Members of the NF-YB transcription factor gene family play important roles in diverse processes related to plant growth and development, such as seed development, drought tolerance, and flowering time. However, the function of NF-YB genes in cotton remains unclear. A total of 23, 24, and 50 NF-YB genes were identified in Gossypium arboreum (G. arboreum), Gossypium raimondii (G. raimondii), and G. hirsutum, respectively. A systematic phylogenetic analysis was carried out in G. arboretum, G. raimondii, G. hirsutum, Arabidopsis thaliana, cacao, rice and, sorghum, where the 150 NF-YB genes were divided into five groups (α–ε). Of these groups, α is the largest clade, and γ contains the LEC1 type NF-YB proteins. Syntenic analyses revealed that paralogues of NF-YB genes in G. hirsutum exhibited good collinearity. Owing to segmental duplication within the A sub-genome (At) and D sub-genome (Dt), there was an expanded set of NF-YB genes in G. hirsutum. Furthermore, we investigated the structures of exons, introns, and conserved motifs of NF-YB genes in upland cotton. Most of the NF-YB genes had only one exon, and the genes from the same clade exhibited a similar motif pattern. Expression data show that most NF-YB genes were expressed ubiquitously, and only a few genes were highly expressed in specific tissues, as confirmed by quantitative real-time PCR (qRT-PCR) analysis. The overexpression of GhDNF-YB22 gene, predominantly expressed in embryonic tissues, indicates that GhDNF-YB22 may affect embryogenesis in cotton. This study is the first comprehensive characterization of the GhNF-YB gene family in cotton, and showed that NF-YB genes could be divided into five clades. The duplication events that occurred over the course of evolution were the major impetus for NF-YB gene expansion in upland cotton. Collectively, this work provides insight into the evolution of NF-YB in cotton and further our knowledge of this commercially important species.
