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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.


Images of 22 Phalaenopsis species.Images (a)-(v) represent samples 1–22 shown in Table 5.
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pone.0141761.g005: Images of 22 Phalaenopsis species.Images (a)-(v) represent samples 1–22 shown in Table 5.

Mentions: Most EST-SSRs are either di- (51.49%) or tri-nucleotide motifs (35.23%). The average number of potential EST-SSRs per unigene is 0.064. Of the detected EST-SSRs, 1,051 EST-SSRs were obtained for suitable primer designation by BatchPrimer3 (Table 2). The information of EST-SSR primers in this study is shown in S1 Table. Of the 421 primer pairs for tri-nucleotide motifs, 30 EST-SSR loci were randomly selected to evaluate the polymorphism and transferability across 22 native Phalaenopsis species that are representative germplasms for breeding most Phalaenopsis cultivars. Of these, ten EST-SSR loci were stably amplified, polymorphic, and transferrable SSR loci across 22 native Phalaenopsis species (S1 Fig). In total, 70 amplifying bands were detected by 10 primer pairs across 22 native Phalaenopsis species, and the number of amplifying bands per primer pairs ranged from 3 to 16, with an average of 7. The polymorphism information content (PIC) value across 22 native Phalaenopsis species ranged from 0.163 to 0.889, with an average of 0.588 (Table 3). The amplified products derived from EST-SSR PCR across 22 native Phalaenopsis species are shown to be one or two bands for most of SSR loci, such as the loci Pap-3222 (Fig 3a) and Pap-4825 (Fig 3b). Genetic similarity between 22 native moth orchids was evaluated by principal coordinate analysis (PCoA), and the three-dimensional representation provided by the plot shows a certain degree of separation between different species (Fig 4a). The resolution of the first, second and third axes show 24.87%, 20.27%, and 16.76% of the variance, respectively. Compared to 22 native taxa (Fig 5) by 10 polymorphic EST-SSR loci, genetic compositions among different species are obviously scattered between taxa but can be grouped at the three axes on Sections Zebrinae, Phalaenopsis, Deliciosae, and Stauroglottis (Fig 4a).


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)

Images of 22 Phalaenopsis species.Images (a)-(v) represent samples 1–22 shown in Table 5.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141761.g005: Images of 22 Phalaenopsis species.Images (a)-(v) represent samples 1–22 shown in Table 5.
Mentions: Most EST-SSRs are either di- (51.49%) or tri-nucleotide motifs (35.23%). The average number of potential EST-SSRs per unigene is 0.064. Of the detected EST-SSRs, 1,051 EST-SSRs were obtained for suitable primer designation by BatchPrimer3 (Table 2). The information of EST-SSR primers in this study is shown in S1 Table. Of the 421 primer pairs for tri-nucleotide motifs, 30 EST-SSR loci were randomly selected to evaluate the polymorphism and transferability across 22 native Phalaenopsis species that are representative germplasms for breeding most Phalaenopsis cultivars. Of these, ten EST-SSR loci were stably amplified, polymorphic, and transferrable SSR loci across 22 native Phalaenopsis species (S1 Fig). In total, 70 amplifying bands were detected by 10 primer pairs across 22 native Phalaenopsis species, and the number of amplifying bands per primer pairs ranged from 3 to 16, with an average of 7. The polymorphism information content (PIC) value across 22 native Phalaenopsis species ranged from 0.163 to 0.889, with an average of 0.588 (Table 3). The amplified products derived from EST-SSR PCR across 22 native Phalaenopsis species are shown to be one or two bands for most of SSR loci, such as the loci Pap-3222 (Fig 3a) and Pap-4825 (Fig 3b). Genetic similarity between 22 native moth orchids was evaluated by principal coordinate analysis (PCoA), and the three-dimensional representation provided by the plot shows a certain degree of separation between different species (Fig 4a). The resolution of the first, second and third axes show 24.87%, 20.27%, and 16.76% of the variance, respectively. Compared to 22 native taxa (Fig 5) by 10 polymorphic EST-SSR loci, genetic compositions among different species are obviously scattered between taxa but can be grouped at the three axes on Sections Zebrinae, Phalaenopsis, Deliciosae, and Stauroglottis (Fig 4a).

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.