多组学整合分析揭示热响应微RNA参与调控高温胁迫下棉花 CMS-D2恢复系雄性育性稳定性

Cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) is the main source of cytoplasm for the "three-line" hybrid cotton varieties currently being promoted and planted in the market. However, the anther and pollen development of cotton CMS-D2 restorer line is susceptible to continuous high-temperature(HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and metabolome data analysis was performed to explore the roles of microRNA(miRNA) in regulating male fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH (HT-tolerant) and SH (Previously named ZBR, HT-sensitive) under HT stress at Anyang, Henan and Jiujiang, Jiangxi environments. A total of 211 known and 248 novel miRNA were identified, of which 159 were differentially expressed miRNA (DEM). Additionally, 45 DEM in 39 miRNA clusters(PmCs) were also identified, and most highly expressed miRNA were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNA.PmC28 was located in the fine-mapped interval of the Rfi gene and contained two DEM, gra-miR482_L-2R+2 and gma-miR2118a-3p_R+1_1ss18TG. Transcriptome sequencing identified 6 281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA-mRNA-gene-KEGG containing 35 pairs of miRNA/target genes involved in the regulation of pollen development in response to HT, among which the mtr-miR167a_ R+1,tcc-miR167c, and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility stability by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with metabolome data revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results obtained in this study provide a new understanding of how heat-responsive miRNA regulate the pollen fertility stability of cotton CMS-D2 restorer line under HT stress, and has important practical significance for solving the breeding bottleneck in which the anther development of CMS-D2 restorer line is sensitive to HT stress in production.