Abstract: Celastrus orbiculatus Thunb. belongs to Celastrus L. of the family Celastraceae. As a kind of traditional Chinese herbal medicine, it has great ornamental value and important medicinal value. It is a dioecious vine, a small amount of them are andromonoecy plants. However, the morphological differences between the male and female plants are not obvious before the maturation of their sex organs, which restricts the early cultivation and breeding. Currently, molecular markers have been widely used in the sex identification of dioecious plants. But the research on the sex differentiation of C.orbiculatus at the molecular level has not been reported. Due to unknown genome sequence, many studies on C.orbiculatus are limited in the field of chemistry and cultivation technology, which greatly limits its utilization. In this study, differentiation among dioecious plants was compared using random amplified polymorphic DNA (RAPD) markers. It is the first time to use the RAPD markers on the study of C.orbiculatus. Using the young leaves of male and female plants as materials, we extracted the DNA using the improved CTAB method. Then through screening of different factors, we established the optimized PCR system. Based on the above PCR system, we selected 100 RAPD primers to conduct the analysis. Significantly different bands were amplified using primer S127, S140, S148, S174 and S111, respectively. These different bands were cloned into the T-vector, and then sequenced. Based on the sequences, the specific SCAR markers were obtained. Only one female-associated dominant SCAR marker from RAPD marker S111 was verified. Sequence analysis showed that this female specific fragment contained nine open reading frames (ORF) between 93-570 bp in length, among which two have more than 100 amino acids. The results of sequence Blast showed that the polypeptide fragment had a higher similarity with plants reverse transcription factor of the gag-POL protein, which belongs to UBN2 super family. We inferred a possible reason of the gender traits is that the insertion of the reversal seat factor may inhibit some important sex-determining factors. In the future, we need to get their full-length sequences using RACE, and then study their function on sex differentiation.

Key words: molecular markers, sex differentiation, RAPD, SCAR