Genetic polymorphism of Japanese cultivated Rheum species in the internal transcribed spacer region of nuclear ribosomal DNA

In order to develop new domestic production of Rhei Rhizoma (RR) from Rheum specimens cultivated in the Sugadaira Medicinal Plant Cultivation Test Field (SMPCF), the ITS sequences of 12 SMPCF specimens and Chinese Rheum specimens of four species, as well as RR samples produced in North Korea, China and Japan, were determined by subcloning and their sequences were compared. As the ITS sequences of 10 SMPCF specimens showed significant intra-individual polymorphism, identification of pseudogenes was conducted by detecting the three motifs of the 5.8S sequence and the stability of the 5.8S secondary structure. Approximately 46% of sequences obtained from the SMPCF specimens were putative pseudogenes. The maximum likelihood tree based on ITS sequences showed three main groups—the outer group and inner clusters I and II; clones from 10 SMPCF specimens including putative pseudogenes belonged to the outer group. Cluster I was composed of two clades, one including clones from R. officinale specimens and R. palmatum-derived samples with matK genotype Rp9, and another including clones from R. coreanum-derived samples. Cluster II consisted of three clades, one including clones from R. palmatum specimens with genotype Rp5, another including clones mainly from R. tanguticum specimens with genotype Rt4, and the third including clones from R. palmatum or R. tanguticum specimens with various matK genotypes. Clones from SMPCF specimen RC5 showed a close relationship with those from R. tanguticum specimens with matK genotype Rt4, whereas those from specimen RC9 related to R. coreanum-derived samples. As a result, specimens RC5 and RC9 were considered as candidates for the development of domestic RR.