Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

[A semimicroquality evaluation method on Panax notoginseng and its application in analysis of continuous cropping obstacles research samples].

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Study on morphological and genetic diversity of Rehmannia glutinosa cultivated in Japan.

It is said that Rehmannia glutinosa is grouped into two types, Akaya and Kaikei, in Japan. However, previous reports of genetic analysis of R. glutinosa in commercial products suggest the existence of varieties other than these two, and therefore, it is inappropriate to simply classify them into these two varieties. In this study, we clarified the diversity of R. glutinosa cultivated in Japan on the basis of morphological observation and genetic analysis. We conducted principal component analysis (PCA) of R. glutinosa morphology, including leaf surface color, leaf undersurface anthocyanin coloration, root shape, and the ratio of string root. We also performed (1) sequence-related amplified polymorphism (SRAP) analysis and (2) polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We were able to separate Akaya type from Kaikei type, and to divide Kaikei type into three small groups. These two gene analysis methods were also useful in estimating the patrilineal and matrilineal strains of a hybrid origin. Our findings revealed that Akaya type and Kaikei type can be distinguished on the basis of morphological and genetic analyses, and that Kaikei type cultivated in Japan exhibited morphological and genetic diversity.

Polymorphism analysis of TCP gene region to intraspecific analysis of Paeonia lactiflora, and application for authentication of Paeoniae Radix.

We attempted to conduct an intraspecific analysis of 30 peony cultivars in Japan and to authenticate five herbal medicine samples derived from Paeoniae Radix by polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We focused on cultivar-dependent differences in leaf margin undulation and analyzed the sequence of the related TCP gene region. As a result, we found that the nucleotide sequences of 29 cultivars of Paeonia lactiflora except 'America' exhibit interspecific variations compared with the nucleotide sequences of Paeonia suffruticosa and Paeonia tenuifolia. Therefore, in the dendrogram constructed on the basis of the sequence similarity in the TCP gene region, the 29 cultivars of P. lactiflora were separated from P. suffruticosa, P. tenuifolia, and 'America', and clustered into three subgroups. There were 16 variations containing heterogenous DNA sequences within P. lactiflora species, and two characteristic variations in subgroup I. Some P. lactiflora cultivars showed the same nucleotide sequence in the TCP gene region, whereas the five herbal medicine samples showed different sequences, although all of them could be authenticated. The results suggest that Paeoniae Radix in the Japanese crude drug market can be authenticated by analysis of the TCP gene region.

[History and the present of cultivation and production of Angelica root, Yamato-Toki, in Japan].

Japanese Angelica root, Toki in Japanese, is prepared from the roots of cultivated Angelica acutiloba or A. acutiloba var. sugiyamae. Since Toki has been frequently used as a crude drug in traditional Chinese formulations, the stable supply and quality of Toki are essential issues in Japanese clinical practice. To clarify the historical and present conditions of Toki, a historical survey on herbal books and a field investigation on the cultivation condition of A. acutiloba in the Fuki area (Wakayama Prefecture) were carried out. From the present historical survey, it was proven that Yamato-Toki produced in the Yamato area, an old local area including the current Nara and Wakayama prefectures in Japan, had been considered to be superior merchandise. It was also proven that a special processing method to prevent flower stalk growth ("Mekuri" in Japanese) is an original method different from Chinese methods. From the present field investigation, it was also proven that the traditional transplant operation of the second year has been handed down since the 1930s and that washing roots in hot water ("Yumomi" in Japanese) is an original method of preparing Yamato-Toki. Toki is one of the precious crude drugs cultivated and prepared in Japan. The present study may help to pass on traditional cultivation culture and contribute to an expansion in the volume of Yamato-Toki produced in Japan.