| Wintersweet (Calycanthaceae, Chimonanthus praecox), a kind of perennial deciduous shrub, is an traditional ornamental flowering plant endemic to China and is of great ornamental and economic value in practice. During its long history of cultivation with more than one thousand years, abundant variations arose, producing a variety of germplasms. However, at present, due to lack of uniformed standards and rules for identification and documented description of wintersweet cultivars, various characters as well as different criteria for their assessment were employed in the current cultivar classification system, which caused considerable confusions in cultivar identification and description. Furthermore, because there existed several different cultivar classification systems, homonyms and synonyms had occurred from time to time, which brought a great deal of inconvenience in the exchange, utilization and conservation of germplasms resources of wintersweet.Wuhan and Nanjing are two regions especially suitable for the cultivation and production of wintersweet. In addition, for the'moment, these two regions appear to hold the most abundant germplasms resources of wintersweet germplasms. In this research, the germplasms resources of wintersweet cultivated in Wuhan and Nanjing were observed and surveyed for years, and as well the genetic diversity of these germplasms was investigated by means of morphological, RAPD and ISSR markers. The purposes of this research were to explore for the rules of variations in morphological traits of wintersweet germplasms, identify the potential relationships between morphological traits and molecular markers with respect to their utilizations in cultivar classification, and eventually provide a basis for germplasm or cultivar identification and classification for wintersweet.Firstly, morphological research was carded out.(1) Based on the analysis of extent of variation and two-way ANOVA among years and genotypes of wintersweet for morphological traits, it was demonstrated that the following 15 characters were suitable to be used for identification, description and evaluation in classification system in that they exhibited significant differences among different germplasm categories but gave stable performance within a category, with minor variance between years. The 15 characters include: flower period, flower color, flower bud color, flower urn-shaped or not, tepal margin crisped or not, the tip of the tepal recurved or not, medium tepal length, medium tepal length/width, area size of medium tepal, inner tepal length, inner tepal length/width, area size of inner tepal, number of medium and inner tepals, stamen number and color of inner tepal(2) There were rich diversities on morphology for the wintersweet germplasms under the study. The variation of 8 numerical characters ranged from 9.54% to 28.3%, with over 2 times difference between maximum and minimum values. The average morphological diversity index for the 15 characters reached 0.59, while the morphological diversity index for the genotypes from Wuhan was higher than that for the genotypes from Nanjing.(3) By means of Q cluster analysis, the wintersweet germplasms collected for this study were categorized into 8 groups, i. e. green flower bud group, big-sized and crisped tepal group, urn-shaped and narrow tepal group, big-sized group, big-sized and urn-shaped group, small-sized group and patens group. They were distinctive on morphology between each other.(4) It was shown by R cluster that there was some degree of correlation between the characters of flower bud (green or not) and tepal margin (crisped or not).(5) In principal component analysis, the first 8 principal components, from which the cumulative contribution reached 86.37%, corresponded to the following 8 characters, i. e. flower size, tepal shape, tepal margin crisped or not, number of tepals and flower urn-shaped or not, tepal recurved or not, flower and flower bud color, color of inner tepal and flower period. Moreover, these principal components basically reflected the major attributes of each of the groups generated in Q cluster analysis. The extremely high correlation (r=0.98) between the results produced by principal component analysis and Q cluster analysis showed that it was reasonable to choose the 15 morphological characters and the correlation among them had little effect on morphological clustering.(6) It was proven by Bayes discriminant analysis that the selected 15 characters had strong discriminative power for classification of wintersweet. The discriminant formulae can be used for the management of wintersweet germplasms in the future.Secondly, RAPD and ISSR molecular marker analyses were carried out.(1) 19 RAPD primers amplified 165 bands, of which 105 (63.63%) were polymorphic, while 11 ISSR primers generated 115 bands, with 90 (78.26%) polymorphic. Either ISSR or RAPD markers were sufficient to distinguish all the genotypes surveyed.(2) The Nei index calculated for RAPD or ISSR was h_RAPD=0.21 or h_ISSR=0.25 respectively, showing the relatively high level of genetic diversity existing in cultivated wintersweet germplasms. Meanwhile, both analyses indicated that wintersweet from Wuhan had higher genetic diversity compared to that from Nanjing. (3) Clustering and principal coordinate analysis using both RAPD and ISSR data revealed that the clustering pattern of wintersweet was tightly linked with their geographical origins.(4) Most of the genetic variation (86.75% with RAPD data and 85.68% with ISSR data) occurred among genotypes within each region, with the remaining existing between two regions. However, it was indicated by further AMOVA and HOMOVA analyses that the genetic difference between Wuhan and Nanjing groups was statistically significant (Φ_ST =0.132, p＜0.001, with RAPD data;Φ_ST=0.143, p＜0.001, with ISSR data).(5) In consistent with the result by the morphological analysis, both RAPD and ISSR analyses revealed that the two morphological characters, flower bud color (green or not) and tepal margin shape (crisped or not), had very close relationship.(6) By comparison, with respect to clustering, while the correlation between morphological and each molecular marker was low, two kinds of molecular markers were closely related.The comprehensive results by the above analyses demonstrated that abundant genetic diversity was produced during the long history of wintersweet cultivation. Due to the distinctive criteria adopted for breeding, the unique gene pools were gradually formed in different geographical regions, resulting in the genetic differentiation of cultivated wintersweet germplasms. Consequently, in the future breeding program, parental lines from diverse regions should be utilized on purpose so as to make the most of the genetic diversity reserved in the current germplasms.
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