title: RNA-Seq SSRs of Moth Orchid and Screening for Molecular Markers across Genus Phalaenopsis (Orchidaceae).
abstract: 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.
  
  
  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.
authors.affiliation: Kaohsiung District Agricultural Research and Extension Station, Pingtung 900, Taiwan; National Pingtung University of Science and Technology, Pingtung 912, Taiwan.
authors.forename: Chi-Chu
authors.initials: CC
authors.lastname: Tsai
authors.affiliation: Department of Nursing, Meiho University, Pingtung 912, Taiwan.
authors.forename: Huei-Chuan
authors.initials: HC
authors.lastname: Shih
authors.affiliation: Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Hao-Ven
authors.initials: HV
authors.lastname: Wang
authors.affiliation: Kaohsiung District Agricultural Research and Extension Station, Pingtung 900, Taiwan.
authors.forename: Yu-Shium
authors.initials: YS
authors.lastname: Lin
authors.affiliation: Kaohsiung District Agricultural Research and Extension Station, Pingtung 900, Taiwan.
authors.forename: Chia-Hung
authors.initials: CH
authors.lastname: Chang
authors.affiliation: Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan; Department of Biomedical Science and Environment Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
authors.forename: Yu-Chung
authors.initials: YC
authors.lastname: Chiang
authors.affiliation: Research Center for Biodiversity, China Medical University, Taichung 404, Taiwan.
authors.forename: Chang-Hung
authors.initials: CH
authors.lastname: Chou
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D018895:Microsatellite Repeats; D029595:Orchidaceae / Q000145:classification / Q000235:genetics*; D000069600:Plant Breeding; D018749:RNA, Plant / Q000032:analysis; D017423:Sequence Analysis, RNA / Q000379:methods*; D013045:Species Specificity
chemical_list: D005819:Genetic Markers; D018749:RNA, Plant