Cytoplasmic male sterility (CMS) conferred by the cytoplasm from Gossypium harknessii (D2) is an important system for hybrid seed production in Upland cotton (G. hirsutum). The male sterility of CMS-D2 (i.e., A line) can be restored to fertility by a restorer (i.e., R line) carrying the restorer gene Rf1 transferred from the D2 nuclear genome. However, the molecular mechanisms of CMS-D2 and its restoration are poorly understood. In this study, a genome-wide comparative transcriptome analysis was performed to identify differentially expressed genes (DEGs) in flower buds among the isogenic fertile R line and sterile A line derived from a backcross population (BC8F1) and the recurrent parent, i.e., the maintainer (B line). A total of 1464 DEGs were identified among the three isogenic lines, and the Rf1-carrying Chr_D05 and its homeologous Chr_A05 had more DEGs than other chromosomes. The results of GO and KEGG enrichment analysis showed differences in circadian rhythm between the fertile and sterile lines. Eleven DEGs were selected for validation using qRT-PCR, confirming the accuracy of the RNA-seq results. Through genome-wide comparative transcriptome analysis, the differential expression profiles of CMS-D2 and its maintainer and restorer lines in Upland cotton were identified. Our results provide an important foundation for further studies into the molecular mechanisms of the interactions between the restorer gene Rf1 and the CMS-D2 cytoplasm.

Cytoplasmic male sterility (CMS) in flowering plants is usually caused by incompatibility between mitochondrial and nuclear genomes, and can be restored by nuclear genes known as restorer-of-fertility (Rf). Although the CMS/Rf system is useful and convenient for economic production of commercial hybrid seed, the molecular mechanisms of CMS occurrence and fertility restoration in cotton are unclear. Here, a combined small RNA and transcriptome sequencing analysis was performed on floral buds at the meiosis stage in three-line hybrid cotton system, and differentially expressed microRNAs (DEMs) and their target genes were identified and further analyzed for a possible involvement in CMS and fertility restoration. Totally 10 and 30 differentially expressed miRNA-target gene pairs were identified in A-B and A-R comparison group, respectively. A putative regulatory network of CMS occurrence and fertility restoration-related miRNA-target pairs during anther development were then constructed. The RLM-RACE analysis showed that gra-miR7505b regulates a PPR gene (Gh_D05G3392) by cleaving precisely at the 643 nt and 748 nt sites. The further analysis indicated that the sequence variation in the binding regions of Gh_D05G3392 and Gh_D05G3356 may cause a lower cleavage efficiency of the PPR genes by miR7505b and miR7505 in R line, respectively, leading to the up-regulation of the PPR genes and fertility restoration. These results have established their genetic involvement in fertility restoration in the CMS-D2 system. Our combined miRNA and transcriptome analysis in three-line hybrid cotton system provides new insights into the molecular mechanisms of CMS occurrence and fertility restoration, which will contribute to further hybrid breeding in cotton.

Cotton (Gossypium spp.) is a major global oilseed crop, ranking sixth in production worldwide and fifth in China. Protein arginine methyltransferases (PRMTs) catalyze arginine methylation, playing pivotal roles in DNA repair in Gossypium. However, their functions in cotton lipid metabolism remain unexplored. In this study, we identified 7, 9, 24, and 32 PRMT genes in Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. Phylogenetic analysis classified these genes into 7 distinct clades, with structural conservation suggesting functional preservation during cotton evolution. Collinearity analysis indicated segmental duplication as a major driver of PRMT family expansion. Expression profiling revealed significantly divergent expression patterns of GhPRMT5 between high-oil and low-oil cotton accessions, particularly during the critical oil accumulation phase. Heterologous expression in yeast showed that GhPRMT5 overexpression significantly increased total lipid content by 14.13 % (p < 0.05), providing direct evidence for its role in promoting lipid biosynthesis. At the same time, GhPRMT5-silenced lines also exhibited a 15.3 % reduction in cottonseed oil content, with significant alterations in fatty acid composition: saturated fatty acids (e.g., myristic acid [C14:0] and stearic acid [C18:0]) increased by 22.67 % and 26.84 %, respectively, whereas unsaturated fatty acids showed elevated oleic acid (C18:1, +20.90 %) and reduced linoleic acid (C18:2, -8.88 %) (p < 0.01).These results not only confirm the critical role of GhPRMT5 in regulating cottonseed oil accumulation but also reveal its role in modulating fatty acid composition. We are the first to report the connection between the PRMT family and lipid biosynthesis in cotton, and our findings provide novel genetic targets for improving cottonseed oil yield and nutritional quality, offering potential applications in industrial oilseed crop breeding.

Cotton seeds as the main by-product of cotton are not only an important raw material for edible oil and feed but also a source of biofuel. The quality of cotton seeds directly affects cotton planting and is closely related to the yield and fiber quality. However, the molecular mechanism governing cotton seed size remains largely unexplored. This study investigates the regulatory mechanisms of cotton seed size by focusing on two cotton genotypes, N10 and N12, which exhibit notable phenotypic variations across multiple environments. Developing seeds were sampled at various stages (5, 20, 30, and 35 DAP) and subjected to RNA-seq. The temporal pattern clustering and WGCNA on differentially expressed genes identified 413 candidate genes, including these related to sugar metabolism that were significantly enriched in transcriptional regulation. A genetic transformation experiment indicated that the overexpression of the GhUXS5 gene encoding UDP-glucuronate decarboxylase 5 significantly increased seed size, suggesting an important role of GhUXS5 in regulating cotton seed size. This discovery provides crucial insights into the molecular mechanisms controlling cotton seed size, helping to unravel the complex regulatory network and offering new strategies and targets for cotton breeding to enhance the economic value of cotton seeds and overall cotton yield.