A rapd primer for distinguishing radish varieties and its application
The technology of a variety and radish, applied in the field of molecular biology, can solve the problems of large amount of operation, inability to visually display primers for distinguishing varieties, and lack of practicability, and achieve the effects of wide versatility, early identification, and simple operation
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Embodiment 1
[0052] Example 1: Extraction of DNA.
[0053] The samples of radish varieties of the present invention are common important radish varieties on the market. These 20 varieties include: Manshenhong, Bawangqing, Duanyeshisan, Akita Yanghua, Akita Dahongpao, Akita Mantanghong, Chuanxinhong, Mianyanghong, Yinmei, Fengqiu Qingwang, Yangzhou Yuanbai, Qingpi Tianshuai, Qiye Yuanhong, Zhejiang University Chang, Qiaotouqing, South Korea Dagen, Han Yuchun, Southwest Hongshuai, Mid-Autumn Red, Bai Yuchun.
[0054] About 0.2 g of young radish leaves were ground with liquid nitrogen, and 500 μl of CTAB lysate (2% CTAB, 2M NaCl 2 , 20mM EDTA, 100mM Tris-HCl (PH=8.0) and 0.2% β-mercaptoethanol), transfer to a 1.5ml centrifuge tube, take out and cool in a 65°C water bath for 0.5-1h, add an equal volume of chloroform: isoamyl alcohol ( 24:1) After shaking the mixture gently, centrifuge the emulsion at 12000r / min for 8-10min, extract the supernatant with chloroform / isoamyl alcohol (24:1) for 1-...
Embodiment 2
[0055] Example 2: RAPD-PCR reaction system and conditions.
[0056] RAPD-PCR reaction system is 20μl, containing 50ng genomic DNA, 2.0mmol L -1 MgCl 2 , 0.15mmol·L - 1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase.
[0057] The RAPD-PCR amplification program was pre-denaturation at 94°C for 2min, denaturation at 94°C for 30S, annealing for 45S, extension at 72°C for 90S, 42 cycles, extension at 72°C for 10min, and storage at 4°C. Amplify on a PCR amplification instrument, and the amplified product adopts agarose gel electrophoresis (containing 1.2% ethidium bromide), 1 × TAE electrophoresis buffer (contains in 1L: 0.242g Tris, 0.057ml glacial acetic acid, 0.2 ml 0.25mol / L EDTA pH 8.0). Add 4 μl of 6× loading buffer (containing 0.25% bromophenol blue, 30% sucrose) to the amplified DNA sample, and perform electrophoresis at a constant voltage of 120V for 0.5-0.8 hours until the amplified DNA bands are clearly separated After that, the amplified bands were ...
Embodiment 3
[0058] Example 3: Stringent screening of specific primers.
[0059] The 4 primers of the present invention are obtained through strict screening of 52 random primers originally designed. First, 52 random primers were designed for the genome of the existing radish varieties, and 11 bases of RAPD random primers with good polymorphism, strong amplification and high stability were selected from them, and then the strips were selected by two consecutive gradient PCRs. Take a consistent temperature as the annealing temperature. In detail, the gradient PCR reaction system is 20 μl, containing 50ng genomic DNA, 2.0mmol·L -1 MgCl 2 , 0.15mmol·L -1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase. The reaction was pre-denatured at 94°C for 2min, denatured at 94°C for 30S, annealed for 45S, extended at 72°C for 90S, 42 cycles, and extended at 72°C for 10min. The random primer sequence and annealing temperature of the present invention are as follows:
[0060] RP13: 5'...
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