Here, cytosine methylation at single-base resolution across the whole genome of cotton (Gossypium hirsutum L.) anthers was mapped using the whole-genome bisulfite sequencing technique, and the methylome changes associated with high-temperature (HT) stress were analysed in two cotton lines of the CMS system with contrasting HT stress tolerance. The cotton anther genome was found to display approximately 31.6%, 68.7%, 61.8%, and 21.8% methylation across all sequenced C sites and in the CG, CHG and CHH sequence contexts, respectively. In an integrated global methylome and transcriptome analysis, only promoter-unmethylated genes showed higher expression levels than promoter-methylated genes, whereas gene body methylation presented an obvious positive correlation with gene expression. The methylation profiles of transposable elements in cotton anthers were characterized, and more differentially methylated transposable elements were demethylated under HT stress. HT-induced promoter methylation changes caused upregulated expression of the mitochondrial respiratory chain enzyme-associated genes GhNDUS7, GhCOX6A, GhCX5B2, and GhATPBM, ultimately promoting a series of redox processes to form ATP for normal anther development under HT stress. In vitro application of the common DNA methylation inhibitor 5-azacytidine and accelerator methyl trifluoromethanesulfonate demonstrated that DNA demethylation promoted anther development, while increased methylation only partially inhibited anther development under HT stress.

Background: Utilization of heterosis has greatly improved the productivity of many crops worldwide. Understanding the potential molecular mechanism about how hybridization produces superior yield in upland cotton is critical for efficient breeding programs. Results: In this study, high, medium, and low hybrids varying in the level of yield heterosis were screened based on field experimentation of different years and locations. Phenotypically, high hybrid produced a mean of 14% more seed cotton yield than its better parent. Whole-genome RNA sequencing of these hybrids and their four inbred parents was performed using different tissues of the squaring stage. Comparative transcriptomic differences in each hybrid parent triad revealed a higher percentage of differentially expressed genes (DEGs) in each tissue. Expression level dominance analysis identified majority of hybrids DEGs were biased towards parent like expressions. An array of DEGs involved in ATP and protein binding, membrane, cell wall, mitochondrion, and protein phosphorylation had more functional annotations in hybrids. Sugar metabolic and plant hormone signal transduction pathways were most enriched in each hybrid. Further, these two pathways had most mapped DEGs on known seed cotton yield QTLs. Integration of transcriptome, QTLs, and gene co-expression network analysis discovered genes Gh_A03G1024, Gh_D08G1440, Gh_A08G2210, Gh_A12G2183, Gh_D07G1312, Gh_D08G1467, Gh_A03G0889, Gh_A08G2199, and Gh_D05G0202 displayed a complex regulatory network of many interconnected genes. qRT-PCR of these DEGs was performed to ensure the accuracy of RNA-Seq data. Conclusions: Through genome-wide comparative transcriptome analysis, the current study identified nine key genes and pathways associated with biological process of yield heterosis in upland cotton. Our results and data resources provide novel insights and will be useful for dissecting the molecular mechanism of yield heterosis in cotton.

Cotton (Gossypium hirsutum L.) is the most important fiber crop worldwide. Characterizing genotype by environment interaction (GEI) is helpful to identify stable genotypes across diverse environments. This study was conducted in six environments to compare the performance and stability of 11 inbred lines and 30 intraspecific hybrids of cotton. Analysis of variance using the additive main effects and multiplicative interaction model revealed that genotype (G), environment (E), and GEI had highly significant effects on yield and fiber quality traits. Mean comparisons among genotypes showed that most hybrids had higher means for yield and fiber quality traits than inbred genotypes. Additionally, a larger portion of the total variability in yield traits was explained by E than G and GEI. However, G and GEI combined contributed more to the total variance in fiber traits than E. The first three interaction principal components explained the majority of GEI in all traits under study. For most traits, the environments were not clustered together, implying contrasting interaction with genotypes. Stability measurements indicated that most hybrids showed more stable performance than inbred lines for all traits. The hybrids SJ48-1 × Z98-15 and L28-2 × A2-10 displayed both better performance and stability in yield and fiber quality traits. Our results show the importance of hybridization for improving cotton yield and fiber quality in a wide range of environments.

Cotton is an important fiber crop worldwide. The yield potential of current genotypes of cotton can be exploited through hybridization. However, to develop superior hybrids with high yield and fiber quality traits, information of genetic control of traits is prerequisite. Therefore, genetic analysis plays pivotal role in plant breeding. In present study, North Carolina II mating design was used to cross 5 female parents with 6 male parents to produce 30 intraspecific F1 cotton hybrids. All plant materials were tested in three different ecological regions of China during the year of 2016–2017. Additive-dominance-environment (ADE) genetic model was used to estimate the genetic effects and genotypic and phenotypic correlation of yield and fiber quality traits. Results showed that yield traits except lint percentage were mainly controlled by genetic and environment interaction effects, whereas lint percentage and fiber quality traits were determined by main genetic effects. Moreover, dominant and additive-environment interaction effects had more influence on yield traits, whereas additive and dominance-environment interaction effects were found to be predominant for fiber traits. Broad-sense and its interaction heritability were significant for all yield and most of fiber quality traits. Narrow-sense and its interaction heritability were non-significant for boll number and seed cotton yield. Correlation analysis indicated that seed cotton yield had significant positive correlation with other yield attributes and non-significant with fiber quality traits. All fiber quality traits had significant positive correlation with each other except micronaire. Results of current study provide important information about genetic control of yield and fiber quality traits. Further, this study identified that parental lines, e.g., SJ48–1, ZB-1, 851–2, and DT-8 can be utilized to improve yield and fiber quality traits in cotton.

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.

Background: Heterosis breeding is the most useful method for yield increase around the globe. Heterosis is an intriguing process to develop superior offspring to either parent in the desired character. The biomass vigor produced during seedling emergence stage has a direct influence on yield heterosis in plants. Unfortunately, the genetic basis of early biomass vigor in cotton is poorly understood.Results: Three stable performing F₁hybrids varying in yield heterosis named as high, medium and low hybrids with their inbred parents were used in this study. Phenotypically, these hybrids established noticeable biomass heterosis during the early stage of seedling growth in the field. Transcriptome analysis of root and leaf revealed that hybrids showed many differentially expressed genes (DEGs) relative to their parents, while the comparison of inbred parents showed limited number of DEGs indicating similarity in their genetic constitution. Further analysis indicated expression patterns of most DEGs were overdominant in both tissues of hybrids. According to GO results, functions of overdominance genes in leaf were enriched for chloroplast, membrane, and protein binding, whereas functions of overdominance genes in root were enriched for plasma membrane, extracellular region, and responses to stress. We found several genes of circadian rhythm pathway related to LATE ELONGATED HYPOCOTYL (LHY) showed downregulated overdominant expressions in both tissues of hybrids. In addition to circadian rhythm, several leaf genes related to Aux/IAA regulation, and many root genes involved in peroxidase activity also showed overdominant expressions in hybrids. Twelve genes involved in circadian rhythm plant were selected to perform qRT-PCR analysis to confirm the accuracy of RNA-seq results.Conclusions: Through genome-wide comparative transcriptome analysis, we strongly predict that overdominance at gene expression level plays a pivotal role in early biomass vigor of hybrids. The combinational contribution of circadian rhythm and other metabolic process may control vigorous growth in hybrids. Our result provides an important foundation for dissecting molecular mechanisms of biomass vigor in hybrid cotton.