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Background Domain of unknown function 668 (DUF668) may play a crucial role in the plant growth and developmental response to adverse stress. However, our knowledge of the function of the DUF668 gene family is limited. Results Our study was conducted based on the DUF668 gene family identified from cotton genome sequencing. Phylogenetic analysis showed that the DUF668 family genes can be classified into four subgroups in cotton. We identified 32 DUF668 genes, which are distributed on 17 chromosomes and most of them located in the nucleus of Gossypium hirsutum. Gene structure and motif analyses revealed that the members of the DUF668 gene family can be clustered in G. hirsutum into two broad groups, which are relatively evolutionarily conserved. Transcriptome data analysis showed that the GhDUF668 genes are differentially expressed in different tissues under various stresses (cold, heat, drought, salt, and Verticillium dahliae), and expression is generally increased in roots and stems. Promoter and expression analyses indicated that Gh_DUF668–05, Gh_DUF668–08, Gh_DUF668–11, Gh_DUF668–23 and Gh_DUF668–28 in G. hirsutum might have evolved resistance to adverse stress. Additionally, qRT-PCR revealed that these 5 genes in four cotton lines, KK1543 (drought resistant), Xinluzao 26 (drought sensitive), Zhongzhimian 2 (disease resistant) and Simian 3 (susceptible), under drought and Verticillium wilt stress were all significantly induced. Roots had the highest expression of these 5 genes before and after the treatment. Among them, the expression levels of Gh_DUF668–08 and Gh_DUF668–23 increased sharply at 6 h and reached a maximum at 12 h under biotic and abiotic stress, which showed that they might be involved in the process of adverse stress resistance in cotton. Conclusion The significant changes in GhDUF668 expression in the roots after adverse stress indicate that GhDUF668 is likely to increase plant resistance to stress. This study provides an important theoretical basis for further research on the function of the DUF668 gene family and the molecular mechanism of adverse stress resistance in cotton.

Background Serine carboxypeptidase-like protein (SCPL) plays an important role in response to stress in plant. However, our knowledge of the function of the SCPL gene family is limited. Results In this study, a comprehensive and systematic analysis of SCPL gene family was conducted to explore the phylogeny and evolution of the SCPL gene in Gossypium hirsutum. The phenotype and molecular mechanism of silencing of the Gh_SCPL42 under Verticillium wilt stress was also studied. Our results showed that 96 SCPL genes were observed in genome of G. hirsutum, which distributed on 25 chromosomes and most of them were located in the nucleus. The phylogenetic tree analysis showed that members of SCPL gene family can be divided into three subgroups in G. hirsutum, which are relatively conservative in evolution. SCPL gene has a wide range of tissue expression types in G. hirsutum. Promoter analysis showed that the most cis-acting elements related to MeJA and ABA were contained. Through RNA-seq combined with genotyping, it was found that 11 GhSCPL genes not only had significant expression changes during Verticillium wilt stress but also had differential SNPs in the upstream, downstream, exonic or intronic regions. The expression of these 11 genes in the resistant (Zhongzhimian 2) and susceptible (Junmian 1) materials was further analyzed by qRT-PCR, it was found that 6 genes showed significant expression differences in the two materials. Among them, Gh_SCPL42 has the most obvious expression change. Furthermore, virus-induced gene silencing (VIGS) showed necrosis and yellowing of leaves and significantly higher disease severity index (DSI) and disease severity rate (DSR) values in VIGS plants than in control silenced Gh_SCPL42 plants. Moreover, the expression levels of genes related to the SA and JA pathways were significantly downregulated. These results show that Gh_SCPL42 might improve resistance to Verticillium wilt through the SA and JA pathways in G. hirsutum. Conclusion In conclusion, our findings indicated that Gh_SCPL42 gene plays an important role in resistance to Verticillium wilt in cotton. It was provided an important theoretical basis for further research on the function of SCPL gene family and the molecular mechanism of resistance to Verticillium wilt in cotton.

Fuzzless Gossypium hirsutum mutants are ideal materials for investigating cotton fiber initiation and development. In this study, we used the fuzzless G. hirsutum mutant Xinluzao 50 FLM as the research material and combined it with other fuzzless materials for verification by RNA sequencing to explore the gene expression patterns and differences between genes in upland cotton during the fuzz period. A gene ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic process, microtubule binding, and other pathways. A weighted gene co-expression network analysis (WGCNA) showed that two modules of Xinluzao 50 and Xinluzao 50 FLM and four modules of CSS386 and Sicala V-2 were highly correlated with fuzz. We selected the hub gene with the highest KME value among the six modules and constructed an interaction network. In addition, we selected some genes with high KME values from the six modules that were highly associated with fuzz in the four materials and found 19 common differential genes produced by the four materials. These 19 genes are likely involved in the formation of fuzz in upland cotton. Several hub genes belong to the arabinogalactan protein and GDSL lipase, which play important roles in fiber development. According to the differences in expression level, 4 genes were selected from the 19 genes and tested for their expression level in some fuzzless materials. The modules, hub genes, and common genes identified in this study can provide new insights into the formation of fiber and fuzz, and provide a reference for molecular design breeding for the genetic improvement of cotton fiber.

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