The orchid, the champagne of flowers, brings luxury, elegance, and novelty to nature. Cymbidium sinense is a symbol of gigantic floral variability on account of wavering shapes and sizes of floral organs, although marker–trait association (MTA) has not been studied for its floral traits. We evaluated markers associated with 14 floral traits of C. sinense through a genome-wide association study (GWAS) of 195 accessions. A total of 65 318 522 single-nucleotide polymorphisms (SNPs) and 3 906 176 insertion/deletion (InDel) events were identified through genotyping-by-sequencing. Among these, 4694 potential SNPs and 477 InDels were identified as MTAs at −log₁₀ P > 5. The genes related to these SNPs and InDels were largely associated with floral regulators, hormonal pathways, cell division, and metabolism, playing essential roles in tailoring floral morphology. Moreover, 20 candidate SNPs/InDels linked to 11 genes were verified, 8 of which were situated on exons, one was located in the 5′-UTR and two were positioned in introns. Here, the multitepal trait-related gene RABBIT EARS (RBE) was found to be the most crucial gene. We analyzed the role of CsRBE in the regulation of flower-related genes via efficient transient overexpression in C. sinense protoplasts, and found that the floral homeotic genes CsAP3 and CsPI, as well as organ boundary regulators, including CsCUC and CsTCP genes, were regulated by CsRBE. Thus, we obtained key gene loci for important ornamental traits of orchids using genome-wide association analysis of populations with natural variation. The findings of this study can do a great deal to expedite orchid breeding programs for shape variability.

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