Studies On Rapid Testing Genetic Puirty Of Maize Seeds With Molecular Technique

Seed sales process often appears tort or illegal business activities. Breeders, operatorsand users pay more and more attention to quality of seeds. The genetic purity of maize seedshas received much attention. There are many methods for testing the genetic purity of seeds,and traditional detection techniques still have an important role. However, they struggle tomeet the requirements of rapid and accurate testing genetic purity of seeds because they havemany disadvantages. The accuracy of testing with molecular technique is very high, but thetesting time is long now. This study based on SSR markers, through rapid DNA extractionmethod from half a maize seed for PCR and universal multiplex PCR technique research anddevelopment, established a rapid testing genetic purity of maize seeds with moleculartechnique. This technique is very important in maize varieties protection, testing geneticpurity and authenticity of seeds, and it is of great significance to ensure the quality of maizeseeds. The main results were as follows:1. Through the study of DNA extraction method, we established a rapid DNA extractionmethod from half a maize seed for PCR. We have studied the effect of concentration andvolume of extracting solution and extracting time on integrity of DNA and PCR products. Wehave also studied the effect of amount of DNA templates and Mix from various companies onPCR products. The parameters of DNA rapid extraction method were confirmed. Slittingmaize seeds along the embryo and then take half of maize seeds (must contain embryo) into1.5mL centrifuge tube. Add200μL–1000μL extracting solution (0.03M–0.2M KOH orNaOH solution) and extract1min–2d, then blending extracting solution. Mixing theextracting solution is the DNA sample.0.5μL–5μL the DNA samples can be used directlyfor PCR amplification.2. Through the study of multiplex PCR technique, we established a universal multiplexPCR technique. We have studied primers, PCR amplification system and PCR procedure, andvalidate the feasibility of the technique. The mode of execution was confirmed. Universal adapter-F (5’-CTCGTAGACTGCGTACCA-3’) and universal adapter-R (5’-TACTCAGGA-CTCATCGTC-3’) were linked to the5’ end of the forward and reverse SSR primers,respectively. U-primers include universal adapters and SSR primers. UM-PCR reactions werein a20μL reaction volume, which contained10μL2×Power Taq PCR MasterMix (BioTeke,Beijing, China),3μL DEPC ddH2O,0.5μL of10μM (a total of five pairs of adapter-primers)adapter-primers (Sangon, Shanghai, China) and2μL DNA. UM-PCR amplification wasimplemented using a novel PCR procedure, termed “Two Rounds Mode”(3and28–32cycles).①Initial denaturation (94°C，5min).②The first round (the first three cycles) wastermed “One by One Annealing Round”. Denaturation (94°C，40s); annealing temperaturesthat are optimized in accordance with the Tm of normal SSR primers are sorted in descendingorder (Annealing temperatures may be different in various combinations of U-primers.),20sat per annealing temperatures; extension (72°C，30s).③The second round (28–32cycles,usually30cycles) combines annealing with extension. Denaturation (94°C，40s); combiningannealing with extension (70°C，50s).④Final extension for10min at72°C. UM-PCRproducts can be electrophoresed in9%polyacrylamide gel. Application of UM-PCR cansimultaneously detect multiple pairs of primers. UM-PCR improves the universality ofmultiplex PCR.3. Rapid DNA extraction method from half a maize seed for PCR and universalmultiplex PCR technique are combined into a rapid testing genetic purity of maize seeds withmolecular technique. Zhengdan958, Xianyu335and Jundan20seed samples were used forverifying rapid testing genetic purity of maize seeds with molecular technique. Therefore, thetechnique improves the testing speed of genetic purity of maize seeds with moleculartechnique.