Optimization and Primers Screening of MSAP Analysis System in Cucurbita
JIANG Yi，JIANG Li-na*，ZHOU Jun-guo*
（School of Horticulture and Landscape Architecture，Henan Institute of Science and Technology，Horticultural Plant Resources
Utilization and Germplasm Enhancement Engineering Research Center of Henan Province，Xinxiang 453003，Henan，China）
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．
