Molecular Identification of Vietnam Paphiopedilum Orchids Using DNA Sequences

Abstract

Paphiopedilum is a valuable genus in Vietnam. The dissertation constructed DNA sequences for genetic characterization and identification of Paphiopedilum species in Vietnam and to determine certain markers for detecting correct orchid origin and genetic diversity. The sequence analyses mainly based on the tree-based method. Species are identified when all sequences of the same species are clustered in the same branch on phylogenetic trees. Nucleotide polymorphism characteristics including variation, parsimony, singleton, mono-indel, insertion, deletion parameters were also recorded for sequence analyses. First, for screening potential sequences, 5 loci ITS, matK, rpoB, rpoC1 and trnH-psbA were sequenced and analyzed on 8 Paphiopedilum. Simultaneously, 37 loci of Paphiopedilum from GenBank were also examined for identification capability. Besides, proper primers for those 5 potential loci, i.e. ACO, LFY, atpB-rbcL, trnL and matK were developed and tested on fresh samples. Finally, the ITS in the nuclear genome and the two loci matK, trnL in chloroplast were selected for large scale applications. Second, the identification of Paphiopedilum population in Vietnam were conducted on a total of 95 samples belonging to 22 species including 20 original species and 2 natural hybrids of Vietnam Paphiopedilum population using 3 short sequences ITS, matK and trnL. The result was that 17 out of 22 species of Vietnam Paphiopedilum species were well identified. Furthermore, the combination of preliminary vegetative classification with later molecular recognition step resulted in the successful identification of up to 20 Paphiopedilum species in the study. Third, for further comparison of species identification capability between whole chloroplast genome and short sequences, the complete chloroplast genome of thexv species Paphiopedilum delenatii was sequenced, assembled, and analyzed with other Orchidaceae species. The results showed that the complete genome could also be used as potential identification sequence, especially for closely related species. All data from the study was upload to GenBank library for extensive research. The endemic species Paphiopedilum x dalatense of Vietnam was first reported. The results were also given back to scientific Institutes which contribute to the conservation and control of the illegal trade of Paphiopedilum species in Vietnam.