Verticillium wilt (VW), also known as “cotton cancer,” is one of the most destructive diseases in global cotton production that seriously impacts fiber yield and quality. Despite numerous attempts, little significant progress has been made in improving the VW resistance of upland cotton. The development of chromosome segment substitution lines (CSSLs) from Gossypium hirsutum × G. barbadense has emerged as a means of simultaneously developing new cotton varieties with high-yield, superior fiber, and resistance to VW. In this study, VW-resistant investigations were first conducted in an artificial greenhouse, a natural field, and diseased nursery conditions, resulting in the identification of one stably VW-resistant CSSL, MBI8255, and one VW-susceptible G. hirsutum, CCRI36, which were subsequently subjected to biochemical tests and transcriptome sequencing during V991 infection (0, 1, and 2 days after inoculation). Eighteen root samples with three replications were collected to perform multiple comparisons of enzyme activity and biochemical substance contents. The findings indicated that VW resistance was positively correlated with peroxidase and polyphenol oxidase activity, but negatively correlated with malondialdehyde content. Additionally, RNA sequencing was used for the same root samples, resulting in a total of 77,412 genes, of which 23,180 differentially expressed genes were identified from multiple comparisons between samples. After Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the expression profiles identified using Short Time-series Expression Miner, we found that the metabolic process in the biological process, as well as the pathways of phenylpropanoid biosynthesis and plant hormone signal transduction, participated significantly in the response to VW. Gene functional annotation and expression quantity analysis indicated the important roles of the phenylpropanoid metabolic pathway and oxidation-reduction process in response to VW, which also provided plenty of candidate genes related to plant resistance. This study concentrates on the preliminary response to V991 infection by comparing the VW-resistant CSSL and its VW-susceptible recurrent parent. Not only do our findings facilitate the culturing of new resistant varieties with high yield and superior performance, but they also broaden our understanding of the mechanisms of cotton resistance to VW.

Polyphenol oxidases (PPOs) are copper-binding metalloproteinases encoded by nuclear genes, ubiquitously existing in the plastids of microorganisms, plants, and animals. As one of the important defense enzymes, PPOs have been reported to participate in the resistant processes that respond to diseases and insect pests in multiple plant species. However, PPO gene identification and characterization in cotton and their expression patterns under Verticillium wilt (VW) treatment have not been clearly studied. In this study, 7, 8, 14, and 16 PPO genes were separately identified from Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively, which were distributed within 23 chromosomes, though mainly gathered in chromosome 6. The phylogenetic tree manifested that all the PPOs from four cotton species and 14 other plants were divided into seven groups, and the analyses of the conserved motifs and nucleotide sequences showed highly similar characteristics of the gene structure and domains in the cotton PPO genes. The dramatically expressed differences were observed among the different organs at various stages of growth and development or under the diverse stresses referred to in the published RNA-seq data. Quantitative real-time PCR (qRT-PCR) experiments were also performed on the GhPPO genes in the roots, stems, and leaves of VW-resistant MBI8255 and VW-susceptible CCRI36 infected with Verticillium dahliae V991, proving the strong correlation between PPO activity and VW resistance. A comprehensive analysis conducted on cotton PPO genes contributes to the screening of the candidate genes for subsequent biological function studies, which is also of great significance for the in-depth understanding of the molecular genetic basis of cotton resistance to VW.

Oxidative Stress 3 (OXS3) encodes a plant-specific protein that makes great contributions to a plant’s stress tolerance. However, reports on genome-wide identification and expression pattern analyses of OXS3 were only found for Arabidopsis, wheat, and rice. The genus Gossypium (cotton) serves as an ideal model for studying allopolyploidy. Therefore, two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study for a bioinformatics analysis, resulting in 12, 12, 22, and 23 OXS3 members, respectively. A phylogenetic tree was constructed using 69 cotton OXS3 genes alongside 8 Arabidopsis, 10 rice, and 9 wheat genes, which were classified into three groups (Group 1–3). A consistent evolutionary relationship with the phylogenetic tree was observed in our structural analysis of the cotton OXS3 genes and the clustering of six conserved motifs. Gene duplication analysis across the four representative Gossypium species suggested that whole-genome duplication, segmental duplication, and tandem duplication might play significant roles in the expansion of the OXS3 gene family. Some existing elements responsive to salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) were identified by cis-regulatory element analysis in the promoter regions, which could influence the expression levels of cotton OXS3 genes. Furthermore, the expression patterns of the GhOXS3 gene were examined in different tissues or organs, as well as in developing ovules and fibers, with the highest expression observed in ovules. GhOXS3 genes exhibited a more pronounced regulatory response to abiotic stresses, of which ten GhOXS3 genes showed similar expression patterns under cold, heat, salt, and drought treatments. These observations were verified by quantitative real-time PCR experiments. These findings enhance our understanding of the evolutionary relationships and expression patterns of the OXS3 gene family and provide valuable insights for the identification of vital candidate genes for trait improvement in cotton breeding.

Cotton fiber strength is a critical property determining fiber qualities, and determined by the secondary cell wall development. Understanding the mechanism of fiber development will provide a way to improvement of fiber strength. In this study, the introgression lines of upland and sea island cotton, and have experience of four generations of backcross with upland parent, and have significant higher fiber strength than their upland parent, and the transcriptome were analyzed and compared between the introgression lines and their upland parent. There were 2201 differentially expressed genes (DEG) identified by comparing two introgression lines with their recurrent parent CCRI45, in different development stages from 15 days post-anthesis (DPA) to 28 DPA. The up-regulated genes regulated the polysaccharide metabolic process, single-organism localization, cell wall organization or biogenesis and so on. The down-regulated genes involved in the microtubule-based process, cellular response to stress, cell cycle process and so on. Further functional analysis revealed three significant functional genes, XLOC_036333 (mannosyl-oligosaccharide-alpha-mannosidase mns1), XLOC_029945 ( FLA8 ) and XLOC_075372 ( snakin-1 ), playing important roles in the regulation of cotton fiber strength. Our results provide important candidates genes and inspirations for the future investigation of the molecular mechanism of fiber quality formation, and improvement of cotton fiber quality in breeding.