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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) |
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Abstract 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.
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Received: 19 February 2019
Published: 11 July 2019
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