SSR molecular marker primer set for identifying purity of variety Shandong cotton research number 29 and application thereof
A technology of molecular markers and species purity, applied in the field of biotechnology, achieves good results, is economical and convenient, and improves the efficiency of electrophoresis detection
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Embodiment 1
[0051] Method: 24 cotton varieties that have been approved by the country and Shandong Province were used as sample materials to obtain SSR core primers, and the primers with high polymorphism, good stability and repeatability, and covering the cotton genome were selected as the characteristic primers of Lumianyan No. 29 Candidate primers were screened, and finally the characteristic primers of Lumianyan 29 were determined according to the molecular marker fingerprint results. The variety clustering diagram of 24 cotton sample materials is as follows Figure 11 shown.
[0052] Table 1
[0053]
[0054]
[0055] 1. Cotton Genomic DNA Extraction
[0056] The experimental materials in this example were 24 cotton varieties approved by the state or Shandong Province, all of which were purchased from the Shandong Provincial Seed Management Station.
[0057] Taking 24 cotton varieties approved by the state or Shandong Province as sample materials, soak dry cotton seeds at ro...
Embodiment 2
[0067] The characteristic primer of Lumianyan No. 29 determined in Example 1 was used to identify the seed purity of the variety to be tested.
[0068] The experimental material of the present embodiment is the market sale seed of No. 29 Lumianyan, and the variety purity detection process is as follows:
[0069] 1. DNA extraction
[0070] The cotton genome DNA extraction method is the same as that in Example 1.
[0071] 2. SSR-PCR amplification
[0072] 5 pairs of characteristic primers (Table 2) of Lumianyan No. 29 were selected for PCR amplification, and the reaction system was:
[0073] 10×PCRBuffer 1 μL, 10mMdNTPMix 0.2 μL, specific SSR forward and reverse primers (10 μmol / L) 0.6 μL each, 5UTaq DNA polymerase 0.1 μL, test sample DNA (20-200ng / μL) 2 μL, ddH 2 O5.6 μL.
[0074] Reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 55°C for 45 s, extension at 72°C for 50 s, and 32 cycles; extension at 72°C for 5 min; storage at ...
Embodiment 3
[0081] Using the Lumianyan No. 29 characteristic primer determined in Example 1 to identify the seed purity of the Lumianyan No. 29 standard variety mixed with other varieties.
[0082] The experimental material of this embodiment is the Lumianyan No. 29 standard variety sold by Shandong Luyi Cotton Industry Technology Co., Ltd. In this experiment, other varieties are mixed in the Lumianyan No. 29 standard variety. The purity of the Lumianyan No. 29 variety The detection process is as follows:
[0083] 1. DNA extraction
[0084] The cotton genome DNA extraction method is the same as that in Example 1.
[0085] 2. SSR-PCR amplification
[0086] 5 pairs of characteristic primers (Table 2) of Lumianyan No. 29 were selected for PCR amplification, and the reaction system was:
[0087] 10×PCRBuffer 1 μL, 10mMdNTPMix 0.2 μL, specific SSR forward and reverse primers (10 μmol / L) 0.6 μL each, 5UTaq DNA polymerase 0.1 μL, test sample DNA (20-200ng / μL) 2 μL, ddH 2 O5.6 μL.
[0088] R...
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