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Background  The moth orchid ( Phalaenopsis  species) is an ornamental crop that is highly commercialized worldwide. Over 30,000 cultivars of moth orchids have been registered at the Royal Horticultural Society (RHS). These cultivars were obtained by artificial pollination of interspecific hybridization. Therefore, the identification of different cultivars is highly important in the worldwide market.    Methods/Results  We used Illumina sequencing technology to analyze an important species for breeding,  Phalaenopsis aphrodite  subsp.  formosana  and develop the expressed sequence tag (EST)-simple sequence repeat (SSR) markers. After  de novo  assembly, the obtained sequence covered 29.1 Mb, approximately 2.2% of the  P .  aphrodite  subsp.  formosana  genome (1,300 Mb), and a total of 1,439 EST-SSR loci were detected. SSR occurs in the exon region, including the 5’ untranslated region (UTR), coding region (CDS), and 3’UTR, on average every 20.22 kb. The di- and tri-nucleotide motifs (51.49% and 35.23%, respectively) were the two most frequent motifs in the  P .  aphrodite  subsp.  formosana . To validate the developed EST-SSR loci and to evaluate the transferability to the genus  Phalaenopsis , thirty tri-nucleotide motifs of the EST-SSR loci were randomly selected to design EST-SSR primers and to evaluate the polymorphism and transferability across 22 native  Phalaenopsis  species that are usually used as parents for moth orchid breeding. Of the 30 EST-SSR loci, ten polymorphic and transferable SSR loci across the 22 native taxa can be obtained. The validated EST-SSR markers were further proven to discriminate 12 closely related  Phalaenopsis  cultivars. The results show that it is not difficult to obtain universal SSR markers by transcriptome deep sequencing in  Phalaenopsis  species.    Conclusions  This study supported that transcriptome analysis based on deep sequencing is a powerful tool to develop SSR loci in non-model species. A large number of EST-SSR loci can be isolated, and about 33.33% EST-SSR loci are universal markers across the  Phalaenopsis  breeding germplasm after preliminary validation. The potential universal EST-SSR markers are highly valuable for identifying all of  Phalaenopsis  cultivars.

RNA-Seq SSRs of Moth Orchid and Screening for Molecular Markers across Genus Phalaenopsis (Orchidaceae)