Method for identification of spring barley varieties/genotypes in the androgenesis process and marker for identification of spring barley varieties/genotypes in the androgenesis process

摘要

The purpose of the notification is to identify the varieties/genotypes of spring barley in the androgenesis process and to identify the varieties/genotypes of spring barley in the androgenesis process. The method of identification of varieties/genotypes of spring barley in the androgenesis process shall be that the stem of the test material and at least one control material favours two control materials after reaching an appropriate stage of development,where they contain micro-spores in the early to medium-to-medium development stage, they are cut and placed in water. Personal test material spike and separate control material spike. At least one variant shall be used as control material,favourable two varieties, with a known level of regeneration, which serves as a negative and positive control of the analysis. The needle is then sterilised with 70-100% ethanol and, under sterile conditions, the logs are removed from which the bone is removed by sterile sieve. The bait shall be taught and stored to provide adequate moisture. It's okay. It's okay. One millimeter fragments and puts in a cooled blender chamber. 0,4 M mannitol is added to the blender chamber and is blended twice at idle speed. Then the faded suspension passes through 100msterile filter for the container on ice. The remaining logs are collected from the filter and blended twice into 0,4 M mannitol to release as many microspores as possible and re-filtered through a 100m sterile filter. The microspora suspension is transferred to a sterile tube and rotates at 4.oC. After swirling, the supernatant is removed.and the microspores suspended in mannitol are transferred to the test tube and rotate at room temperature. The supernatant is then removed by pipette to completely dry the micro-spore sediment. Immediately isolate the RNA from the micro-spore sediment using the appropriate set and obtain the RNA isolate. DNA contamination is removed from the RNA isolate by using the DNA and buffer and the entire digestive response is supplemented by ddH2O. The amount of RNA solution to be added depends on the concentration of the RNA isolate. The reaction is then incubated in a thermocycle, preferably at temperature 37oC,and then inactivates the enzyme. Then synthesize cDNA using the appropriate set using random starters. So prepared cDNA is frozen,favourable at temperature -21oC until the test and control material is evaluated. The identification of the test and control material shall be performed in the RT-PCR (Reverse transcriptase polymerase chain reaction) and/or RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction). The method is characterised by the RT-PCR and/or RT-qPCR reaction using buffer, DNA polymerase, dNTPs, starters and cDNA. The reaction is performed for the GBSSI (Granulebound old synthases) using starters and an internal control gene.