Specific Primers Related to Low Protein of Weak Gluten Wheat Ningmai No. 9 and Its Application
A low-protein, specific technology, applied in biochemical equipment and methods, microbial determination/testing, DNA/RNA fragments, etc., can solve problems that have not been reported
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Embodiment 1
[0024] Example 1 Screening of SNP sites related to low protein of weak gluten wheat variety Ningmai No. 9 and designing primers
[0025] In this example, 101 wheat varieties from the winter wheat region in the middle and lower reaches of the Yangtze River were used as materials (Table 1), and all materials were planted in the experimental base of the Jiangsu Academy of Agricultural Sciences and the Liuhe experimental base in two consecutive growing seasons in 2015-2016 and 2016-2017 , Field planting in 3 rows of plots. The seeds were harvested and dried in 2016 and 2017 respectively, and the protein content of the grain was determined according to the method of GB / T 24899-2010.
[0026] Table 1 Wheat Varieties
[0027]
[0028] The CTAB method was used to isolate DNA from the young leaves of the test material plants, and the genome scan of the test material was performed using the Illumina 9k gene chip (for details, see: Cavanagh et al., 2013, Genome-wide comparative diver...
Embodiment 2
[0035] Embodiment 2 wheat genotype detection
[0036] The detection steps are as follows:
[0037]From the 101 test materials described in Example 1, randomly select 24 varieties (see Table 2) to carry out PCR amplification and enzyme digestion reaction successively, and the enzyme digestion product is carried out agarose electrophoresis detection, and the detection results are shown in figure 1 , and compared with the gene chip detection results.
[0038] Table 2 Testing materials
[0039]
[0040] 1. PCR reaction
[0041] The PCR reaction system is 10μL, including 10×buffer 1μL, 15mmol·L –1 MgCl 2 0.6 μL, 2mmol·L –1 dNTP 0.8μL (Takara), 20ng·μL –1 1 μL each of upstream and downstream primers, 0.1 μL Taq enzyme (Takara), 50 ng·μL –1 Template DNA 3 μL (CTAB method extraction template) 3 μL, deionized water 3.5 μL;
[0042] The PCR amplification program was: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension a...
Embodiment 3
[0047] Example 3 Correlation Detection of Protein Content in Ningmai No. 9 Advanced Generation Breeding Material
[0048] From the high-generation breeding population (F7 generation), all of its parents have Ningmai 9, and its combination information, phenotype and genotype performance are shown in Table 3:
[0049] Table 3 Names of high-generation breeding population materials
[0050]
[0051] The detection method is as follows:
[0052] 1. PCR reaction
[0053] The PCR reaction system is 10μL, including 10×buffer 1μL, 15mmol·L –1 MgCl 2 0.6 μL, 2mmol·L –1 dNTP 0.8μL (Takara), 20ng·μL –1 1 μL each of upstream and downstream primers, 0.1 μL Taq enzyme (Takara), 50 ng·μL –1 Template DNA (CTAB method) 3 μL, deionized water 3.5 μL;
[0054] The PCR amplification program was: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 90 s, 36 cycles; extension at 72°C for 10 min;
[0055] The PCR amplificati...
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