玉米自交系SSR标记技术的优化  

Optimization of SSR Marking Technique for Maize Inbred Lines

作　　者：姜涛[1] 李凯[1] 才源[1] 刘炜 刘敬泽[3] 王丕武[1] 

机构地区：[1]吉林农业大学农学院,长春130118 [2]东辽县园艺特产中心,东辽136600 [3]辽源市农业科学院,辽源136200

出　　处：《吉林农业大学学报》2014年第6期653-659,共7页Journal of Jilin Agricultural University

基　　金：吉林省主要作物分子标记辅助育种技术平台建设;农业部"948"高效作物分子育种体系核心技术引进(2013-Z47)

摘　　要：选用7个玉米自交系为材料,分别研究了PCR反应体系中Mg2+浓度、d NTPs浓度、Taq DNA聚合酶量、引物浓度、模板DNA量,退火温度,PCR产物变性处理及银染方法对玉米自交系SSR标记结果的影响,优化了玉米自交系SSR标记技术。结果表明:优化后的20μL PCR反应体系为1.5 mmol/L Mg2+、0.05 mmol/L d NTPs、0.5 U Taq DNA聚合酶、0.05μmol/L引物、10倍PCR buffer 2.0μL和80 ng模板DNA,最优退火温度为60℃。PCR产物在95℃条件下变性5 min后上样电泳,电泳结束后胶板用蒸馏水快速漂洗,洗后用0.1%的硝酸银溶液银染8 min,再用蒸馏水洗30 s,最后用3%氢氧化钠与1%甲醛显影。In this study, the factors influencing the results of SSR marking technique were investigated and optimized, such as the concentration of Mg2＋, dNTPs and primers, content of Taq DNA polymerase and DNA template, annealing temperature, PCR product denaturing treatment and silver staining method by selecting 7 maize inbred lines as tested materials. The results show that the optimized PCR reaction system is 15 mmol/L Mg2＋,0.05 mmol/L dNTPs,05 U Taq DNA polymerase,0.05 μmol/L primer. 10×PCR buffer 20 μL and 80 ng DNA template in the 20 μL reaction volume. The optimal annealing temperature is 60℃. The sample electrophoresis was proceeded after the PCR product was denatured for 5 minutes under the condition of 95 ℃, then the rubber plate was rinsed quickly with distilled water. The optimal image develop was obtained by using 3% sodium hydroxide and 1% formaldehyde after silver staining for 8 minutes with 01% silver nitrate and then distilled water rinsing for 30 minutes.

关 键 词：玉米 自交系 PCR反应条件 退火温度 染色方法 

分 类 号：S513.032[农业科学—作物学]