Technical Report on the Molecular Phylogeny of Camellia with nrITS: The Need for High Quality DNA and PCR Amplification with Pfu-DNA Polymerase

Internal transcribed spacer (ITS) of nrDNA has been widely employed for reconstructing phylogenetic relationships in plants, especially at the species level. Previous attempts to reconstruct the molecular phylogeny of Camellia based on nrITS, however, have not succeeded due to technical difficulties. In order to identify the major factors responsible for these difficulties and also to assess the efficacy of nrITS in elucidating the interspecific relationships of Camellia, the present investigation was conducted with seven closely or distantly related species. The purity of the DNA was found to be one of the major factors affecting the success of PCR amplification and the errors in the sequences. Therefore, an efficient protocol has been developed for extracting genomic DNA from dried leaf samples of Camellia. The purity of the DNA, extracted using this method, was quite good as revealed by the A260/A280 ratio, which ranged from 1.84 to 1.89. Further investigation on the effect of DNA polymerases on PCR induced variations revealed that the PCR error rate was much higher in Taq-amplified sequences than Pfu-amplified sequences. The effect of the error on phylogenetic analysis was evident from the wide dispersal of Taq-amplified sequences across the gene tree while the Pfu-amplified sequences from the same sample joined together to form a single clade. Our extensive study of Camellia based on Pfu-amplified ITS sequences showed well-resolved interspecies relationships. Since the results of the molecular phylogenetic investigation of Camellia needs to be reported in a series, due to the technical and scientific complexity of the work, in this first report, we provide technical and scientific insights into the major factors responsible for the failure of the PCR amplification, the occurrence of high sequencing errors, and their effect on the phylogenetic interpretations. The results further stress the potential of nrITS in deducing the phylogenetic relationships in Camellia.