A method and primers for rapidly identifying the purity of hybrid seeds of watermelon variety 'Exigua 16'
A technology for hybrid seed purity and watermelon varieties, which is applied in the field of agricultural melon and vegetable breeding and application, can solve the problems of long cycle, high cost, and inaccuracy, and achieve the effects of high accuracy, labor saving, and low cost
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Embodiment 1
[0034] Embodiment 1 is used for the SSR primer of watermelon variety ' Exigua No. 16 ' hybrid seed purity identification development, comprises steps:
[0035] First, download the SSR sequence according to the published watermelon genome sequence (www.icugi.org / );
[0036] Then, DNAstar software was used to design 100 pairs of PCR primers spanning the SSR site, which were synthesized by Invitrogen.
Embodiment 2
[0037] The extraction of the genome DNA of embodiment 2 watermelon varieties ' No. 16 of Exigua' comprises the steps:
[0038] Select 30mg-40mg of tender tissue of the watermelon hybrid variety 'E Xigua 16' after 3-4 days of germination, put it in a 2.0ml pre-cooled centrifuge tube, and quickly sprinkle about 0.5mg of DIECA crystals on the tissue, Add liquid nitrogen, crush the sample into powder with a tissue masher, add 700 μL 2% CTAB preheating buffer and 0.5 mg of activated carbon, bathe in water at 65°C for 1 hour, add 700 μL 24:1 chloroform-isoamyl alcohol, mix well, Centrifuge at 12,000rpm for 10min; absorb the supernatant and add it to a 1.5mL centrifuge tube pre-filled with 700μL isopropanol, mix well and put it in a -20°C refrigerator for 30min, then centrifuge at 12,000rpm for 10min; discard the supernatant, and use 70% ethanol solution After washing twice and drying under natural conditions, add 100 μL ddH 2 O, check the concentration after fully dissolved, store ...
Embodiment 3
[0039] Example 3 Utilize the upstream primer: EXGF: 5'-CATTTCCGTTTCCATTTTCTTCAC-3' and the downstream primer: EXGR: 5'-AAGTAACATCAAGCGATTCGCCAT-3' to prepare the standard map of the seed purity of the watermelon variety 'E Xigua No. 16':
[0040] (1) Extraction of DNA: Take 5 grains of 'E Xigua No. 16' F1, 1 grain of the male parent, and 1 grain of the female parent, and extract genomic DNA respectively with reference to the improved CTAB method of Example 2;
[0041] (2) PCR reaction system and amplification program: Add dNTP 0.25mmol / L, forward and reverse primers 0.2μmol / L, TaqDNA polymerase 1.0U, 10×PCR buffer (containing Mg 2+ ), 'E Xigua No. 16' sample genomic DNA 60ng, sterilized double distilled water to add to 15μL, use fluorescently labeled primers when detecting with a DNA analyzer; set up a blank control experiment at the same time, and use sterile water instead of genomic DNA in the blank control experiment; After mixing, put it on a PCR machine for amplification....
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