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Background: Molecular identification based on microsatellite loci is an important technology to improve the commercial breeding of the moth orchid. There are more than 30,000 cultivars have been enrolled at the Royal Horticultural Society (RHS). In this study, genomic microsatellite primer sets were developed from Phalaenopsis aphrodite subsp. formosana to further examine the transferability of across 21 Phalaenopsis species. Methods and results: Twenty-eight polymorphic microsatellite markers were obtained using the magnetic bead enrichment method, with high transferability of the 21 species of the genus Phalaenopsis, especially in the subgenus Phalaenopsis. The 28 newly developed polymorphic microsatellite markers with high polymorphism information content values. The best and second fit grouping ( K) are inferred as two and four by the ΔK evaluation in the assignment test. This result indicates that these microsatellite markers are discernible to subgenus Phalaenopsis. Conclusions: Our results indicate that these new microsatellite markers are useful for delimiting species within genus Phalaenopsis. As expected, the genetic relationships between species of subgenus Phalaenopsis can be well distinguished based on the assignment test. These molecular markers could apply to assess the paternity of Phalaenopsis as well as investigating hybridization among species of genus Phalaenopsis. [ABSTRACT FROM AUTHOR]

Background: The intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status. Methods/Results: GISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype. Conclusions: Both GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]