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

Tsai CC, Shih HC, Wang HV, Lin YS, Chang CH, Chiang YC, Chou CH - PLoS ONE (2015)

Bottom Line: 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.The potential universal EST-SSR markers are highly valuable for identifying all of Phalaenopsis cultivars.

View Article: PubMed Central - PubMed

Affiliation: Kaohsiung District Agricultural Research and Extension Station, Pingtung 900, Taiwan; National Pingtung University of Science and Technology, Pingtung 912, Taiwan.

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.

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.

No MeSH data available.


The polymorphism of 12 Phalaenopsis varieties by SSR-PCR analysis.The polymorphism of 12 Phalaenopsis varieties at (a) Pap-3222 SSR, (b) Pap-4825 SSR, and (c) Pap-4282 SSR loci. Lanes 1–12 represent 12 Phalaenopsis varieties/lines listed in Table 4. Lanes 1–4 represent four similar commercialized cultivars with white floral color; Lanes 5–8 represent four similar commercialized cultivars with yellow floral color; Lanes 9–12 represent four similar commercialized cultivars with red floral color.
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pone.0141761.g006: The polymorphism of 12 Phalaenopsis varieties by SSR-PCR analysis.The polymorphism of 12 Phalaenopsis varieties at (a) Pap-3222 SSR, (b) Pap-4825 SSR, and (c) Pap-4282 SSR loci. Lanes 1–12 represent 12 Phalaenopsis varieties/lines listed in Table 4. Lanes 1–4 represent four similar commercialized cultivars with white floral color; Lanes 5–8 represent four similar commercialized cultivars with yellow floral color; Lanes 9–12 represent four similar commercialized cultivars with red floral color.

Mentions: The EST-SSRs studied were further used to identify 12 commercialized Phalaenopsis cultivars, including white, red and yellow floral color groups (Table 4). The morphological characters of the same floral color of plant materials studied are very similar. It is not easy to identify them based on either vegetative or reproductive characters (such as floral color, size, and morphology). Three validated polymorphic and transferable primer pairs (i.e., SSR loci Pap-3222, Pap-4825, and Pap-4282) for EST-SSR were selected to discriminate 12 commercialized Phalaenopsis cultivars. According to the amplified PCR products, more than two bands can be found within an individual (Fig 6). In white floral color group (Fig 7a–7d), each of cultivars can be identified according to both SSR loci Pap-3222 and Pap-4282 (Fig 6a). In red floral color group (Fig 7e–7h), each of cultivars can be identified by using SSR locus Pap-4825 (Fig 6b). In yellow floral color group (Fig 7i–7l), each of cultivars can be identified by using SSR locus Pap-3222 (Fig 6c). Using the aforementioned three EST-SSR markers, each of the 12 commercialized Phalaenopsis cultivars can be discriminated.


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

Tsai CC, Shih HC, Wang HV, Lin YS, Chang CH, Chiang YC, Chou CH - PLoS ONE (2015)

The polymorphism of 12 Phalaenopsis varieties by SSR-PCR analysis.The polymorphism of 12 Phalaenopsis varieties at (a) Pap-3222 SSR, (b) Pap-4825 SSR, and (c) Pap-4282 SSR loci. Lanes 1–12 represent 12 Phalaenopsis varieties/lines listed in Table 4. Lanes 1–4 represent four similar commercialized cultivars with white floral color; Lanes 5–8 represent four similar commercialized cultivars with yellow floral color; Lanes 9–12 represent four similar commercialized cultivars with red floral color.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4629892&req=5

pone.0141761.g006: The polymorphism of 12 Phalaenopsis varieties by SSR-PCR analysis.The polymorphism of 12 Phalaenopsis varieties at (a) Pap-3222 SSR, (b) Pap-4825 SSR, and (c) Pap-4282 SSR loci. Lanes 1–12 represent 12 Phalaenopsis varieties/lines listed in Table 4. Lanes 1–4 represent four similar commercialized cultivars with white floral color; Lanes 5–8 represent four similar commercialized cultivars with yellow floral color; Lanes 9–12 represent four similar commercialized cultivars with red floral color.
Mentions: The EST-SSRs studied were further used to identify 12 commercialized Phalaenopsis cultivars, including white, red and yellow floral color groups (Table 4). The morphological characters of the same floral color of plant materials studied are very similar. It is not easy to identify them based on either vegetative or reproductive characters (such as floral color, size, and morphology). Three validated polymorphic and transferable primer pairs (i.e., SSR loci Pap-3222, Pap-4825, and Pap-4282) for EST-SSR were selected to discriminate 12 commercialized Phalaenopsis cultivars. According to the amplified PCR products, more than two bands can be found within an individual (Fig 6). In white floral color group (Fig 7a–7d), each of cultivars can be identified according to both SSR loci Pap-3222 and Pap-4282 (Fig 6a). In red floral color group (Fig 7e–7h), each of cultivars can be identified by using SSR locus Pap-4825 (Fig 6b). In yellow floral color group (Fig 7i–7l), each of cultivars can be identified by using SSR locus Pap-3222 (Fig 6c). Using the aforementioned three EST-SSR markers, each of the 12 commercialized Phalaenopsis cultivars can be discriminated.

Bottom Line: 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.The potential universal EST-SSR markers are highly valuable for identifying all of Phalaenopsis cultivars.

View Article: PubMed Central - PubMed

Affiliation: Kaohsiung District Agricultural Research and Extension Station, Pingtung 900, Taiwan; National Pingtung University of Science and Technology, Pingtung 912, Taiwan.

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.

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.

No MeSH data available.