Small RNA and Degradome Sequencing in Floral Bud Reveal Roles of miRNAs in Dormancy Release of Chimonanthus praecox

Abstract

Chimonanthus praecox (wintersweet) is highly valued ornamentally and economically. Floral bud dormancy is an important biological characteristic in the life cycle of wintersweet, and a certain period of chilling accumulation is necessary for breaking floral bud dormancy. Understanding the mechanism of floral bud dormancy release is essential for developing measures against the effects of global warming. miRNAs play important roles in low-temperature regulation of flower bud dormancy through mechanisms that are unclear. In this study, small RNA and degradome sequencing were performed for wintersweet floral buds in dormancy and break stages for the first time. Small RNA sequencing identified 862 known and 402 novel miRNAs; 23 differentially expressed miRNAs (10 known and 13 novel) were screened via comparative analysis of breaking and other dormant floral bud samples. Degradome sequencing identified 1707 target genes of 21 differentially expressed miRNAs. The annotations of the predicted target genes showed that these miRNAs were mainly involved in the regulation of phytohormone metabolism and signal transduction, epigenetic modification, transcription factors, amino acid metabolism, and stress response, etc., during the dormancy release of wintersweet floral buds. These data provide an important foundation for further research on the mechanism of floral bud dormancy in wintersweet.

Keywords: Chimonanthus praecox; floral bud dormancy; high-throughput sequencing technology; miRNAs; target identification.

Figure 3

Differentially expressed miRNAs (DEMs). (a) The distribution of DEMs. (b) Venn diagram of DEMs from IB570/FB.Nov, IB570/FB150, IB570/FB300, IB570/FB450, and IB570/nF16. The numbers in the figure represent the number of DEMs. (c) Heatmap of the 23 DEMs. Red and blue represent upregulation and downregulation, respectively. (a,b) were completed with TBtools [51] software with default parameters. (c) was completed with TBtools software; the values of miRNA expression were normalized using Z-score normalization.

Figure 1

Sequence length distribution of sRNAs in the floral buds of wintersweet. FB.Nov: FBs in November before treatment; nF16: FBs collected after treatment for −300 CU chilling accumulation at 16 °C; FB150, FB300, FB450, and IB570: FBs collected after treatment for 150, 300, 450, and 570 CU chilling accumulations at 12 °C, respectively.

Figure 2

Sequence information of all microRNAs (miRNAs) in wintersweet floral buds. (a) Sequence length distribution of miRNAs. The numbers in the figure represent the percentage of miRNAs sequences with different lengths in the total miRNA sequences. (b) First base distribution of miRNAs of different lengths. A, U, C, and G represent adenine, uracil, cytosine, and guanine, respectively. (a,b) were generated with GraphPad Prism 9 software [48].

Figure 4

miRNA–target pairs. (a,b) Classification statistics of miRNA–target pairs. Numbers in parentheses indicate number of miRNA–target pairs. (c,d) The T-plots of two target genes confirmed by degradome sequencing. The T-plots were completed with CleaveLand 3.0.

Figure 5

Gene Ontology (GO) enrichment analysis of differentially expressed miRNAs (DEMs) target genes. The figure shows significantly enriched GO terms (p-value ≤ 0.05) in three categories: Cellular component (a), Molecular function (b), and Biological process (c). All of the GO terms are contained in (a,b); the twenty most dominant GO terms are contained in (c). GO analysis was performed with the GOseq R package. The results were visualized on level 3.