Method for quickly identifying purity of watermelon variety Zhentian 1217 by using EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker
A watermelon variety and molecular marker technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as affecting the efficiency and accuracy of variety identification, inability to meet, poor accuracy, etc., to save manpower and overcome errors , the effect of high accuracy
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Embodiment 1
[0032] Example 1 Obtaining Genomic DNA of Zhentian 1217 Variety Leaves
[0033] The specific operation method is as follows:
[0034] (1) Put the "Zhentian 1217" parents and F1 seeds into a petri dish covered with damp filter paper, cultivate in the dark at 30°C for 3 days, and then take the young roots;
[0035] Among them, in the process of hybrid seed production of this variety, the female parent needs to be artificially detasselled, and the pollen of the male parent is given to complete the hybridization process, and finally the F1 generation seeds on the female parent are harvested;
[0036] (2) Put a young watermelon root 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 ;
[0037] The extraction buffer described therein is 2×CTAB extraction solution, and its proportioning is as follows:
[0038]
[0039] Sterilize at high temperature, cool to ro...
Embodiment 2
[0045] Embodiment 2PCR amplification
[0046] Using the RNA enzymatic mixture obtained above as a template, use ZT1217 primers for PCR amplification:
[0047] The PCR reaction system is 10 μL, including 1 μL of 10×PCRBuffer, 0.2 mMdNTPs, 5 μM of primers, 0.5 UTaq DNA polymerase, 100 ng of template, and make up to 10 μL with sterile ultrapure water;
[0048] According to the present invention, the amplification program is set as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 20 s, 35 cycles; final extension at 72°C for 5 min; PCR products were stored at 10°C.
[0049] Example 3 Amplification product detection
[0050] 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 12%, 180v constant voltage electrophoresis for 1.5h, and silver staining for band pattern statistics;
[00...
Embodiment 4
[0065] Example 4 Identification of the purity of "Zhentian 1217" hybrids
[0066] According to the electrophoresis results of the specific bands, the purity of the "Zhentian 1217" hybrid was identified, and the judgment criteria were:
[0067] The female parent has a specific band of 159bp; the male parent has a specific band of 147bp; the hybrid has two specific bands of 159bp and 147bp;
[0068] According to the above identification results, the purity of Zhentian 1217 variety (%)=(1-n / N)×100% can be converted, where N is the number of watermelon seeds to be tested, and n is the band characteristic of the male parent alone or the female parent alone The number of plants whose band characteristics were either different from the band characteristics of "Zhentian 1217" or the parents.
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