Cotton Genomics and Genetics 2024, Vol.15, No.2, 112-126 http://cropscipublisher.com/index.php/cgg 117 the proportions of different lncRNA categories, such as long intergenic non-coding RNAs (lincRNAs) and long intronic non-coding RNAs (lnc-Intronic), with different colors representing each lncRNA type. Additionally, the figure includes information on CPC (Coding Potential Calculator) and CNCI (Coding-Non-Coding Index) scores, which assess the coding potential of lncRNAs, where lower scores indicate weaker protein-coding potential. By integrating these data, they developed an analysis pipeline named PULL, successfully identifying 9 240 lncRNAs. The study found that many lncRNAs regulate adjacent protein-coding genes (PCGs) in cis. For instance, some lncRNAs modulate gene expression by influencing the selection of transcription start sites (TSS) of PCGs. Additionally, the research explored the structural characteristics of lncRNAs, such as their exon number, transcript length, and GC content, and how these features impact their function and expression. This study not only provides new insights into the biological functions and regulatory mechanisms of cotton lncRNAs but also establishes a high-resolution lncRNA map, laying a foundation for future functional research. 3.2.2 Differential gene expression Differential gene expression analysis using RNA-Seq has provided critical insights into the regulatory networks controlling key biological processes in cotton. By comparing gene expression profiles between different tissues, developmental stages, or treatment conditions, researchers can identify genes that are differentially expressed and potentially involved in specific pathways. This information is essential for understanding the genetic basis of complex traits and for developing strategies to improve cotton breeding (Begum and Banerjee, 2021). The ability to perform differential gene expression analysis with high precision and accuracy has made RNA-Seq a cornerstone of functional genomics studies in cotton. 3.3 Epigenomics and methylation studies Epigenomic studies focus on modifications to the genome that do not change the DNA sequence but can affect gene expression. NGS technologies have made significant contributions to epigenomics, particularly in the study of DNA methylation, which plays a crucial role in gene regulation and plant development. 3.3.1 Whole-Genome bisulfite sequencing Whole-genome bisulfite sequencing (WGBS) is an NGS-based technique used to study DNA methylation patterns across the entire genome. In cotton, WGBS has been employed to investigate the epigenetic modifications that regulate gene expression and contribute to phenotypic variation. This technique provides a comprehensive view of the methylome, allowing researchers to identify differentially methylated regions and their potential roles in gene regulation (Ferros et al., 2022). The insights gained from WGBS studies are crucial for understanding the epigenetic mechanisms underlying important agronomic traits in cotton. Lu et al. (2022) successfully assembled a new genome for CRI-12, a major cotton variety in China, using Pacific Biosciences and Hi-C sequencing technologies (Figure 3). The results showed that the CRI-12 genome is of high quality, with a total length of approximately 2.31 Gb and a contig N50 of 19.65 Mb, outperforming previously reported cotton genomes. Comparative analysis with other reported genomes revealed that CRI-12 has 7 966 structural variations and 7 378 presence/absence variations, contributing to its ability to adapt to different environments. Lu et al. (2022) investigated the phenotype of CRI-12 cotton and the homology relationships between different cotton genomes. Figure 3A shows the actual plant morphology of CRI-12. Figure 3B presents the chromosome information of CRI-12 using Hi-C mapping, with chromosomes numbered from 1 to 26 from left to right. Figure 3C, through synteny analysis, reveals the high collinearity between the A subgenome and D subgenome of CRI-12 and other cotton species. Figure 3D, using 4DTV analysis, illustrates the evolutionary distance of CRI-12 from other species, indicating genome duplication and divergence at the genomic level. The researchers also used whole-genome bisulfite sequencing (WGBS) to analyze the DNA methylation patterns of CRI-12, exploring its molecular mechanisms for environmental adaptation. The WGBS results indicated that methylation variations might enhance CRI-12's adaptability to various biotic and abiotic stress conditions by regulating gene expression. Notably, the study found significant changes in methylation levels of several
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