Application of specific primer for identifying physiological race THTS of puccinia triticcina
A technology for wheat leaf rust and physiological races, which is applied in the application field of identifying the physiological race of wheat leaf rust THTS specific primers, and can solve the problems of few molecular markers and inability to identify races with DNA molecular markers.
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[0025] Obtaining of Physiological race THTS and EST-SSR marker Ptssr0125 of Phytophthora triticifolia:
[0026] 21 pairs of EST-SSR primers were selected, and all primer sequences were derived from Wang et al. The EST-SSR primers for wheat leaf rust developed in 2010, use the genomic DNA of 213 wheat leaf rust strains as templates for PCR detection, the 20μL PCR reaction system is: template DNA 60 ng, Taq enzyme 0.5 U, upstream and downstream Primers (5 μmol L -1 ) each 0.4 μL, dNTP (10 mmol·L -1 ) 0.4 μL, 10 × PCR buffer 2 μL, supplement the reaction system with sterile ultrapure water to 20 μL. The PCR reaction program was as follows: first at 94°C for 5 min; then at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 1 min, a total of 35 cycles; finally at 72°C for 5 min, and stored at 4°C.
[0027] Then carry out 10% (w / v) non-denaturing polyacrylamide gel electrophoresis detection on the PCR amplification product according to the following method: take 20 μL of the ampl...
Embodiment 1
[0030] 1. Extract the genomic DNA of 213 leaf rust strains as templates;
[0031] 2. Using Ptssr0125 as a primer and 213 genomic DNAs of leaf rust as a template, carry out PCR amplification. The 20 μl PCR reaction system is: template DNA 60 ng, Taq enzyme 0.5 U, downstream primer (5 μmol L -1 ) each 0.4 μL, dNTP (10 mmol·L -1 ) 0.4 μL, 10 × PCR buffer 2 μL, supplement the reaction system with sterile ultrapure water to 20 μL. The reaction program was: first at 94 °C for 5 min; then 35 cycles: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; finally 72 °C for 5 min, and stored at 4 °C.
[0032] 3. Separation and detection of the amplified products: add non-denaturing loading indicator to the amplified products, and then separate them by electrophoresis in a denatured polyacrylamide gel with a concentration of 10% (w / v), and develop the color by silver staining. In the PCR amplification pattern detected by polyacrylamide gel electrophoresis, the bands with 200bp and 178bp at ...
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