Indel (Insertion-deletion) molecular marker primer for exocarpium benincasae color gene and application thereof
A wax gourd skin color and molecular marker technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as indistinguishability, achieve simple operation, short identification cycle, and broad application prospects Effect
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Embodiment 1
[0038] The development and verification process of the indel molecular marker primers that control the wax gourd skin color gene provided by this example are as follows:
[0039] According to the preliminary research results of wax gourd skin color gene mapping and candidate gene screening, a pair of specific indel markers black-skinned parent B227, yellow-skinned parent B214 and their F 1 (the first generation hybrid between B214 and B227) was amplified, and as a result, the marker band pattern was clear and reproducible, and the sequence was as follows:
[0040] PC-F: 5'-GGATGACAACGCCTCCATGA-3' (SEQ ID NO.1);
[0041] PC-R: 5'-GACGGGTTTACTCGGGTCAG-3' (SEQ ID NO.2);
[0042]Using the wax gourd genomic DNA as a template, use the above-mentioned indel molecular marker primers for PCR amplification—PAGE polyacrylamide gel electrophoresis for the amplified products—recover DNA from specific fragments (polyacrylamide non-denaturing gel)—PCR amplification—agarose Gel recovery—aft...
Embodiment 2
[0044] The identification method of the wax gourd skin color that the present embodiment provides, comprises the following steps:
[0045] (1) Extraction of wax gourd DNA
[0046] The experimental materials are fresh leaves of B227, B214 and F1, and the steps of extracting genomic DNA are as follows:
[0047] ①Put a small amount of fresh leaves into a 2mL centrifuge tube, add liquid nitrogen and grind with a grinding pestle, quickly add 800 μL 2% CTAB extract when the liquid nitrogen is quickly evaporated, mix well and place in a 65°C water bath for 45 minutes (shaking every 10 minutes) uniform once);
[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 supernatant to absolute ethanol (pre-cool...
Embodiment 3
[0067] The method in Example 2 was used to verify the individual plants of the F4:5 family population of B227 and B214 (the first selfed generation of the F2 population is the F2:3 family, the next selfed generation is F3:4, and then F4:5). There are a total of 288 strains in this group, and the individual strains are numbered, and the individual strain DNA is extracted for detection (see figure 2 , image 3 , Figure 4 );according to Figure 2-4 According to the description in , two bands are heterozygous band type is black skin, one band, the large segment is black skin, and the small segment is yellow skin.
[0068] According to the results of marker detection, there are 124 individual plants with 142bp band type (homozygous black skin), 120 individual plants with 136bp band type (homozygous yellow skin), and 44 plants with heterozygous band type (heterozygous black skin) .
[0069] The results of field character purity identification showed that among 288 individual p...
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