaichun Zhang,Tianzhong Li,Jing Wang,Guohua Yan,Chuanbao Wu,Wenqi Fan,Chunsheng Liu

doi:10.1038/s41438-019-0218-3

Abstract

Sweet cherry (Prunus avium) is a popular fruit with high nutritional value and excellent flavor. Although pollen plays an important role in the double fertilization and subsequent fruit production of this species, little is known about its pollen tube transcriptome. In this study, we identified 16,409 transcripts using single-molecule sequencing. After filtering 292 transposable elements, we conducted further analyses including mRNA classification, gene function prediction, alternative splicing (AS) analysis, and long noncoding RNA (lncRNA) identification to gain insight into the pollen transcriptome. The filtered transcripts could be matched with 3,438 coding region sequences from the sweet cherry genome. GO and KEGG analyses revealed complex biological processes during pollen tube elongation. A total of 2043 AS events were predicted, 7 of which were identified in different organs, such as the leaf, pistil and pollen tube. Using BLASTnt and the Coding-Potential Assessment Tool (CPAT), we distinguished a total of 284 lncRNAs, among which 154 qualified as natural antisense transcripts (NATs). As the NATs could be the reverse complements of coding mRNA sequences, they might bind to coding sequences. Antisense transfection assays showed that the NATs could regulate the expression levels of their complementary sequences and even affect the growth conditions of pollen tubes. In summary, this research characterizes the transcripts of P. avium pollen and lays the foundation for elucidating the physiological and biochemical mechanisms underlying sexual reproduction in the male gametes of this species.

Highlights

During plant sexual reproduction, pollen plays a vital role in the transportation of male gametes to the ovule, and the cellular and regulatory mechanisms underlying pollen tube elongation have been an ongoing focus of investigation[1]
As the FL reads are useful in transcript assembly, we determined the length distribution statistics of the FL reads in different sizes of libraries
To further elucidate the functions of different transcripts in sweet cherry pollen tubes, our data were first annotated by using the WEGO database (Fig. 3), and 40 Gene Ontology (GO) terms were observed to be enriched

Summary

Introduction

Pollen plays a vital role in the transportation of male gametes to the ovule, and the cellular and regulatory mechanisms underlying pollen tube elongation have been an ongoing focus of investigation[1]. The mechanism of complicated process of pollen tube elongation remains largely unknown. RNA sequencing of pollen tubes is a useful method for obtaining global transcriptional information as a starting point for pollen-related research. Shortread sequence data from pollen tubes have been obtained for a variety of species[4,5,6,7] in studies that have focused mainly on the differential expression of genes in different varieties of pollen. To further investigate the molecular mechanism of pollen tube growth, relative entire transcriptome data and additional transcript isoforms are necessary; single-molecule long-read sequencing (PacBio sequencing) technology is well suited for addressing such questions[8]

Methods

Results

Conclusion