Optimization of SRAP-PCR system using orthogonal design and its application to genetic analysis in Fenneropenaeus chinensis

• Published in: Yuye kexue jinzhan Vol. 31; no. 6; pp. 43 - 53
• Main Authors: Chen, Huanzeng, Li, Jian, He, Yuying, Li, Jitao, Wang, Qingyin
• Format: Journal Article
• Language: Chinese
• Published: 01.01.2010
• Subjects: Fenneropenaeus chinensis; Marine
• ISSN: 2095-9869

The orthogonal design L16(45)was used to optimize SRAP-PCR amplification system in marine shrimp Fenneropenaeus chinensis on four levels of five factors(Taq DNA polymerase, Mg super(2+), DNA template, dNTP, and primer). The results showed that the optimum concentrations were 1.0 U Taq enzyme, 2.0 mmol/L Mg super(2+), 40.0 ng DNA template, 0.125 mmol/L dNTP, and 0.4 mu mol/L primer, respectively, in 20 mu l SRAP-PCR system, and the annealing temperature was 53.5 degree C via setting temperature gradient. Significant effects of each factor in different levels were observed and the concentration of Mg super(2+) was the most dominant factor affecting the result of PCR, while Taq polymerse, primer, dNTP, and template DNA followed in terms of dominance. Then, the established amplification system was used to analyze the genetic diversity of the 12th generation of selected 'Huanghai No.1' of F. chinensis. As a result, 171 bands were generated with 8 primer sets, of which 154 bands were polymorphic bands, accounting f