Development of gene-based identification markers for Phalaenopsis ‘KS Little Gem’ based on comparative genome analysis

Gene-based markers are useful tools for genetic background surveys and comparative genome analysis due to their ease of application across closely related species. We developed gene-based DNA markers for Phalaenopsis ‘KS Little Gem’, a new Phalaenopsis variety with excellent ornamental traits, based on a genome-wide comparison of chloroplast and nuclear sequences with those of orchids as well as monocot species, including rice and banana, to establish a molecular basis for species identification. Sequence comparisons of chloroplast DNA from P. ‘KS Little Gem’ with those of P. aphrodite and P. equestris identified six variable genic regions of the chloroplast genome in which an intron of rpl16 was the most polymorphic between related orchid species. To develop conserved ortholog set (COS) markers, we compared transcriptome unigenes of P. ‘KS Little Gem’ with the P. equestris, rice, and banana genomes and identified 582 Phalaenopsis COS candidates with at least two exons. PCR application using primer sets targeting six variable regions of the chloroplast genome and introns of 45 randomly selected COS candidate genes showed 92-98% amplification in three Phalaenopsis species. Among the candidate genes, we developed rpl16 and three COS genes as diagnostic cross-species markers for P. ‘KS Little Gem’ and diverse, related orchids. The gene-based molecular markers developed in this study will play an important role in species identification to facilitate the protection of P. ‘KS Little Gem’ variety rights, as well as breeding and genetic studies of Phalaenopsis orchids.