Comparison of Extraction Methods of Total DNA and Optimization of SSR Reaction Systems from Docynia delavayi

PurposeTo extract high-quality total DNA from the leaves of Docynia delavayi, establishing a stable SSR-PCR reaction system.

MethodsUsing young leaves of D. delavayi as materials, total DNA was extracted by urea method, modified urea method, SDS method, CTAB method, MAGEN kit method, and magnetic beads kit method to select the best extraction method. The SSR-PCR reaction system was established by an orthogonal design L₁₆(4⁵) to optimize the factors affecting the SSR-PCR amplification system of D. delavayi.

ResultsModified urea method was the best extraction method to extract the total DNA, and the mass concentration of total DNA, total amount, and A₂₆₀/A₂₈₀ extracted were 382.64 ng/μL, 38.26 μg and 1.87, respectively, and the bands of agarose gel electrophoresis were clear and bright, indicating good DNA integrity and high mass concentration. The optimized reaction system for SSR-PCR amplification was: template DNA 30 ng, Taq DNA polymerase 0.5 U, primers 1.25 μmol/L, Mg²⁺ 2.0 mmol/L, dNTPs 2.5 mmol/L, 10×PCR buffer 2.5 μL, and make-up water to 25 μL.

ConclusionThe modified urea method can be used to extract high-quality total DNA from D. delavayi, and the established SSR-PCR reaction system provides technical support for genetic diversity analysis and molecular marker-assisted breeding of D. delavayi.