Pan-Genome Analysis of the Tubulin Gene Family Reveals Candidates for Fiber Strength in Gossypium barbadense

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.