Sequence analysis of matK gene of Pueraria Candollei and Butea Superba and the application of PCR-RFLP genetic marker for identification

Abstract

The accurate identification and quality control of plant material is, therefore, an essential prerequisite for ensuring the quality, safety, and efficacy of herbal medicines. The purpose of this study was to examine the matK gene sequences of White Kwao Khruea [(Pueraria candollei Graham ex Benth. var. mirifica (Airy Shaw et Suvatabandhu) Niyomdham and Pueraria candollei Graham ex Benth. var. candollei)] and red Kwao Khruea (Butea superba Roxb.) and used the results for their classification and phylogenetic studies, as well as developed PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method in order to use as a convenient tool for identification. The complete matK gene of White Kwao Khruea was 1,521 bp in length, whereas that in Red Kwao Khruea was found to be 1,527 bp due to a 6-bp indels (insertions or deletions). Five sites of nucleotide substitutions were detected in 10 specimens of White Kwao Khruea. A total of 83 sites of substitutions were observed in 14 specimens of Both Kwao Khruea. Their phylogenetic analysis using parsimony analysis showed that the specimens of White Kwao Khruea were divided into two clades and were separated from Red Kwao Khruea. Based on the differences among the sequences, the PCR-RFLP analysis was performed. The restriction patterns of DNA amplified from partial matK gene with two restriction enzymes, EcoRI and DdeI showed distinct and polymorphic fingerprints between White Kwao Khruea and Red Kwao Khruea. These results suggest that the matK gene sequences of White Kwao Khruea and Red Kwao Khruea can be used to study of phylogenetic relationships. Moreover, PCR-RFLP genetic markers developed here can be used as a convenient tool for identification of both types of Kwao Khruea and can be applied to their commercial products.