Primer for screening wheat leaf rust resistance QTL and application of primer
A technology for leaf rust and wheat, which is applied to primers for screening wheat leaf rust resistance QTL and its application field, can solve the problems of loss, affect disease prevention effect and quality, and single-gene resistance is not persistent, and achieves long-term extension. The effect of disease resistance
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[0033] A new QTL for wheat leaf rust resistance- QLr.zh-4B The SSR markers wmc692 and wmc657 are obtained:
[0034] During the three consecutive growth years of 2011-2012, 2012-2013 and 2013-2014, Zhoumai 22, Chinese Spring and their hybrids were planted in the experimental field of Hebei Agricultural University in Baoding by using a completely randomized block design method and self-crossed year by year. f 2:3 family lineage. Two replicates were planted each year, and the first two rows of each replicate were planted with the parents Zhoumai 22 and China Spring respectively, so as to facilitate the observation of the disease status of the parents, and then the 215 families planted were randomly arranged in sequence. Plant 50 grains in each family, plant in a single row, the row length is 1.5m, and the row spacing is 0.25m. Zhengzhou 5389 is planted in every ten rows, which is convenient for counting and used as a susceptible control. Zhengzhou 5389 is planted on both sides...
Embodiment 1
[0041] 1. Extract Chinese spring genomic DNA as a template;
[0042] 2. Using wmc692 and wmc657 as primers and Chinese spring genome DNA as template, carry out PCR amplification. 10μL PCR reaction system: 10×buffer 1 μL, 10 mmol L -1 dNTPs 0.2 μL, 4 μmol L -1 Primer 1 μL, 40 ng μL -1 DNA template 1 μL, Taq Enzyme 1 U, ddH 2 O 6.7 μL.
[0043] The PCR reaction program was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; the annealing temperatures of primers wmc692 and wmc657 were 51°C and 61°C respectively, the annealing time was 1 min, and extension at 72°C for 1 min; 35 cycles; Extend for 10 min at ℃ and store at 4℃.
[0044] 3. Separation and detection of the amplified products: add 2.5 μL 6×Loading buffer to the amplified products, take 5 μL for each amplified sample, and run it on a non-denaturing polyacrylamide gel with a concentration of 12% (w / v). Separated by electrophoresis and visualized by silver staining. Such as figure 1 and figur...
Embodiment 2
[0046] 1. Extract Zhoumai 22 genomic DNA as a template;
[0047] 2. Using wmc692 and wmc657 as primers and Zhoumai 22 genomic DNA as template, carry out PCR amplification. 10μL PCR reaction system: 10×buffer 1 μL, 10 mmol L -1 dNTPs 0.2 μL, 4 μmol L -1 Primer 1 μL, 40 ng μL -1 DNA template 1 μL, Taq Enzyme 1 U, ddH 2 O 6.7 μL.
[0048] The PCR reaction program was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; the annealing temperatures of primers wmc692 and wmc657 were 51°C and 61°C respectively, the annealing time was 1 min, and extension at 72°C for 1 min; 35 cycles; Extend for 10 min at ℃ and store at 4℃.
[0049] 3. Separation and detection of the amplified products: add 2.5 μL of 6×Loading buffer to the amplified products, separate by electrophoresis in a non-denaturing polyacrylamide gel with a concentration of 12% (w / v), and develop the color by silver staining. Such as figure 1 and figure 2 shown ( figure 1 Non-denaturing polyacryl...
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