The Orchidaceae family is a massive plant family with exceptional adaptations that could have ensured its evolutionary success. Flavonoids are a class of secondary metabolites that help plants respond to changing climates. Chalcone synthase (CHS) plays a vital role in accumulating flavonoid compounds in the plant; it involves the first step in the biosynthesis pathway. Nevertheless, the CHS gene family in Erycina pusilla has not been isolated and characterized yet. The study aimed to investigate the structure and essential residues of EpCHS and the evolution of the chalcone synthase gene family in E. pusilla and relative orchids. The complete CHS genes were determined based on the transcriptomic data by PCR technique. The sequences of three EpCHS were constructed; these sequences were used for analyzing the structure, amino acid sequence and evolution. Three EpCHS genes were identified with a length range from 1250 bp to 1500 bp. Among the CHS family in E. pusilla, EpCHS1 is the lowest identity and lacking 30 amino acids may affect the functions. Analysis of the essential residues in three EpCHS indicates that EpCHS2 and EpCHS3 are conserved during the evolution process, and deletion mutation in EpCHS1 may affect the functions of the protein. Moreover, the phylogenetic tree shows the CHS family in the orchid were classified into two clades, the EpCHS in Clade 2 duplicated before E. pusilla divergent from Oncidium sp. During the evolution period, some mutations were occurred lead to change in sequences in individual genes and affect the function. The results contributed to studying the CHS gene family's regulation, function, and evolution in the orchid.

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