IDENTIFICATION OF ENDEMIC CURCUMA ALBIFLORA THW . BY DNA BARCODING METHOD

Harankaha is important medicinal plant, which is used in Sri Lankan traditional medicine. However, three plants are reported under the same vernacular name (e.g. Curcuma albiflora, Curcuma zedoaria, and Zingiber zerumbet) and therefore the raw material may be adulterated or substituted with each other. Phylogenetic analysis of gene sequences and combining with complete genomic sequences helps to identify genetic basis of plants. Standard CTAB method with little modifications was used for the extraction and purification. Extracted DNA was amplified using universal primers for matK genes in chloroplast genome rbcL gene in chloroplast genome by PCR (polymerase chain reaction). The rbcL primers amplified about 600 bp, while matK was about 850 bp. Amplified fragments were sequenced and obtained the DNA sequences for the matK and rbcL genes. Sequenced fragments were analyzed and used for DNA barcoding. DNA barcode was submitted to BOLD (Barcoding of Life Database) online database and then uploaded to the GenBank through the BOLD system (Accession No KF 521885). The distance of interspecific were 0.0010 in rbcL and 0.05 in matK, which is less than or equal to 0.05 matK sequences were selected for identification of C. albiflora, C. zedoaria and Z. zerumbet. C. albiflora appeared as a different group as per the Neighbor-Joining method and therefore, it can be identified as a new group. The matK gene of C. albiflora and other species showed, totally 212 variable sites. However, rbcL gene of C. albiflora and other species showed only 2 variable sites. Therefore, matK gene is suggested to identify C. albiflora from other two species claimed as Harankaha plants.


Introduction
Curcuma is a genus of family Zingiberaceae, many species are important in Sri Lankan traditional medicine.By plant morphology, five species of Curcuma available in Sri Lanka can be identified.But C. albiflora, C. zedoaria, and Z. zerumbet are named locally under same vernacular name Harankaha, which is likely to be adulterated for each other.These plants are used for inflammatory joint disorders, anti-venom for snake bites etc. Comparatively many researches have been conducted on genera Alpinia, combined data sets produce a highly resolved tree.Curcuma species of C. albiflora and C. oligantha, which are available in Sri Lanka, should be included in NCBI database for phylogenetic analysis.Phylogenetic analysis of the matK coding and noncoding regions is used to derive relationship among genera (Kress et al., 2002).DNA sequencing is one of reliable methods in biological identification which can be performed by many methods such as Maxam-Gilbert sequencing, Sanger's chain-termination methods and new sequencing methods including Lynx therapeutics massively parallel signature sequencing (MPSS), polony-sequencing, pyro-sequencing, solexa-sequencing, solid-sequencing, DNA nanoball sequencing etc.These techniques are applied for reliable identification of microbes, DNA barcode of plant species, and identification of new species of insects etc. (Kumar, 2012).Phylogenetic tree building is a process necessary for an understanding of broad patterns of biological diversity in plant species.MaturaseK (matK) gene of chloroplast was used in DNA barcoding of family Zingiberaceae (Selvaraj et al., 2008) and studies of angiosperm specie using ribulosebisphosphate carboxylase/oxygenace (rbcL) large subunit gene extracted from Ceratophyllum genus were reported (Chase et al., 1993;Chase et al., 2001).
Dry rhizomes of C. zedoaria, C. aromatica, and Z. zerumbet are hard to differentiate because they have very similar external appearance.Main objective of this study was to identify efficient and reliable method to differentiate these herbal materials.Morphological and microscopic identification were carried out in previous studies (Herath et al., 2016).Morphologically, C. albiflora can be differentiated by 35 cm plant height, green colour glabrous leaf, absence of coma and fertile bract spreading at lower one third of inflorescence from other two species of Harankaha.Microscopically, three sizes of simple starch grains, prismatic crystal available only in leaf of C. albiflora.However, the chemical compositions are varying for different geographical areas, harvesting season, way of storage etc (Yu et al., 2016).Due to stability of tissue macromolecule against above external factors, authentication by DNA barcode is a reliable method to identify similar plant raw materials for medicinal purposes.

Material and Methods
Matured plant samples of C. albiflora were collected from natural habitats at Kitulgala of Kegalle and Eratna of Rathnapura districts in the flowering season in 2015.Voucher specimens were authenticated from National Herbarium, Peradeniya.Standard hexadecyltrimethylammonium bromide (CTAB) method with little modification was used for the extraction and purification of DNA (Doyle, 1987;Dhanya et al., 2007).Preheated buffer (1 ml) containing 3% CTAB 100 μl of 10% PVP and 3 μl of β-Mercaptoethanol was added to plant material (100 mg) and ground.Proteinase K (10 μl) was also added and the suspension was incubated at 65 o C water bath for 30 min.Then 1/3 V of 5 M potassium acetate was added and incubated in ice for 1 h.Equal volume of Chloroform: Isoamyl (24:1) was added and mixed by inversion for 15 min, and centrifuged at 10,000 g for 15 min at 4 o C. The supernatant was transferred and equal volume of 30% PEG was added.The mixture was incubated on ice for 30 min, centrifuged at 12,000 g for 20 min at 4 o C.  (Kumar, 2015).Tajima test statistic (Tajima, 1989) was performed according to Nei et al. 1987.The phylogenetic tree was obtained using the Neighbor-Joining method (Saitou et al., 1987).The sum of branch length was computed using the maximum composite likelihood method (Tamura et al., 2004).The genetic distance was calculated based on Kimura-2-parameter model (Kimura, 1980).BioRad MyCycler thermal cycler was used for current study.

Results
DNA barcode was submitted to BOLD (Barcoding of Life Database) online database and then uploaded to the GenBank through the BOLD system.The interspecific distance is 0.0010 in rbcL and 0.05 in matK, which is less than or equal to 0.05.Multiple sequence alignment shows that, there are variable numbers of InDels in the gene matK.The alignment of matK gene of combined nucleotide sequence shows 212 variable sites, zero parsim-info sites, 212 singleton sides, and 571 conserved sites.Nucletide compositions of uncoded, 1 st , 2 nd , and 3 rd codon positions were reported in Table 1.Pairwise distances of matK gene analysis were mentioned in Table 2.There were a total of 777 positions in the final dataset.Phylogenetic tree of matK gene was shown in Figure 1.The optimal tree with the sum of branch length was 0.4738.Results of Tajima's Neutrality Test were mentioned in Table 6.The interspecies distance (dt) between C. albiflora and group 2 was 0.4857 and the intra-species distance (di) was 0.0043 on matK gene.Chen (2012) has reported that higher interspecific divergences and lower intraspecific variations are desirable for DNA barcode.In this study, di/dt was 112.9.Therefore, interspecific divergence is significantly large and suitable for DNA barcoding (Yu et al., 2016).Moreover, the neighbor-joining tree also showed that C. albiflora can cluster into new group.C. albiflora matK gene showed total of 859 nucleotide positions, but other two species showed less than 800 nucletide positions(C.zedoaria-783 positions and Z. zerumbet-777 positions).While, pairwise distances of matK between C. albiflora and other species was around 0.46, pairwise distance of rbcL was about 0.008.Cytosine nucleotide percentage of C. albiflora of matK was significantly higher (15.7%) at 3 rd codon position than other two species.However, rbcL was not showed significant difference of nucleotide composition.Therefore,

Figure 1 .
Figure 1.Evolutionary relationships on matK of C. albiflora, C. zedoaria, Z. zerumbet and their phenotypic appearance; A: C. albiflora, B: C. zedoaria, C: Z. zerumbet The resulting pellet was washed with 70% ethanol, DNA pellet was dissolved in 20 μl of PCR grade water and stored at -20 o C.

Table 6 .
Results of Tajima's Neutrality Test on rbcL