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.