Genetic variability and relationships among interspecific hybrid cultivars and parental species of Paphiopedilum via ribosomal DNA sequence analysis

Abstract

The aim of the present study was to investigate genetic variability and relationships among several hybrid cultivars and parental species of Paphiopedilum for the purposes of further breeding. Genetic information was obtained by means of phylogenetic analysis, using nuclear ribosomal DNA (rDNA) nucleotide sequence data. Nuclear rDNA was derived from internal transcribed spacer (ITS) regions, and the 5.8S regions were amplified by polymerase chain reaction. The resulting nucleotide sequences were examined and compared for genetic variability, and the relationships were analyzed by pair-wise multiple alignment. ITS region analysis revealed the following lengths of the ITS1, ITS2, and 5.8S regions: 382–428, 207–215, and 170 bp, respectively. Sequence identity at the ITS site, including the 5.8S region, ranged from 75.9 % between P. emersonii and P. sanderianum, to 99.4 % between Paphiopedilum ‘Hamana Wave’ and Paphiopedilum ‘Kolosand’. Next, we investigated whether each of the analyzed hybrid cultivars could be identified and differentiated at the interspecies or intraspecies levels. Our phylogenetic tree revealed that hybrids derived from the subgenus Brachypetalum were clearly differentiated from those derived from the subgenus Paphiopedilum. Moreover, interspecific hybrids showed unique phylogenetic positioning among parental species, according to the ITS sequence.