The CONSTANS (CO)-like gene family has been well studied for its role in the regulation of plant flowering time. However, their role remains poorly understood in cotton. To better understand the possible roles of CO-like in cotton, we performed a comprehensive genome-wide analysis of CO-like genes in cotton. Phylogenetic tree analysis showed that CO-like genes naturally clustered into three groups. Segmental duplication and whole genome duplication (WGD), which occurred before polyploidy, were important contributors to its expansion within the At (“t” indicates tetraploid) and Dt subgenomes, particularly in Group III. Long-terminal repeat retroelements were identified as the main transposable elements accompanying 18 genes. The genotype of GhCOL12_Dt displayed low diversity; it was a candidate involved in domestication. Selection pressure analyses indicated that relaxed purifying selection might have provided the main impetus during the evolution of CO-like genes in upland cotton. In addition, the high expression in the torus and calycle indicated that CO-like genes might affect flowering. The genes from Group II, and those from Group III involved in segmental duplication or WGD, might play important roles in response to drought and salt stress. Overall, this comprehensive genome-wide study of the CO-like gene family would facilitate further detailed studies in cotton.

EPSPS is a key gene in the shikimic acid synthesis pathway that has been widely used in breeding crops with herbicide resistance. However, its role in regulating cell elongation is poorly understood. Through the overexpression of EPSPS genes, we generated lines resistant to glyphosate that exhibit an unexpected dwarf phenotype. A representative line, DHR1, exhibits a stable dwarf phenotype throughout its entire growth period. Except for plant height, the other agronomic traits of DHR1 are similar to its transgenic explants ZM24. Paraffin section observations showed that DHR1 internodes are shortened due to reduced elongation and division of the internode cells. Exogenous hormones confirmed that DHR1 is not a classical brassinolide (BR)- or gibberellin (GA)-related dwarfing mutant. Hybridization analysis and fine mapping confirmed that the EPSPS gene is the causal gene for dwarfism, and the phenotype can be inherited in different genotypes. Transcriptome and metabolome analyses showed that genes associated with the phenylpropanoid synthesis pathway are enriched in DHR1 compared with ZM24. Flavonoid metabolites are enriched in DHR1, whereas lignin metabolites are reduced. The enhancement of flavonoids likely results in differential expression of auxin signal pathway genes and alters the auxin response, subsequently affecting cell elongation. This study provides a new strategy for generating dwarfs and will accelerate advancements in light simplification in the cultivation and mechanized harvesting of cotton.