Optimization of SSRPCR system on Caragana microphylla and its application
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Abstract
An orthogonal design was applied to optimize the SSRPCR system on Caragana microphylla, which employed four levels and five factors (DNA, Mg2+, dNTP, primers, and Taq DNA polymerase, respectively). The optimum SSR reaction included 40 ng template DNA, 3 mmol/L Mg2+, 300 mol/L dNTP, 0.6 mol/L primer, 1 U Taq DNA polymerase, 1Buffer with total 25 L reaction volumes. The PCR procedure was an initial step of 94℃ for 10 min, followed by 10 cycles of 94℃ for 45 s, 65℃ for 1 min (decreasing 1℃ every cycle), 72℃ for 1 min; 30 cycles of 94℃ for 45 s, 55℃ for 1 min, 72℃ for 1 min, and a final extension at 72℃ for 10 min. The optimized SSRPCR system was stable and universality, which was proved by application on C. microphylla germplasm and different SSR primers. This work could be used in analysis of genetic diversity, construction of a genetic map, and fingerprint in Caragana spp..
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