Studies of the Molecular Markers for genus Paphiopedilum
Date Issued
2008
Date
2008
Author(s)
Hsu, Ya-Wen
Abstract
DNA molecular markers based on PCR are divided into two groups: one is to detect the whole genome variation and the other is to focus on target DNA sequence. In this research, Inter-simple sequence repeat (ISSR) was employed to study the molecular fingerprinting of Paphiopedilum genome and rDNA internal transcribed spacer (ITS) and chloroplast intergenic spacers (IGS) were selected to do phylogenetic analysis. According to the results of these different techniques, two processes were established for differentiation. ISSR technique was applied to detect the whole genome variation of 30 species in Paphiopedilum. Seven out of hundred primers from University of British Columbia (UBC) were selected. The unweighted pair-group method using arithmetic average (UPGMA) was employed to analyze the phylogenetic relationships based on ISSR data. The phylogenetic trees subdivide Paphiopedilum species into six groups. The result was consistent with the subgenus classification of Karasawa and Saito (1982), which include Parvisepalum, Brachypetalum, Paphiopedilum, Sigmatopetalum, Polyantha and Cochlopetalum. nother part of this study was to analyze the chloroplast intergenic spacers and rDNA ITS of genus Paphiopedilum such that the phylogenetic tree can be constructed. Cleaved Amplified Polymorphic Sequence (CAPS) was employed to study the polymorphism pattern of genus Paphiopedilum. The trnS-trnM IGS treated by TspRI could separate three subgenra: Parvisepalum, Brachypetalum and Paphiopedilum. The restriction pattern of rDNA ITS cleaved by SfaNI could separate sectionPardalopetalumand Barbata. There was a 140bp deletion in the sequence of trnR-trnN IGS of the section Paphiopedilum and Barbata. To further distinguish section Paphiopedilum and Barbata, specific primers were used to amplify trnR-trnN IGS. The results built a different system to differentiate genus Paphiopedilum. The variation regions of ITS and trnS-trnM IGS are selected for PCR amplification and the products were analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE can detect the small variation, even one nucleotide difference, in DNA sequence of the same length. Among five fragments: ITS-a, ITS-b, SM1, SM2 and SM3, ITS-a was found to exhibit a better resolution than others.
Subjects
Paphiopedilum
intergenic spacer
inter-simple sequence repeat
cleaved amplified polymorphic sequence
denaturing gradient gel electrophoresis
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