南瓜MSAP 体系的优化及引物筛选

以南瓜自交系北观（Cucurbita maxima）为材料，利用正交设计L₁₆（4⁵）优化南瓜MSAP 预扩增和选择性扩增体系的主要因素。结果表明，第一步最佳酶切时间2 h，Hap Ⅱ（HpaⅡ，MspⅠ）用量0.5 μL；第二步酶切时间6 h，EcoR Ⅰ用量0.4 μL；连接体系包括酶切产物21 μL，EcoR Ⅰ接头（5 μmol · L^-1）、Hap Ⅱ接头（5 μmol · L^-1）各1.0 μL，连接时间12 h，T4 DNA Ligase 用量为0.5 μL；最佳预扩增反应体系（25 μL）包含2.0 μL 连接产物，1.5 μL 10 × PCR Buffer（Mg²⁺ plus），2.0 μL dNTP（2.5 mmol · L^-1），0.6 μL Taq 酶（5 U · μL^-1），0.4 μL 上下游引物（10 μmol · L^-1）；最佳选择性扩增反应体系为预扩增产物稀释120 倍的模板4.0 μL，其他同预扩增体系。最后，利用建立好的MSAP 体系进行6% 聚丙烯酰胺凝胶电泳验证并筛选出适于南瓜MSAP 分析的36 对引物，表明优化后的MSAP 体系多态性好，体系稳定，可重复，为后续进行南瓜MSAP 分析奠定了基础。

Optimization and Primers Screening of MSAP Analysis System in Cucurbita

Taking pumpkin ‘Beiguan’（Cucurbita maxima） as material，this experiment used orthogonal design L₁₆ （4⁵） to optimize main factors of pumpkin MSAP pre-amplification and selective amplification system． The results showed that in the first step：the best digestion time was 2 hours，Hap Ⅱ（Hpa Ⅱ，MspⅠ） dosage was 0.5 μL；in the second step：digestion time was 6 hours，EcoR Ⅰ dosage was 0.4 μL．The digestligation system，including 21 μL digestion product，1 μL EcoR Ⅰcontact，and 1 μL Hap Ⅱ contact．The connection time was 12 hours．The T4 DNA Ligase usage was 0.5 μL．The optimal pre-amplification reaction system contained 2.0 μL digest-ligation products，1.5 μL 10 × PCR Buffer（Mg²⁺ plus），2.0 μL dNTP（2.5 mmol · L^-1），0.6 μL Taq enzyme（5 U · μL ^-1），0.4 μL upstream and downstream primers（10 μmol · L^-1）． The optimal selective amplification reaction system was template 4 μL diluted 120 times from the preamplification products．The rest was identical with pre-amplification system．Finally，36 pairs of primers suitable for pumpkin MSAP analysis were screened out and verified with 6% PAGE by the established MSAP system．The result indicated that the optimized MSAP system had good polymorphism，the system was stable and repeatable．This experiment has laid a foundation for subsequent MSAP analysis on pumpkin．