Central China Sex Differentiation Of Fructus Schisandrae Physiological And Molecular Markers

Schisandra sphenanthera Rehd. et Wils., which belongs to Schisandra Michx. of Schisandraceae, is a kind of woody and deciduous liana. It is commonly used in traditional Chinese medicine with the main biological active ingredient of lignans. S. sphenanthera has been considered to be one of the important groups to solve the problem of the origin and evolution in primitive angiosperms, therefore, the investigation of its sex differentiation mechanism has very important significance to the evolutionary studies of plant gender diversity; The economic value of female plants was greater than the male plants, therefore, in the introduction, cultivation and breeding of S. sphenanthera, seeking early identification of gender from different perspectives of gender studies, has important practical significance.Currently, the researches of gender differences in S. sphenanthera were few. The studies in morphology and isozyme were affected by developmental stage, external environment and tissue specificity, and can not provide accurate, reliable informations of identification, so they can only be used as the reference of S. sphenanthera gender identification. In this study, female and male plants of S. sphenanthera as the material, which were studied in gender differences by physiological and molecular markers for the first time, in order to seek its early gender identification methods and preliminary explore the sex differentiation process. The main conclusions as follows:1. From vegetative stage to flowering end, the change trends of endogenous hormone in male and female plants of S. sphenanthera were consistent. ABA content showed a significant upward trend in the flower formation stage, IAA, GA3, and ZR content then showed a significant decrease trends, the changes tend to smooth from flowering to the end of flowering.2. The male and female differences of ABA content achieved to the most significant at the beginning of female flowering stage. In female plants, ABA contents were significantly higher than male plants, it may play a dominant role in the sex differentiation process of S. sphenanthera. In male plants, IAA, GA3and ZR contents were significantly higher than female plants, and the male and female differences were highest at the beginning of male flowering stage. The male and female difference of ZR is small relatively, the change is not obvious. The trends of ABA/IAA, ABA/GA3and ABA/ZR ratio is similar. Overall increased gradually, showing a higher ratio, while the ratio of female plants are always higher than male plants. The upward trend is obvious at the flower formation stage, and the differences in male and female plants were significantly or very significant. From flowering to the end of flowering, the ratio remained stable, the male and female differences decreased relatively.3. The beginning of flowering stage is a critical point about endogenous hormone levels change of S. sphenanthera. During this period, the synthesis of a high level of ABA is conducive to the development of the female plants, while maintaining a high level of IAA, GA3, and ZR is conducive to the development of the male plants, which provide an experimental evidence for the early identification of the male and female plants in S. sphenanthera wild tending.4. The optimal RAPD reaction system of S. sphenanthera, that is25μL total volume included Mg2+of2.5mmol·L-1, dNTPs of0.08mmol·L-1, primer of0.6μmol·L-1, Taq enzyme1.5U, DNA template60ng, annealing temperature41.3℃,35cycles. The system had high stability and good reproducibility.5. In400random primers, only by S353, the male plants were amplified a band about500bp while the female plants were not with this band in female DNA poll, male DNA pool, and all individual DNA of S. sphenanthera. This band belongs to a male-linked specific bands, both ends of the sequence contains the binding sites of S353, the size was541bp and the AT content was64.3%. There were no highly homologous sequence to found by Blast search. The RAPD markers can be used in early sex identification of S. sphenanthera.6. Respectively using three pairs of primers for SCAR amplification of S. sphenanthera under different annealing temperatures, both of the female and male DNA amplified target fragment. It showed the specificity was lost when RAPD marker converted into SCAR marker, the conversion was failed.