Extraction of Seed DNA from Yunnan Perilla Using Seed DNA Extractor

Twenty perennial varieties of perilla were collected in Yunnan and evaluated for many years of cultivation and economic traits. It was found that there are abundant genetic variations in Perilla species, and the method of morphological cluster analysis has been used to plant perilla in Yunnan. The variation was explored and divided into five variants. As morphological characteristics are the product of interaction between genotype and environment, the results of morphological cluster analysis of Perilla frutescens only indirectly reflect the genetic diversity of Perilla frutescens in Yunnan. The experiment used Yunnan perilla seed as material to study the extraction of genomic DNA and the selection of RAPD primers, which laid a foundation for directly revealing the genetic diversity of Yunnan perilla. The seed DNA extractor is an important instrument for the extraction of seed DNA.

The seed DNA extraction instrument played an important role in this research. The extraction of the seed DNA of Perilla frutescens was based on the improved CTAB method for extraction of tobacco DNA, with slight modifications. The reagents were all domestic analytically pure. Take 0.1~0.2g seeds, place them in a mortar cooled at -20°C, and grind them into a 1.5mL centrifuge tube. Add 0.35mL of 1×DNA preheated 55°C extract (50mmol). /LTris-HCl pH7.5, 10mmol/LEDTApH8.0, 0.7mmol/L NaCl, 1% β-mercaptoethanol, 1% CTAB), 3 min later, 2× DNA extract (concentration twice as much as the former) was placed in 55 Incubate overnight in the incubator, invert several times between them, add 0.7mL saturated phenol, mix and extract for 3h on the homemade mixer (2r/min), centrifuge for 10min at 6000r/min, transfer the supernatant to another In a tube, 0.35 mL of saturated phenol and 0.35 mL of chloroform-isoamyl alcohol (24:1) were extracted for 2 h, centrifuged at 6000 r/min for 10 min, the supernatant was taken to another tube, and 0.7 mL of chloroform-isopentyl was added. Alcohol (24:1) was extracted for 2 h, centrifuged at 6000r/min for 10 min, supernatant was taken to another tube, 0.07 mL of 3 mol/L NaAc and 0.7 mL of isopropanol were added, and precipitated at room temperature for 30 min and centrifuged at 3000 r/min for 5 min. The supernatant was discarded and the DNA was washed with 70% alcohol, dried, and then dissolved in 100 μ LTE.

In the study of plant molecular markers, the genomic DNA extracted from the seed DNA extractor generally uses fresh young leaves as a material, and rarely using mature seeds as materials for the extraction of genomic DNA. Since perilla leaves are rich in perilla pigments and phenolic substances, it is difficult to obtain high-quality genomic DNA without special treatment. Therefore, we tried to extract the genomic DNA of Perilla frutescens with modified CTAB method using perilla seed as material. .

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