DNA fingerprinting and genetic diversity analysis of 79 early-maturing upland cotton (Gossypium hirsutum L.) cultivars were performed using Simple Sequence Repeat (SSR) molecular markers. Out of 126 pairs of SSR primers, we selected 71 pairs that gave good polymorphisms and clear bands, had good stability, and showed even distribution on the cotton chromosomes, and 142 polymorphic genotypes were amplified. The average number of alleles amplified with the SSR primers was 2.01. The polymorphism information content (PIC) of the markers ranged from 0.1841 to 0.9043, with an average of 0.6494. The results of fingerprint analysis showed that nine varieties had characteristic bands, and at least six primer pairs could be used to completely distinguish all 79 cotton accessions. Using NTSYS-pc 2.11 cluster analysis, the genetic similarity coefficients between the cotton genotypes ranged from 0.3310 to 0.8705, with an average of 0.5861. All cotton accessions were grouped into five categories at a similarity coefficient of 0.57, which was consistent with the pedigree sources. At the same time, the average genetic similarity coefficients of early-maturing upland cotton varieties in China showed a low-high-low pattern of variation over time, revealing the development history of early-maturing upland cotton varieties from the 1980s to the present. This also indirectly reflects that in recent years, China’s cotton breeders have focused on innovation and have continuously broadened the genetic resources for early-maturing upland cotton.

Background/Objectives: The terrestrialization of plants has been a significant driver of plant evolution over hundreds of millions of years, and epidermal development plays a crucial role in adapting to terrestrial environments. It is therefore of great interest that the HD-Zip IV subfamily serves as a key regulator of epidermal and cuticle development in plants. However, research on their expansion trajectory, adaptive evolution, and functional divergence remains scarce. Methods: We conducted a functional divergence and adaptive evolution analysis in the plant HD-Zip IV subfamily and confirmed the functional differentiation caused by positive selection site mutations through binding affinity prediction analysis and EMSA experiments. Results: Our findings revealed that the HD-Zip IV subfamily has diversified into five distinct branches, with progressive expansion throughout plant evolution. These variations in the HD (homeodomain) and START drive the functional differentiation among the evolutionary branch, in particular, a distinct leucine-rich motif in HD and lipid-binding pockets. Furthermore, we identified several amino acid sites within the START domain that have been under selective pressure during plant evolution, as well as convergent evolutionary sites shared between early land plants and seed plants. Conclusions: Our findings show that the HD-Zip IV subfamily experienced a significant expansion in gene number and diversification of evolutionary branch from streptophyte algae to seed plants. During plant evolution, genomic duplication events and variations in the HD and START have contributed to its expansion in gene number and diversification, respectively. We suggest that the balanced coexistence of functional robustness and relaxed constraints in the HD-Zip IV subfamily may have underpinned its successful response to the challenges of land environment.