Spathoglottis is one of the most popular terrestrial orchids because it is attractive, easy to cultivate and continuously blooms throughout the year. It is a native orchid of Malaysia and listed as an endangered species. Therefore, this species needs to be conserved for commercial and environmental purposes. DNA barcoding is a technique for species identification, which uses a short DNA sequence from a standard and agreed-upon position in the genome. This technique plays an important role in biodiversity conservation for animals but is not yet well established in plants. Chloroplast DNA was chosen for this study aimed at building a DNA barcoding of Spathoglottis species. Seven species of Spathoglottis were used to carry out this study. They were Spathoglottis plicata, Spathoglottis gracilis, Spathoglottis aurea, Spathoglottis plicata alba, Spathoglottis unguiculata, Spathoglottis kimbaliana and a Spathoglottis hybrid. Eight regions (i.e., accD, matK, ndhJ, rpoB, rpoC1, ycf5, rbcL-a and trnH-psbA) in the chloroplast genome and two regions (i.e., ITS1 and ITS2) in the nuclear genome were selected and screened in order to define a universal barcoding region across all the seven selected species. The study found that the four chloroplast regions (i.e., matK, rbcL-a, rpoB and rpoC1) were successfully amplified from all the tested species. The DNA sequencing from each chloroplast region was compared among the species and analyzed to differentiate the intra-and inter-genetic variations. The analysis of chloroplast regions was done for single regions and as a combination of the four regions. The haplotypes for the multiple analyses showed sufficiently high resolution to enable differentiation between the selected Spathoglottis species. In conclusion, this study showed that the chloroplast DNA regions had high potential to be developed for DNA barcoding of Spathoglottis species. This is the first step towards the development of universal DNA barcoding technique for all native orchids in Malaysia.

Spathoglottis plicata is an endangered terrestrial orchid species that have experienced severe threats to its habitat as wild forest sites come under pressure from industrialisation and natural disasters. This orchid species chosen to evaluate their levels of genetic diversity and population genetic structure, which 25-30 accession collected in the different location with different geographical, altitude and habitat. Genomic DNA was extracted from six natural populations (n=172) in Peninsular Malaysia using eleven AFLP markers of EcoRI+3 bases/MseI+3 base primer combinations. Based on 279 polymorphic bands, a significant degree of genetic population differentiation was found, with a 78.5% variation within populations as measured by AMOVA, indicating a potential restricted gene flow. Two distinct clades generated from a UPGMA dendrogram were further investigated through a Bayesian analysis using STRUCTURE software, producing an estimated population structure at optimal value K=4. These results point to the presence of four genetic structures in the Spathoglottis plicata population. The Pahang and Terengganu population revealed a higher than average genetic variation (60.25%), indicating that there may be a robust structural division between the population samples and a possible hybridisation between the Northern (Kedah), Southern (Negeri Sembilan and Johor) and Central (Selangor) region populations. In sum, these results suggest that geographical distance is the primary factor contributing to differences among populations and the need for conservation measures to protect the Spathoglottis plicata species.

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