Assessment of the DNA Barcodes Characteristic of Phalaenopsis deliciosa based on matK, rbcL, and ITS

Indonesia has high biodiversity for plant species, including orchids with medicinal potential such as Phalaenopsis deliciosa. Generally, morphological characters, especially in flowers are used for orchids identification. However, when the plants are not in the flowering period, the identification becomes difficult. Therefore an alternative method, such as molecular identification (DNA barcoding) needs to be applied for the best solution. This research aims to identify and compare three markers (matK, rbcL, ITS) for their function as potential barcodes for Phalaenopsis deliciosa. This study was conducted by DNA amplification using three different markers set. The data were analyzed using Bioedit, BLAST, and ClustalX. The result found that the identity level of matK, rbcL, and ITS to other orchids species was 9998%, 98%, and 94-96%, respectively. Furthermore, matK and ITS showed high specificity for Phalaenopsis deliciosa, and are therefore recommended as the best molecular identification marker of genus Phalaenopsis.


INTRODUCTION
Indonesia is a country with high biodiversity. The government declared through PERMENHUT No. 35 of 2007(KLHK, 2007 that one of the potential biodiversity to be developed are ornamental plants, particularly orchids and those with medicinal properties (Hani et al., 2014;Wahyudiningsih & Nion, 2017;Utami & Hariyanto, 2019). Orchids belong to Orchidaceae family and has the most species-rich angiosperm when compared to other flowering plants group (Givnish et al., 2016;Fay, 2018;Tsiftsis et al., 2019). Furthermore, Indonesia has about 5000 species of orchids, about 1500 species have been identified (Schuiteman et al., 2010;Semiarti, 2012), and reports that some have the potential to be used as raw material for medicines (Hani et al., 2014;Teoh, 2016;Perwitasari et al., 2020). Some contain chemicals that are useful for therapeutic, such as glycerides, alkaloids, and others. When these plants are handled appropriately, they are useful as herbal medicines and have the potential to be used as a substitute for synthetic drugs. Herbal medicines are acceptably safe for health and have a dramatically fewer side effects (Taheri et al., 2011;Ekor, 2014;Moreira et al., 2014;Yao et al., 2016).
In general, morphological characteristics are used when identifying orchids and other plants. However, this approach sometimes meet obstacle since the vegetative characters of orchids are almost identical. Therefore, the existence of flowers as generative organs for identification is indispensable (Su et al., 2013;Feng et al., 2015;Dirks-Mulder et al., 2017). For precision and also to support the conventional approach, the molecular markers through DNA barcoding used short fragments to identify the specimens is an alternative method (Jinbo et al., 2011;Li et al., 2011;Kim et al., 2014). DNA barcoding use marker selection that distinguish specific differences between species (Takamiya et al., 2011;Saddhe & Kumar, 2018). The CBOL Plant Working Group (2009) suggests the use of two plastid genes, namely maturase-K (matK) and ribulose-1.5-bisphosphate carboxylase (rbcL) as the standard barcodes for plant DNA, and one potential nuclear ribosome DNA, namely the Internal Transcribed Spacer (ITS) region (Feng et al., 2015). Previously, the potential barcodes for Paphiopedilum and Thrixspermum Vol 8(2), December 2020 Biogenesis: Jurnal Ilmiah Biologi 139 orchids were investigated using in silico approaches . Therefore, this research aims to identify and compare three markers (matK, rbcL, ITS) for their function as potential barcodes for Phalaenopsis deliciosa. The core barcode for Phalaenopsis deliciosa thus can evaluate the tropical biodiversity especially in Indonesia.

MATERIALS AND METHODS
The samples were obtained from Kebon Agung orchids nursery located in Gebang district, Jember. They were morphologically identified using several identification books such as Orchids of Papua New Guinea (Millar, 1978), Lowland Orchids of Papua New Guinea (O'Byrne, 1994), Key to the genera of Orchidaceae of New Guinea (Schuiteman, 1995), and Flora Malesiana: Orchids of New Guinea (Schuiteman et al., 2010). For DNA extraction, the procedures were based on the standard protocol of genomic isolation kit (GeneAll Exgene TM Korea). Briefly, 0.1 g leaf sample was mixed with extraction buffer containing RNAse and crushed using mortar and pestle until homogenized. The mixture was centrifuged and the supernatant was recovered, which was then processed through filter column and dissolved with elution buffer in a total volume of 50 μL. The genomic DNA was then stored until used as a template in PCR amplification.
The PCR product was then processed for sequencing using Macrogen Service Korea. Subsequently, the data were analyzed using several bioinformatics software such as Bioedit, BLAST, and ClustalX. The phylogenetic tree was constructed using MEGA5.05 (Zhai et al., 2013).

RESULTS AND DISCUSSION
Based on morphological characteristics, Phalaenopsis deliciosa has shown several characters such as growing epiphyte with a very short stem, monopodial, oblong-lanceolate leaves with undulate margins, and the flowers consist of two lateral sepals, one dorsal sepal, two petals and one labellum (Fig. 1). Furthermore, the flowers bloom sequentially to all directions. These characters are consistent with previous research on the morphology of Phalaenopsis deliciosa (Teoh, 2016  In addition to the attractive flower character, Phalaenopsis deliciosa is used as a herbal ingredient, therefore, it is very important in orchid biodiversity (Ming, 2000;Huda et al., 2017). However, there are limited data related to genetic biodiversity for the DNA barcodes. As evidence, only matK is available in NCBI's GenBank for Phalaenopsis deliciosa (Hidayat et al., 2005). Furthermore, the samples were used to complete the existing data and its DNA barcodes were explored using three molecular markers. All primer sets for each marker was tested for amplification reaction through PCR. The results showed that the bands that appeared corresponded to the proposed sizes, which include ± 450 bp, ± 600 bp, and ± 500 bp for matK, rbcL, and ITS, respectively (Fig. 2).  These results are consistent with related research that the proposed matK used for barcoding is in the range of 450 bp (Khew & Chia, 2011;Azofeifa-Bolaños et al., 2017). Relevant results were also illustrated in the amplification of PCR band using the rbcL and ITS primer set (Fig. 2), which shows the appropriate range size and are also consistent with the previous reports (Costion et al., 2011;Martin et al., 2014).
The sequences from the three different primer sets were analyzed with BLAST for alignment, and MEGA5.05 for phylogenetic tree construction. Table 1 summarizes the BLAST results of matK sequence from the sample with the existing data in NCBI GenBank. The result showed that the matK sequence from samples have high similarity with those from Phalaenopsis deliciosa in China, USA, and Japan with an identity percentage greater than 99%. This indicates that matK is able to discriminate Phalaenopsis deliciosa down to the species level. The BLAST result is also in mutual agreement with the phylogenetic tree created (Fig. 3).   Fig. 3. Phylogenetic tree constructed from: a. matK sequences; b. rbcL sequences; c. ITS sequences of Phalaenopsis deliciosa by MEGA5.05 software Contrary to matK, the sequence of rbcL and ITS originating from Phalaenopsis deliciosa are not available in NCBI GenBank. Therefore, the sequences originating from this research are newly established. Table 2 and Table 3 illustrate the BLAST results of rbcL and ITS sequences, from Phalaenopsis deliciosa compared to several orchids species available in GenBank.   Fig. 3b and Fig. 3c. The molecular markers used are from two different loci, matK, and rbcL from plastid/chloroplast while ITS is derived from nuclei. This difference led to the strength and weakness of each marker. The study showed that the identity level of matK, rbcL, and ITS were 99, 98, and 94-96%, respectively. Although all markers demonstrated the ability to discriminate against orchid species, the level of identity/similarity derived from sequence alignment has also become a crucial consideration. Furthermore, one of the best benchmarks of molecular markers in DNA barcoding is the existence of sequence variability, which indicates a high specificity level for identification. Consequently, the matK and ITS showed prominent character to be the recommended molecular marker for the identification of Phalaenopsis deliciosa.

CONCLUSION
The matK and ITS markers showed high specificity for Phalaenopsis deliciosa. Therefore, they are recommended as the best marker for the molecular identification of genus Phalaenopsis.