Yajie Duan

Background/Objectives: Tubulins (Tub) are central components of microtubules, but intraspecific variation and developmental expression of the Tub family in Gossypium barbadense remain poorly characterized. This study aimed to characterize the GbTub family using a pan-genome framework and identify candidates associated with fiber development and strength. Methods: A total of 50 GbTub genes were identified in the G. barbadense 3-79 reference genome, and their orthologous presence–absence patterns were subsequently assessed across 12 additional G. barbadense accessions. Phylogenetic, presence–absence variation (PAV), Ka/Ks, structural variation (SV), RNA-seq, RT-qPCR, co-expression, and GO enrichment analyses were integrated. Results: Among the 50 reference-defined GbTub genes, 43 were classified as core genes, 6 as near-core genes, and 1 as an accessory gene, and the encoded proteins were classified into α-, β-, and γ-tubulin clades. All genes showed Ka/Ks < 1. Twenty-three GbTub genes differed between the fiber-strength-contrasting accessions 5917 and PimaS-7, and representative expression trends were supported by RT-qPCR. Network analysis prioritized 10 GbTub candidates based on degree centrality. GbTub21 was the sole SV-associated GbTub gene displaying significant differential expression between accessions harboring versus lacking the corresponding SV. Non-Tub neighbors of the candidate hub genes were enriched for cytoskeletal, intracellular-transport, and plasma-membrane functions. Conclusions: The pan-genome analysis reveals strong conservation with limited intraspecific variation in the GbTub family. Co-expression profiles nominate candidates associated with fiber secondary-wall development, and their causal contribution to fiber strength awaits functional dissection.

Background/Objectives: Among cotton species, Gossypium barbadense produces the strongest fibers. Examining cytoskeletal dynamics in single epidermal cells of G. barbadense ovules offers a direct approach to investigating fiber quality. Microtubules are major cytoskeletal components whose organization and dynamics are precisely regulated by microtubule-associated proteins (MAPs). However, information on the TPX2 family remains limited, and characterizing its features in G. barbadense is critical to clarifying the role of TPX2 family members in fiber strength formation. Methods: Using the Arabidopsis thaliana TPX2 sequence as a reference, 40, 49, 26, and 26 TPX2 family members were identified in the genomes of G. barbadense, Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii, respectively. We further analyzed the expression pattern of GbTPX2-35 and validated its function via virus-induced gene silencing (VIGS). Results: In G. barbadense, GbTPX2-35 (Gbar_D11G59825.1) was significantly upregulated in fiber samples of the parental lines at 25 days post-anthesis, and this expression pattern was further validated in G. barbadense lines with extreme fiber strength phenotypes. Next, VIGS-mediated silencing of GbTPX2-35 downregulated the transcript levels of cellulose synthase and microtubule-related protein genes, a finding further validated by mature fiber strength phenotypic data. Conclusions: This study preliminarily validated a pathway in which GbTPX2-35 regulates fiber strength by coordinating cellulose biosynthesis with microtubule cytoskeleton dynamics, providing valuable candidate genes and theoretical support for molecular breeding of high-strength cotton fibers.

Cyclophilins (CYPs) are a member of the immunophilin superfamily (in addition to FKBPs and parvulins) and play a significant role in peptidyl-prolyl cis-trans isomerase (PPIase) activity. Previous studies have shown that CYPs have important functions in plants, but no genome-wide analysis of the cotton CYP gene family has been reported, and the specific biological function of this gene is still elusive. Based on the release of the cotton genome sequence, we identified 75, 78, 40 and 38 CYP gene sequences from G. barbadense, G. hirsutum, G. arboreum, and G. raimondii, respectively; 221 CYP genes were unequally located on chromosomes. Phylogenetic analysis showed that 231 CYP genes clustered into three major groups and eight subgroups. Collinearity analysis showed that segmental duplications played a significant role in the expansion of CYP members in cotton. There were light-responsiveness, abiotic-stress and hormone-response elements upstream of most of the CYPs. In addition, the motif composition analysis revealed that 49 cyclophilin proteins had extra domains, including TPR (tetratricopeptide repeat), coiled coil, U-box, RRM (RNA recognition motif), WD40 (RNA recognition motif) and zinc finger domains, along with the cyclophilin-like domain (CLD). The expression patterns based on qRT-PCR showed that six CYP expression levels showed greater differences between Xinhai21 (long fibres, G. barbadense) and Ashmon (short fibres, G. barbadense) at 10 and 20 days postanthesis (DPA). These results signified that CYP genes are involved in the elongation stage of cotton fibre development. This study provides a valuable resource for further investigations of CYP gene functions and molecular mechanisms in cotton.