Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers
A molecular marker, golden pepper technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of limited information, difficult hybridization, long cycle, etc., to achieve good repeatability and stability, popularization Promising, easy-to-use effects
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Embodiment 1
[0031] The acquisition of embodiment 1 golden pepper variety leaf genomic DNA
[0032] The specific operation method is as follows:
[0033] (1) Put the "Golden Pepper" parent and F1 seeds into a petri dish covered with damp filter paper, light intensity 2000lx, light cycle 16h light / 8h dark, light culture at 28°C for 6 days and then take the leaves;
[0034] The process of obtaining the F1 seeds described therein is as follows: after the female parent plant with pollen is pulled out during flowering, the female parent plant and the male parent plant are 4:1, through artificial pollination, the male parent pollen is pollinated to the female parent, and finally harvested F1 generation seeds on the female parent;
[0035](2) Put a young pepper leaf in each centrifuge tube, add 500 μL extraction buffer preheated at 60-65°C, put 4 steel balls, crush the material on a crusher, and bathe in water at 60-65°C for 20-30min ;
[0036] The extraction buffer described therein is 2×CTAB...
Embodiment 2
[0044] Embodiment 2 PCR amplification
[0045] Using the RNA enzymatic digestion mixture obtained above as a template, PCR amplification was performed using ClSH1 primers:
[0046] The PCR reaction system is 20 μL, including 2 μL of 10×PCR Buffer, 0.4 mM dNTPs, 10 μM of primers, 1U Taq DNA polymerase, 100 ng of template, and make up to 20 μL with sterile ultrapure water;
[0047] The amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min; PCR products were stored at 10°C.
[0048] Example 3 Amplification product detection
[0049] 5 μL of amplified product was mixed with 2 μL of loading buffer, and then subjected to non-denaturing polyacrylamide gel electrophoresis with acrylamide mass volume ratio of 10%, 160v constant voltage electrophoresis for 1.5h, and silver staining for band pattern statistics;
[0050] The loading buffer used i...
Embodiment 4
[0064] Embodiment 4 identifies the purity of " golden pepper " hybrid
[0065] According to the electrophoresis results of the specific bands, the purity of the "Golden Pepper" hybrid is identified, and the judgment standard is:
[0066] The female parent has specific bands of 120bp, 200bp, 250bp, and 270bp; the male parent has specific bands of 120bp, 200bp, 250bp, and 260bp; the hybrid has specific bands of 120bp, 200bp, 250bp, 270bp, 400bp, and 450bp;
[0067] According to the above-mentioned identification results, the purity of the pepper variety (%)=(1-n / N)×100% can be converted, where N is the number of pepper seeds to be tested, and n is the band characteristic of the male parent alone or the spectrum of the female parent alone The number of plants with band characteristics or different band characteristics from both "Golden Pepper" and parent parent.
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