Four DEF-like and two AGL6-like MADS-box genes display possible roles in perianth formation in Rhynchostylis retusa

Rhynchostylis retusa (L.) Blume is a popular and scarce orchid species with attractive flowers arranged in a racemose cluster. To study this flower’s complex floral perianth formation, MADS-box genes that play an important role in flower morphogenesis were investigated. Four B-class DEF-like MADS-box genes, RrDEF1, RrDEF2, RrDEF3, and RrDEF4, and two E-class AGL6-like MADS-box genes, RrAGL6-1 and RrAGL6-2, were identified, and their expression was characterized by qRT-PCR. These genes were expressed in every developmental stage of reproductive organs but not in the vegetative leaves, except for RrDEF2. Most of these genes revealed similar expression patterns in stage 3 and mature flowers. Higher RrDEF1 and RrAGL6-1 expression was detected in the sepals and petals rather than the lips, while RrDEF3, RrDEF4, and RrAGL6-2 were highly expressed in the lips but exhibited low expression in the sepals. RrDEF3 was also strongly expressed in petals. These gene expression patterns supported current profiles involving sepal/petal/lip determination, with RrDEF1 and RrAGL6-1 promoting sepal/petal formation, while RrDEF3, RrDEF4, and RrAGL6-2 promoted lip formation. Our findings support revised ‘orchid code’ and ‘perianth code’ hypotheses in which floral organs are regulated by combined levels of expression from each clade of DEF-like genes and AGL6-like genes. These four RrDEF and two RrAGL6 genes promote and enrich our knowledge and understanding concerning the floral development of orchids.