Application Of Predigestin SSR Analysis Protocol For The Breed Purity Test Of Wheat

Several methods of breed purity test were summarized and Several general markers technique of advantage and disadvantage in breed purity test in the thesis. And we have studied extraction methods of DNA, established the optimum PCR system, selected SSR primer and established a good method of staining used seeds of 9 general varieties in the experiment The study aimed to establish a more economical, simple and precise protocol of wheat SSR analysis for service of seed purity test of wheat. The results were as follows:1) A DNA extraction method for PCR amplification has been established: 300μL of CTAB extracting buffer were added into a fresh 1.5mL Eppenddorf tube .An embryo was added and ground to plasm in the tube. 300μL mixture of chloroform and iso-amyl alcohol (volume ratio 24:1) were added and mixed. Separate the organic and aqueous phases by centrifuge at 10000r/min at 4℃. Transfer the upper aqueous phase to a fresh 1.5mL Eppenddorf tube and add an equal volume of isopropanol and mix. Separate the DNA and aqueous phase by centrifuge at the same conditions and discard the aqueous phase. Dry the DNA in room temperature. Dissolve the DNA in 50μL of ddH2O.2) An optimum reaction system for PCR was set up: In the 10μL reaction system, the concentration of DNA is 1.5μL, PCR buffer (Mg2+ 20 mM) is 1.0μL, Taq DNA polymerase is 0.25μL, dNTPs is 0.2μL and 1.25mM SSR primer is 2.0μL.3) A staining method was selected: Stain 5 min in the mixture of 10% ethanol, 0.5% acetic acid and 0.2% silver nitrate. Wash 25s in distilled water. Develop in the mixture of 2.0% sodium hydroxide and 0.5% formaldehyde. Wash 5 min in distilled water.4) 12 pairs core SSR primers were confirmed from 100 pairs SSR primers of wheat according to the location and DNA band spectrum of these primers. These SSR primers were used for building various multiplex PCR-sets according to their amplified bands size range. Under the conditions as touch down amplification program, 32 two-PCR-sets appeared 5 different amplification behaviors, of which 19 were amplified normally. These screened normally-amplified multiplex-PCR-sets could be applied in purity testing for wheat. The results showed that most multiplex-PCR-sets could be amplified normally under touch down amplification program. Based on our experience, we proposed a simplified protocol for developing a multiplex PCR assay. And these core SSR primers were used for building various multiplex electrophoresis according to their amplified bands size range. 12 pairs core SSR primers appeared 4 different behaviors.5) The experiment system was applied to test seed purity of 61 general varieties and 18 hybrid varieties. The simplified SSR protocol saved 50% time and 75% money than before.