InDel molecular marker for identifying and/or distinguishing brassica oleracea and application of InDel molecular marker
A molecular marker, cabbage technology, applied in the field of molecular genetics and breeding, can solve the problem of too many primers
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Embodiment 1
[0031] Example 1 Identification of polymorphic sites and development of InDel molecular markers
[0032] 1. Material handling
[0033] In this example of the present application, 48 parts of wild cabbage, 84 parts of cauliflower, 168 parts of broccoli, 105 parts of cabbage, 6 parts of cabbage and 10 parts of kale (421 parts in total) identification.
[0034] These 421 materials were sown and planted normally in the experimental base of Wuhan Institute of Vegetable Science. Leaf tissue sampling was performed at the seedling stage, and the samples were quickly stored at -20°C.
[0035] The whole genome DNA of leaves of each material was extracted by CTAB method. The specific method is:
[0036] 1) Put 0.1-0.2 g of leaves into a 2 ml centrifuge tube containing one steel plant, add 700 μl of 2% CTAB, and fully grind for 5 min using a grinder at 60HZ.
[0037] 2) 65°C water bath for 1h (shake every 20min); 60-65°C water bath for 45min-1h is acceptable.
[0038] 3) Add an equa...
Embodiment 2I
[0051] The application verification of embodiment 2InDel labelling primer
[0052] Using the whole genome DNA of the sample as a template, using the developed InDel-labeled primer PCR amplification reaction system and amplification program to carry out PCR amplification, and subjecting the PCR products to polypropylene gel electrophoresis, different allelic variations can be detected and distinguished. Validation was performed on six cabbage species (421 total), 48 wild cabbage, 84 cauliflower, 168 broccoli, 105 head cabbage, 6 bulbs, and 10 kale. The total volume of the PCR amplification reaction system was 25 μl, consisting of 9.5 μl ddH 2 O, 12.5 μl Green Taq Mix, 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer and 1 μl whole genome DNA. The PCR amplification reaction program was: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 15 s, 35 cycles; extension at 72 °C for 7 min.
[0053] Polyacrylami...
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