Osmanthus fragrans (Thunb.) Lour. (Oleaceae), a valuable fragrant plant, is found to be functionally androdioecious (the presence of males and hermaphrodites in a population) (Hao et al., 2011). As one of the top 10 traditional flowers in China, O. fragrans has been cultivated for about 2500 yr and more than 120 cultivars have been identified. These cultivars are categorized into four groups (Asiaticus, Albus, Luteus, and Aurantiacus) based on morphological and phenological traits (e.g., flower color, peduncle, and flowering period), and there are male and hermaphroditic cultivars in each group (Xiang and Liu, 2008). Moreover, it is thought that the cultivar groups Asiaticus and Albus are less differentiated from wild O. fragrans than the other two groups, based on morphological features and research data (Xiang and Liu, 2008).
Several dominant molecular markers have been used for cultivar identification and classification of O. fragrans (Xiang and Liu, 2008; Yuan et al., 2011). However, codominant microsatellite markers, which have become preferred markers as they are polymorphic, highly abundant, analytically simple, and transferable, have not been reported in O. fragrans cultivars. In this study, microsatellite markers were developed to analyze the genetic diversity of androdioecious O. fragrans cultivars, which will provide new molecular tools for cultivar classification, identification, and genetic improvement.
METHODS AND RESULTS
Through careful field investigation, O. fragrans cultivars and their genders were identified during the 2009 to 2011 flowering seasons. A total of 62 typical O. fragrans cultivars (nine hermaphrodites and 53 males) and six closely related species that were used as outgroup taxa were collected (Appendix 1). Genomic DNA was extracted from young and fully expanded leaves of study materials using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). Microsatellite sequences of O. fragrans were downloaded from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/nuccore), then analyzed with the Simple Sequence Repeat Identification Tool (SSRIT; http://www.gramene.org/gramene/searches/ssrtool) to identify simple sequence repeat (SSR) loci with a minimum length of 10 bp for all repeats. Twenty-nine SSR sequences were selected, and flanking primer sets were designed using the software Primer 5.0 (Clarke and Gorley, 2001). Primers had an optimum length of 22 nucleotides (18 bp minimum, 27 bp maximum) and CG contents ranged from 20% to 80%. The designed primers were synthesized at Generay Biotech Co. Ltd. (Shanghai, China), and 15 microsatellite markers were selected based on amplification and reproducibility in all accessions (Table 1). The final 12.5-µL reaction volume for PCR contained 6.25 µL 2× Taq Master mix (100 U/mL Taq polymerase, 400 µM dNTPs, and 4 mM MgCl2 [Generay Biotech Co. Ltd.]), 0.3 µM of each forward and reverse primer (Generay Biotech Co. Ltd.), and 20 ng of DNA template. Amplification was performed with a 5-min initial denaturation at 94°C, followed by 35 cycles of 94°C for 30 s, annealing at 46–52°C for 30 s, and an extension at 72°C for 30 s. A final extension was performed at 72°C for 8 min. A pBR322 DNA-MspI digest marker (Tiangen, Beijing, China) yielding 26 fragments from nine to 622 bp was used as the molecular size standard. PCR products were separated on 8% denaturing Polyacrylamide gels and stained with a silverstaining method.
Characteristics of 15 Osmanthus fragrans microsatellite loci.
The presence of null alleles was tested with the program MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004), which suggested loci OPF022 and OPF029 showed evidence of null alleles. These loci were excluded from subsequent data analyses. POPGENE version 1.32 (Yeh et al., 1999) was used to calculate the number of alleles per locus, observed heterozygosity, and expected heterozygosity. The number of alleles ranged from two to six, with a mean of 3.7 per locus. The observed and expected heterozygosities ranged from 0.1000 to 0.9091 and from 0.1287 to 0.9167, respectively (Table 2).
The genetic structure of study materials was inferred using the program STRUCTURE 2.3.1 (Pritchard et al., 2000), with a burn-in length of 30,000 followed by 500,000 cycles, and each run was iterated five times. The number of subgroups (K) was determined to be K = 4 using Evanno's method (Evanno et al., 2005). The results of the structure analyses are presented in Fig. 1. The outgroup (represented in Fig. 1 in yellow) was genetically distinct from all O. fragrans cultivar groups. Cultivar groups Luteus and Aurantiacus were somewhat genetically differentiated (mainly represented in Fig. 1 in red and blue, respectively), but gene exchange was evident among many cultivars (indicated in Fig. 1 by the presence of the same color in different groups) and was extensive for Asiaticus and Albus cultivars. The results indicate that Asiaticus and Albus were genetically mixed and incompletely differentiated. Thus, the cultivar groups Asiaticus and Albus possibly have diverged more recently, as they were less genetically differentiated, while the cultivar groups Luteus and Aurantiacus, which displayed greater genetic differentiation, might have diverged earlier. In sum, the molecular results provide some support for the morphological classification of O. fragrans cultivars groups Luteus and Aurantiacus (Xiang and Liu, 2008).
Results of screening of 13 microsatellite loci in Osmanthus fragrans cultivars.
The 13 microsatellite markers developed for O. fragrans are highly polymorphic and informative. These loci will be useful for genetic study of androdioecious O. fragrans cultivars and for cultivar classification, particularly for cultivar groups Luteus and Aurantiacus. They also hold potential for further genetic study of O. fragrans cultivars.
 The authors are grateful to Dr. Xia Tao, Prof. Hao Ri-Ming, Dr. Xie Chun-Ping, Dr. Hou Bei-Wei, Dr. Wang Nian, and Dr. Ding Guang-Hui for valuable input, data analysis advice, and helpful comments. The authors thank the two anonymous reviewers for their valuable comments on the manuscript. The research was supported by the Doctorate Fellowship Foundation of Nanjing Forestry University.