SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS AND DUPLEX POLYMERASE CHAIN REACTION-BASED FOR THAI CALANTHE ORCHIDS AND INTERSPECIES HYBRIDS DETECTION

Calanthe orchids have too similar morphological characteristics to identify in species or variety level using either vegetative or reproductive parts including their hybrids. Molecular markers are the appropriate choice to perform the differences. Therefore, this research aimed to develop the sequence characterized amplified region (SCAR) [1] markers for species delimitation and test hybrids using multiplex polymerase chain reaction (PCR) technique. There were 18 species of orchids in Calanthe group which were studied, consisting of 3 genera: Calanthe (11 species), Phaius (4 species) and Cephalanteropsis (2 species). The 10 pairs of SCAR primers from sequence-related amplified polymorphism (SRAP) and 2 pairs of SCAR primers from random amplified polymorphic DNA (RAPD) were chosen. Based on simplex PCR, only 3 SRAP-SCAR markers were successful to amplify and specific to C. densiflora, C. masuca and P. flavus. The result showed all 3 combinations could be amplify and clarify the expected size with their mixed DNA after testing duplex PCR of 3 SRAP-SCAR markers combination. Therefore, 3 SRAP-SCAR markers were effective to identify Calanthe spp. and examined the varieties and hybrids in duplex reaction as well. Multiplex PCR for these 3 species should be developed.