A kind of ssr primer and its application for the purity identification of Mobao wax gourd hybrid seeds
A hybrid seed purity, wax gourd technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of short identification period, broad application prospect and low cost
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Embodiment 1
[0038] The screening process of the SSR primer SSR11c160 of the Mobao wax gourd hybrid seed purity identification provided by the present embodiment is as follows:
[0039] Several pairs of primers designed according to the genome sequencing results of wax gourd were found in the parents (male and female parents of Mobao wax gourd) and F 1 The primer SSR11c160 for the co-dominant differential marker band was selected, and the marker band was clear and reproducible. The sequence is as follows:
[0040] SSR11c160-F: 5'-GCCCACACTCACCTACACA-3' (SEQ ID NO.1);
[0041] SSR11c160-R: 5'-AGCCCCACCGAATTAATGCT-3' (SEQ ID NO. 2).
[0042] Using wax gourd genomic DNA as a template, use the above SSR primer SSR11c160 for PCR amplification—PAGE for polyacrylamide gel electrophoresis of the amplified product—recover DNA from specific fragments (polyacrylamide non-denaturing gel)—PCR amplification—agarose gel Recovery—After TA clone sequencing, the analysis results showed that the primer SSR...
Embodiment 2
[0044] The Mobao wax gourd hybrid seed purity identification method provided by the present embodiment comprises the following steps:
[0045] (1) Extraction of wax gourd DNA
[0046] The experimental material is the cotyledon of Mobao wax gourd hybrid and its male and female parents. The steps for extracting cotyledon DNA are as follows:
[0047] ①Put a piece of cotyledon into a 2mL centrifuge tube, add liquid nitrogen and grind it with a grinding pestle, quickly add 800 μL 2% CTAB extract when the liquid nitrogen is quickly evaporated to dryness, mix well and place in a 65°C water bath for 45 minutes (shake well once every 10 minutes) );
[0048] ② After standing at room temperature, add 800 μL of chloroform:isoamyl alcohol (24:1), mix gently, let stand for 2 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant (about 510 μL) to a new 1.5 mL centrifuge tube ;
[0049]③ Add 1 / 3 volume of NaAc (3mol / l) to the supernatant, add 1.5 times the volume of sup...
Embodiment 3
[0067] The method of embodiment 2 is adopted to carry out field planting and sampling to Mobao wax gourd hybrid, the sample is 80 in total, its single plant is numbered, and the single plant DNA is extracted and detected (see figure 2 ); Simultaneously, the purity identification was carried out by morphological detection method. Test results: 74 hybrid seeds, 6 female plants (the one in the red box is the female parent plant), the seed purity is 92.5%, which is consistent with the field test results.
[0068] The above examples show that the method of the present invention can effectively distinguish Mobao wax gourd seeds from their parent seeds, and accurately and quickly detect the purity of the seeds.
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