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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, the DNA of ’Gui Gui Gui’ (Osmanthus fragrans) was used as the material, and the 2 × EsTaq Master Mix was used as the basic system to optimize the SCoT-PCR reaction such as annealing temperature, template DNA dosage and primer concentration. SCoT molecular marker reaction system. 12μL reaction system contains 30ng template DNA, primer concentration of 0.4μmol·L -1, different primer annealing temperature were 48,49.9,54.3 or 56 ℃. Using this system, 12 sweet-scented osmanthus cultivars were analyzed, and the amplified bands were clear. The amplification products ranged from 150 to 2200bp. A total of 244 bands were amplified with 12 primers, of which 238 were polymorphic, with a polymorphism rate of 97.47%. At the similarity coefficient of 0.57, ’Jiulonggui’ formed Group I, and the remaining 11 cultivars formed Group II. In Group II, ’Liu Yegui’, ’Xiangyun’, ’Early Seeds Yellow’, ’Grand Flower Jinui’, ’Laurel Jingui’, ’Goose Huang’, ’Seeds Jinui’ and ’Zunyun’ 8 A variety of colors are not exactly the same together; ’Jin Gui’, ’Zao Yin Gui’ and ’Seed Silver Gui’ three different varieties of color together, indicating that the relationship between species and color is not completely related, which Consistent with other molecular markers.