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Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.

Previously, Chinese gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene-trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and beta-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.