桂花SCoT标记体系的建立及其在遗传多样性分析中的应用

Establishment and Genetic Diversity Analysis of SCoT Molecular Marker System for Osmanthus fragrans

以‘籽银桂’桂花叶片DNA为材料,以2×EsTaq Master Mix预混液为基本体系,针对影响SCoT-PCR反应的退火温度、模板DNA用量、引物浓度等因素进行优化,建立了适于桂花SCoT分子标记的反应体系。12μL反应体系含有30ng模板DNA,引物浓度为0.4μmol·L^(-1),不同的引物退火温度分别为48、49.9、54.3或56℃。利用该体系对12个桂花品种进行分析,扩增条带清晰,扩增产物在150~2200bp之间。12条引物共扩增出244条带,其中多态性条带238条,多态性比率为97.47%。在相似系数0.57水平处,‘九龙桂’形成第Ⅰ组,其余11个品种形成第Ⅱ组。第Ⅱ组中‘柳叶桂’、‘香云’、‘早籽黄’、‘大花金桂’、‘桂冠籽金桂’、‘鹅黄’、‘籽金桂’和‘醉云’8个花色不完全相同的品种聚在一起;‘金桂’、‘早银桂’和‘籽银桂’3个不同花色的品种聚在一起,说明品种间的亲缘关系与花色不完全相关,这与其它分子标记结果一致。

In this study,based on the‘Ziyingui'DNA and 2 × EsTaq Master Mix,the SCoT-PCR amplification system of Osmanthus fragrans was established by optimizing reaction annealing temperature,the amount of template DNA,primer concentration and other factors. This system contained 30 ng template DNA,0.4 μmol · L^(-1) primers in 12μL mixture,and the most suitable annealing temperature of different primers was 48,49.9,54.3 and 56 respectively. Twelve ℃ Osmanthus fragrans cultivars were analyzed using this system,and the amplified bands were very clear. The 12 primers generated a total of 244 fragments from 12 Osmanthus fragrans cultivars,and 97.47% of fragments showed polymorphic. At the level of similarity coefficient 0.57,‘Jiulonggui'was to one group and other cultivars were to another group. In the second group,‘Jingui',‘Zaoyingui'and‘Ziyingui'were in one subgroup,and the other eight cultivars were in another subgroup. Our results indicated that genetic relationship between color and variety was not perfectly correlated,which is consistent with the results of other molecular markers.