Optimization and Primer Selection of SSR-PCR System of Parrotia subaequalis
In order to effectively use the SSR-PCR syste m to analyze the genetic diversity of the plant Parrotia subaequalis which is a national primary protection plant, an orthogonal design (L16 (43) ) was applied to optimize and screen the influencing factors of SSR-PCR amplification results, including primer, template DNA concentration and cycle numbers. Based on this analysis, the loading amount of polyacrylamide gel was optimized, and the optimal SSR-PCR system suitable for P. subaequalis was established as follows: 10 μL 2×PCR Mix, 40 ng template DNA, and 10 μmol/L primer, and the rest was supplemented with dd H2O to 20 μL. The PCR amplification was performed according to the following procedure: an initial denaturation for 15 min at 95℃, denaturing for 30 s at 95℃, annealing for 1.5 min at 57.5℃, extending for 1.0 min at 72℃. After 30 cycles, a final extension at 60℃ for 30 min was applied, and the amplified DNA was stored at 4℃. The optimal loading amount of polyacrylamide gel electrophoresis was 1.5～2.0 μL. Through the optimized SSR-PCR system used for primer screening, 11 pairs of polymorphic primers were selected from 18 pairs of primers, and the result showed that the reaction system had excellent stability. Our results indicated that the establishment of SSR-PCR system would provide theoretic and technical support for genetic diversity analysis, phylogenic stud y, gen etic maps establishment, gene localizations and molecular marker assisted breeding of Parrotia subaequalis based on SSR marker.
Parrotia subaequalisOrthogonal designSSR-PCRThe best loading amount of samplePrimer selection
NETL
NSTL
万方数据
维普
