Evaluation of the Influence of Genetic and Environmental Factors on the Ephedrine Alkaloids Content of Ephedra sinica.

Ephedra herb, a dried terrestrial stem of Ephedra sinica, is used in traditional Japanese medicine (Kampo) and Chinese medicine to treat the common cold, headaches, bronchial asthma, and nasal inflammation. E. sinica predominantly contains two ephedrine alkaloids-(-)-ephedrine and (+)-pseudoephedrine-which are crucial for its medicinal effects. This study aimed to reveal the influence of genetic and environmental factors on ephedrine alkaloids content using statistical genetic analyses. To evaluate the influence of genetic factors on ephedrine alkaloids content, 25 clonal lines were cultivated in Ibaraki and the broad-sense heritability of the traits was estimated. The heritabilities of (-)-ephedrine, (+)-pseudoephedrine, and "total alkaloids" (TA) content were 0.871, 0.969, and 0.865, respectively. The heritabilities of ephedrine alkaloids content were high. To evaluate the influence of environmental factors on ephedrine alkaloids content, four clonal lines which have different genotypes were cultivated in three locations (Ibaraki, Shizuoka, and Yamanashi prefectures). The effects of genotype (G), location (L), and genotype by environment (G × E) interactions on ephedrine alkaloids content were found to be significant (p < 0.05) by two-way ANOVA, and, in particular, the genotypic effects were found to be the largest. Our results indicate that the ephedrine alkaloids content in E. sinica is under relatively strong genetic control and remains stable under various environments. These findings suggest that E. sinica with a higher and stable ephedrine alkaloids content could be cultivated in different locations through selective breeding.

Stability and Reproducibility of Ephedra sinica Ephedrine Alkaloid Content and Terrestrial Stem Dry Weight.

Dried terrestrial stems of Ephedra sinica are known as 'Ephedra herb.' The pharmacological effects are mainly related to two major ingredients, (-)-ephedrine and (+)-pseudoephedrine (total alkaloids which are defined in Japanese Pharmacopoeia, TA). In this study, in order to aid in cultivation and breeding, the stability of TA content and stem dry weight of 46 E. sinica genets was evaluated from the first year of transplantation to the sixth year. TA content and composition ratio of these genets were stable after the second year, and dry weight was stable after the fourth year. These traits showed high inter-genet variability but low annual variability for each genet. Additionally, rank correlation coefficients of each trait among the genets were high. There was no significant correlation between these traits. Furthermore, to assess the reproducibility of these traits in clones, we evaluated TA content and dry weight of three clonal lines with high TA contents. TA content and composition ratio of the clonal lines were also stable after the second year of transplantation, and dry weight of the clonal lines was also stable after the fourth year. Moreover, TA content and composition ratio in each clonal line were comparable with those of each original genet after the second year. These results suggested that ephedrine alkaloids content and dry weight of E. sinica plants are stable, and that these traits are highly reproducible in clones. Therefore, selection breeding of E. sinica using vegetative propagation can be effective for high and stable quality of Ephedra herb.

Validity of Selection Breeding for High Ephedrine-Alkaloid Content in E. sinica under Different Environments in Japan.

Dried terrestrial stems of Ephedra sinica are called 'Ephedra herb,' whose pharmacological effects are due mainly to two major ingredients, (-)-ephedrine and (+)-pseudoephedrine (total alkaloids which are defined in Japanese Pharmacopoeia (TA)). Ephedra herb is an important crude drug in Japan. However, E. sinica is widely distributed in arid areas of northeastern China and Mongolia. Recently, E. sinica has started to be cultivated in Japan. This study aimed to assess the validity of selection breeding on TA content of E. sinica in several locations in Japan. In this experiment, we grew approximately 350 seedlings and divided them randomly into seven groups. Nearly fifty plants were cultivated at each of seven locations. In Ibaraki, Yamanashi, and Shizuoka, average TA content of whole samples satisfied the criteria for Ephedra herb defined in Japanese Pharmacopoeia (7.0 mg/g of dry weight (DW)). Plants with high and intermediate TA content at four locations were selected and transplanted to Ibaraki. There were significant differences in TA content between selected plants with high and intermediate TA content before and after transplanting (p < 0.05). TA content of high-TA plants was significantly higher than that of control plants cultivated continuously at Ibaraki (p < 0.05). These results suggest that the selection on content of ephedrine alkaloids in E. sinica under various locations in Japan is valid, and high- TA E. sinica plants can be selected at various locations.

Stabilization of Ephedrine Alkaloid Content in Ephedra sinica by Selective Breeding and Stolon Propagation.

The Ephedra herb, which has been used in Kampo medicines, originates from terrestrial stems of Ephedra species. It is important to establish cultivation methods and cultivars to secure a stable supply of the Ephedra herb that would meet the quality standards for the ephedrine alkaloids content. In this study, we first grew Ephedra sinica plants derived from seeds in the field for 5 years. Then, for selective breeding of cultivars that could meet the quality standards for the ephedrine alkaloids content, we measured the content of total alkaloids (TAs), ephedrine (Eph), and pseudoephedrine (PEph) in individual plants derived from seedlings and grown for 4 years in the field. The range of the TA content in each individual plant was narrower than that among individual plants grown in the field. Therefore, individual plants were selected according to their TA content, Eph/PEph ratio, and stolon-formation capability. The selected individuals were propagated using stolons, and their TA content was studied for 2 years. In the second year, the TA content in terrestrial stems derived from stolons of the selected individuals was as high as that of their parents. Therefore, it was confirmed that the selected individuals that were propagated using stolons could produce TA reproducibly. This study suggested that selective breeding using stolon propagation is effective for stabilizing Ephedra herb TA content.

Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele.

KEY MESSAGE: By genetically eliminating the major restorer - of - fertility gene ( Rf ), a weak Rf gene was unveiled. It is an allele of Z , long known as an elusive Rf gene in sugar beet. In the hybrid breeding of sugar beet, maintainer-genotype selection is a laborious process because of the dependence on test crossing, despite the very low occurrence of this genotype. Marker-assisted selection (MAS) of the maintainer genotype is highly desired by sugar beet breeders. The major restorer-of-fertility gene (Rf) was identified as Rf1, and its non-restoring allele (rf1) was discriminated at the DNA level; however, some of the rf1rf1 selections retained an as yet unidentified Rf, another target locus for MAS. The objective of this study was to identify this Rf. An rfrf1 plant was crossed to a cytoplasmic male-sterile sugar beet and then backcrossed to obtain progeny segregating the unidentified Rf. The progeny exhibited partial male-fertility restoration that was unstable in single plants. The segregation ratio of restored vs. non-restored plants suggested the involvement of a single Rf in this male-fertility restoration, designated as Rf2. We confirmed the feasibility of molecular tagging of Rf2 by identifying four shared amplified fragment length polymorphism (AFLP) fragments specific to 17 restored plants. Bulked segregant analysis also was performed to screen the Rf2-linked AFLP markers, which were subsequently converted into 17 sequence-tagged site markers. All the markers, as well two additional chromosome-IV-assigned markers, were linked to each other to form a single linkage map, on which Rf2 was located. Our data suggested that Rf2 is likely an allele of Z, long known as an elusive Rf gene in sugar beet. We also discuss the importance of Rf2 for sugar beet breeding.