Primer, kit and method for flux identification of major dormant genes in wheat seeds
A technology for wheat seeds and genes, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Good, reliable results
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Embodiment 1
[0023] Embodiment 1: Extraction of DNA and design of PCR primers
[0024] (1) Extraction and detection of DNA
[0025] The total DNA of wheat seeds was extracted by CTAB method, and the quality and concentration of DNA were detected by ultraviolet spectrophotometer.
[0026] (2) PCR primer design
[0027] The mutation characteristics of the allelic sequences were analyzed and compared, and Primer5 biological software was used to design gene-specific molecular marker primers for the gene amplification region, ensuring that the continuous complementarity between the primers did not exceed 4 bp to prevent cross-mismatching. The primer sequences are shown in Table 1. The first PCR reaction uses a primer combination consisting of Vp1-B1 gene forward primer 1 and reverse primer, TaPHS1 gene forward and reverse primer pair and PM19-A1 gene forward and reverse primer pair. The essence of the first PCR is multiplex PCR, which is improved on the basis of traditional PCR principles T...
Embodiment 2
[0034] Example 2: Establishment of PCR system and sequencing verification
[0035] Further optimize the PCR reaction system and thermal cycle conditions, and use the primer combination of Example 1 to carry out PCR amplification and detection of wheat materials containing different alleles:
[0036] The total volume of the first (multiple) PCR reaction system is 20ul, including 1×PCR Buffer (Mg2+Free), 2.5mMMgCl 2 , 300uM dNTP, 2U of Taq enzyme and 400ng of template DNA, the upstream and downstream primers of the PHS1 gene are 10pmol, the upstream and downstream primers of the VP1-B1 gene are 12pmol, and the upstream and downstream primers of the PM19-A1 gene are 2pmol.
[0037] The conditions for the first (multiple) PCR amplification were: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 1 minute, and 10 cycles. Denaturation at 94°C for 30S, annealing at 56°C for 30S, extension at 72°C for 1mi...
Embodiment 3
[0042] Embodiment 3: the identification of main effect dormant allele of wheat variety bred in Sichuan
[0043] The molecular markers of the present invention were used to identify the allelic composition of 98 Sichuan wheat cultivars at three major dormant sites. The DNA extraction and molecular marker PCR detection methods are the same as in Example 1 and Example 2. The result is as image 3 , Figure 4 as shown, image 3 Indicates the identification results of the main dormant genes in 15 of the test materials by the first (multiple) PCR. For the VP1-B1e and VP1-B1c alleles that cannot be distinguished in the detection materials in lanes 12 to 15, continue to use the second The second PCR was used to identify, and the results showed that they were all alleles VP1-B1e, such as Figure 4 shown. The experimental data showed that there were 5 allele types at the VP1-B1 locus in 98 Sichuan wheat cultivars. Among them, 64 materials contained VP1-B1e with a frequency of 65.3...
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