Fluorescence in situ hybridization (FISH) is an efficient cytogenetic technology to study chromosome structure. Transposable element (TE) is an important component in eukaryotic genomes and can provide insights in the structure and evolution of eukaryotic genomes. A FISH probe derived from bacterial artificial chromosome (BAC) clone 299N22 generated striking signals on all 26 chromosomes of the cotton diploid A genome (AA, 2x=26) but very few on the diploid D genome (DD, 2x=26). All 26 chromosomes of the A sub genome (At) of tetraploid cotton (AADD, 2n=4x=52) also gave positive signals with this FISH probe, whereas very few signals were observed on the D sub genome (Dt). Sequencing and annotation of BAC clone 299N22, revealed a novel Ty3/gypsy transposon family, which was named as ‘CICR’. This family is a significant contributor to size expansion in the A (sub) genome but not in the D (sub) genome. Further FISH analysis with the LTR of CICR as a probe revealed that CICR is lineage-specific, since massive repeats were found in A and B genomic groups, but not in C–G genomic groups within the Gossypium genus. Molecular evolutionary analysis of CICR suggested that tetraploid cottons evolved after silence of the transposon family 1–1.5 million years ago (Mya). Furthermore, A genomes are more homologous with B genomes, and the C, E, F, and G genomes likely diverged from a common ancestor prior to 3.5–4 Mya, the time when CICR appeared. The genomic variation caused by the insertion of CICR in the A (sub) genome may have played an important role in the speciation of organisms with A genomes. The CICR family is highly repetitive in A and B genomes of Gossypium, but not amplified in the C–G genomes. The differential amount of CICR family in At and Dt will aid in partitioning sub genome sequences for chromosome assemblies during tetraploid genome sequencing and will act as a method for assessing the accuracy of tetraploid genomes by looking at the proportion of CICR elements in resulting pseudochromosome sequences. The timeline of the expansion of CICR family provides a new reference for cotton evolutionary analysis, while the impact on gene function caused by the insertion of CICR elements will be a target for further analysis of investigating phenotypic differences between A genome and D genome species.

The Dof (DNA-binding one zinc finger) transcription factor family is a representative of plant-specific classes of transcription factors. In this study, we performed a genome-wide screening and characterization of the Dof gene family within two tetraploid species Gossypium barbadense, Gossypium hirsutum, and two diploid species Gossypium arboreum, Gossypium raimondii. 115, 116, 55 and 56 Dof genes were identified respectively and all of the genes contain a sequence encoding the Dof DNA-binding domain. Those genes were unevenly distributed across 13/26 chromosomes of the cotton. Genome comparison revealed that segmental duplication may have played crucial roles in the expansion of the cotton Dof gene family, and tandem duplication also played a minor role. Analysis of RNA-Seq data indicated that cotton Dof gene expression levels varied across different tissues and in response to different abiotic stress. Overall, our results could provide valuable information for better understanding the evolution of cotton Dof genes, and lays a foundation for future investigation in cotton.

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.

Background: YABBY transcription factors play important roles in plant growth and development. Although this gene family has been characterized in many plant species, a comprehensive analysis in cotton remains unavailable. Methods: In this study, we investigated the YABBY gene family in cotton by integrating multiple bioinformatics methods. Results: YABBY genes were identified in the genomes of four cotton species (Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium raimondii) and these identified genes were further classified into six groups. Following this classification, the expansion of the cotton YABBY gene family was examined, and we found that the family exhibits diverse expansion patterns during evolution, with segmental duplication acting as the primary driving force. In contrast, the notably larger repertoire of YABBY genes in G. raimondii is likely attributable to transposon activity. Regarding their evolutionary trajectory, Ka/Ks analysis showed that the YABBY gene family has undergone purifying selection. Beyond evolution, expression and cis-element analyses further demonstrated that YABBY genes possess diverse functions. In addition, we identified YABBY genes involved in different developmental stages of cotton fibers. Conclusions: We clarify the function and evolution of the cotton YABBY gene family in this work, and these results will provide a critical resource for further research on YABBY genes.