Pepper hybrid seed purity testing EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker and application thereof
A hybrid hybrid purity and molecular marker technology is applied in the field of pepper hybrid purity detection EST-SSR molecular markers, which can solve the problems of poor identification result accuracy, long identification cycle, time-consuming and labor-intensive, etc., and achieve labor saving, low cost, and DNA quality. less demanding effects
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[0028] (1) Sow 186 seeds of 'Rela No. 1' in a nutrient bowl, and when the plants grow to 5-6 true leaves, use the improved CTAB method (Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc, 2006, 1:2320–2325) to extract genomic DNA from leaves of a single plant.
[0029] (2) The PCR amplification reaction system and procedure are as follows: PCR reaction system is 10 μL, including 10×PCR Buffer 1 μL, 0.2 mM dNTPs, primer (HE4) 50 ng, 0.5 U Taq DNA polymerase, 'Ran La No. 1' genomic DNA 20 ng , make up to 10 μL with sterile ultrapure water. The amplification program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 1 min, 35 cycles; 72°C final extension for 10 min. PCR products were stored at 10°C.
[0030] (3) Detection of amplified products: 5 μL of amplified products mixed with 2 μL of loadi...
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