Development of molecular markers for the conservation of phalaenopsis gigantea, paphiopedilum rothschildianum and other endangered endemic orchids of Sabah, Malaysia

Paphiopedilum lowii are endangered orchids endemic to Sabah, Malaysia, which are currently accorded protection under the provisions of the CITES. The molecular characterization of these species, is the most significant step in the development of a conservation and management strategy for wild orchid populations. This constituted the basis for this investigation, which was directed towards elucidating the population genetic structure of the endangered orchids using microsatellite loci. Eighty three individuals representing three populations of P. rothschildianum derived from Bukit Ampuan, Melangkap and Telupid were sampled. DNA was extracted and authenticated by amplification of the ITS1 and ITS2 rDNA intergenic spacer region followed by sequencing and comparative analysis using the blastn program. Concurrently, nineteen individuals representing the extant population of P. gigantea were collected from the Tawau Hills Park, DNA was extracted and authenticated using the chloroplast DNA tmL-tmF intergenic region. One individual each from P. dayanum and P. lowii was sampled from Kinabalu National Park. A genomic library enriched for microsatellite lod from these four orchids was constructed using the 5' anchored PeR technique, followed by ligation of the PCR amplicons onto a TOPO TA pCR 2.1 plasmid, transformation of constructs. into competent TOP 10F' E. coli, screening for positive transformants, plasmid extraction, purification and DNA sequendng. A total of 95 sequences containing 212 microsatellite loci and cryptic simple repeats were isolated and deposited at the NCBI GenBank. Specific primer pairs were designed to amplify the microsatellite loci, and applied to characterize the population genetic structure. In P. gigantea, 30 polymorphic primer pairs defined 78 alleles. The averages of the observed and expected heterozygosity were 0.3544 to 0.4910. The Fsr value ranged from 0.1174 to 1.000 with an average of 0.6294, indicating a high level of genetic variability within the mixed population. In the case study of P. rothschildianum, 24 of the 30 primer pairs exhibited polymorphism. The averages of the observed and expected heterozygosity were 0.3800 and 0.4533 respectively. The mean FsT value was 0.5098 indicating genetic diversity. within the total population, however the mean F1s value was 0.8766, implying that there is a deficiency of level of heterozygosity. A test for cross amplification was conducted to determine the degree of genomic similarity within the genus Paphiopedilum indicated that very little homology exists between genomes of the three species examined. This is in accordance with karyotype analysis data, which indicates that the species In island ecosystems of. which one representative is Borneo, have evolved into distinct species as a result of speciation events involving reduction in chromosome numbers. The study has concluded that both the endangered species being investigated exhibit a reduced genetic diversity and warrant categorization as ESUs for the purpose of conservation and has recommended a strategy for the conservation and maintenance of current diversity levels by complying with the scientific breeding strategies that are delineated within the contents of this study. The focus of this investigation has been to develop a strategic approach for the conservation of the endangered endemic species of Sabah, Malaysia with the objective of developing a scientific and pragmatic approach for the conservation of a diverse range of species in addition to orchids. Developing a statutory framework for conservation involves the consolidation of legislative guidelines and scientific data. This comprehensive study will form the basis of future research investigations into the wide range of genetically diverse Malaysian endemic species.