Molecular Identification of Cycas by Restriction Fragment Length Polymorphism (RFLP) and Random Amplified Polymorphic DNA (RAPD)

Abstract

                RAPD and RFLP were used to identify nineteen species of Cycas. Ten species of these Cycas namely C. chamaoensis, C. macrocarpa, C. pectinata, C. clivicola, C. pranburiensis, C. litoralis, C. tansachana, C. siamensis, C. nongnoochiae and C. simplicipinna are locally found in Thailand while the nine remaining species of C. seemannii, C. wadei, C. bougainvilleana, C. chevalieri, C. diannanensis, C. nathorstii, C. edentata, C. parvulus and C. micholitzii are from several countries around the world but collectively planted at Nong Nooch Tropical Botanical Garden.

                In the RAPD study, twenty random primers were screened to amplify the genomic DNA of nineteen species of Cycas. Only five primers, i.e., OPB-1, OPB-8, OPB-14, OPB-15 and OPB-17 of ten nucleotides long were found to give polymorphic DNA patterns. These eighty-seven bands of Cycas DNA at the size of 0.35 -2.5 kb could be used to indicate the differences of these Cycas. As for RFLP, three probes were synthesized from 5S rRNA gene, 5S rRNA repeat unit gene of C. clivicola and 18S rRNA gene of C. pranburiensis. The probes were hybridized with the genomic DNA of Cycas which had been digested with restriction enzymes BamHI, EcoRI and DraI. The phylogenetic trees were constructed based on their similarity index derived from DNA polymorphism of RAPD and RFLP separately. The RAPD data classified nineteen species of Cycas into two major groups which mostly corresponded to their geographic origins, i.e., one group of Thailand origin and another of other countries. However, the RFLP data gave a different set of grouping showing more to their morphological characteristics but less on their geographic origins.