光皮桦SSR分子标记体系的建立

采用十六烷基三甲基溴化铵（CTAB）-硅珠法提取光皮桦Betula luminifera嫩叶DNA，利用近缘种日本白桦B. platyphylla var. japonica和欧洲白桦B. pendula的引物，建立光皮桦简单重复序列标记（SSR）反应体系。在体系建立过程中，采用单因素法分别对影响聚合酶链式反应（PCR）反应的因素进行分析。结果表明：在20 μL反应体系中，模板DNA用量、Mg²⁺浓度、脱氧核糖核苷酸（dNTPs）浓度、引物浓度、T_aq DNA聚合酶用量分别为60 ng，1.500 mmol·L^－1，0.175 mmol·L^－1，2.500 mmol·L^－1，1.0 × 16.67 nkat时结果最好；引物退火温度比日本白桦扩增时提高1 ℃时效果佳。PCR扩增产物经聚丙烯酰胺凝胶电泳后随机挑取其中3对引物扩增的多态性片段进行克隆测序，对引物的通用性做进一步检验，序列经比对后得到的与原物种序列相似度（max ident值）均在95.0%以上。可见，近缘种日本白桦和欧洲白桦的SSR引物可以在光皮桦上应用。图7表2参17

An SSR molecular labeling technique system for Betula luminifera

To find the optimal simple sequence repeats（SSR） system for Betula luminifera,the cetyltrimethyl-ammonium bromide （CTAB） method was used to extract DNA from tender leaves,using the known primers from related species：B. platyphylla var. japonica and B. pendula. Then,one factor was changed at a time to optimize the polymerase chain reaction （PCR）. Next,Polyacrylamide Gel Electrophoresis （PAGE） was used to detect the amplified products with three primers＇ segments randomly selected from thirty-six pairs of primers and sequenced to further check the universality of the primer. Results showed that in a volume of 20 μL,the optimal reaction system was 60 ng template DNA,1.500 mmol·L-1 Mg2＋,0.175 mmol·L-1 deoxyri-bonucleotide triphosphate （dNTP）s,2.500 mmol·L-1 primer,and 1.0 × 16.67 nkat Taq polymerase. Compared with B. platyphylla var. japonica and B. pendula,the optimal annealing temperature of the primer was 1°C higher. The blast results from primer AF310851,AB084479,and AB084480 showed that the values of max ident were all above 95.0%. This indicated that the SSR primers of birch could be used with B. luminifera.