A method for rapid identification of Phytophthora sojae resistance to dimethomorph and special primer pair
A technology of Phytophthora sojae and dimethomorph, applied in the field of molecular biology, can solve the problems of long detection period, heavy workload, and low detection sensitivity, and achieve the effect of delaying further development
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Embodiment 1
[0025] Embodiment 1, Phytophthora sojae is sensitive to dimethomorph
[0026] Three sensitive strains Ps6, Ps13 and PsJMS2; two resistant strains RX9-1 and RX9-2.
[0027] Carrot culture medium: 200g carrots, squeeze the juice, filter through 4 layers of gauze, distill water to 1L, and sterilize with damp heat at 121°C for 20min.
[0028] 1. The experimental steps are as follows:
[0029] 1) Dimethomorph was prepared into 5×10 with dimethyl sulfoxide (DMSO) 4 μg / ml stock solution. For the sensitivity determination of three Phytophthora soybean strains, dimethomorph was diluted to a concentration gradient of 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL and 200 μg / mL . For the sensitivity determination of two Phytophthora sojae resistant strains, dimethomorph was serially diluted to 5×10 3 μg / mL, 1×10 4 μg / mL, 1.5×10 4 μg / mL, 2×10 4 μg / mL and 2.5×10 4 Concentration gradient of μg / mL.
[0030] 2) In the order from low to high, use a pipette to draw 480 ...
Embodiment 2
[0038] Example 2. Discovery of cellulose synthase-related gene CesA3 and its corresponding protein mutation site in Phytophthora soybean
[0039] 1. Strains: resistant strains RX9-1 and RX9-2, sensitive strains Ps6, Ps13 and PsJMS2.
[0040] 2. Method:
[0041] 1) Template: Genomic DNA and cDNA of resistant strains and sensitive strains were used as templates respectively.
[0042] 2) PCR amplification of the full sequence of the cellulose synthase-related gene CesA3
[0043] Primer (Table 2): Ps CesA3F (5'-GTTCGTCGTTGCCTTCACC-3')
[0044] Ps CesA3R(5'-TACTGCCTTCCCGTTTCCT-3')
[0045] Reagents from Beijing Quanshijin Biotechnology Co., Ltd. were used. The 50 μL reaction system included 1 μL template DNA (30-50 ng), 4 μL 10 mM dNTP, 5 μL 10× amplification buffer (containing 20 mM Mg 2+ ), 1 μL Taq DNA polymerase (5U / μL), 1 μL of each primer (10 μM), 37 μL ddH 2 O, the reaction system is multiplied or decreased according to the needs of subsequent experiments. The reaction...
Embodiment 3
[0050] Embodiment 3, the method for detecting mutation site in Phytophthora soybean
[0051] 1. Primer design
[0052] The strains are the strains Ps6, RX2-1 and RX9-1 used in Example 1.
[0053] AS-PCR primers were designed according to the sequence of mutant RX9-1 cellulose synthase-related gene CesA3. The primer pair Ps3070F-Ps3070A / T / C / G.R was used to detect the mutation site of G3070C. Wherein, the last base of the 3'-end of the reverse primer Ps3070A.R is the mutated base, and the forward primer is Ps3070F (Table 2). In order to increase the specificity of the primers, a mismatched base was introduced at the second base at the 3'-end of the reverse primer (Table 2, primers Ps3070T / C / G.R).
[0054] Table 2 detects the PCR primers used for Phytophthora sojae strains showing resistance to dimethomorph
[0055]
[0056] The specific reaction system is the same as that in Example 2, and temperature gradient PCR with different annealing temperatures is performed on the ...
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