T/CQAAS 006-2023 Active

Main Technical Content: 3. Terms and Definitions The following terms and definitions apply to this document. 3.1 Standard Samples Standard samples are representative samples of known varieties, certified by authoritative institutions, and used in this standard for crop seeds, i.e., planting or propagation materials, including grains, fruits, roots, stems, seedlings, buds, leaves, etc. 3.2 Molecular Markers Molecular markers refer to specific DNA fragments that reflect genetic differences between individuals or populations. 3.3 SSR Markers SSR markers are tandem repeat sequences composed of several nucleotides (generally 1–6), with polymorphism in SSR length due to the number of repeat units. 3.4 DNA Fingerprints DNA fingerprints refer to the polymorphism amplified by molecular markers such as SSR. 3.5 Variety ID Cards Variety ID cards reflect the commercial attributes and DNA fingerprints of a variety. 4 Methods 4.1 DNA Extraction The modified CTAB method was used to extract DNA from fresh pepper leaves, involving the following steps: (1) Preheat the pre-prepared 2×CTAB extraction buffer (0.1 M Tris-HCl, 0.02 M EDTA, 1.4 M NaCl, 2% PVP, 2% CTAB) at 65°C in a water bath. (2) Grind the pomegranate leaves to a powder and transfer them to a 2.0 mL centrifuge tube. (3) Add 1.0 mL of 2×CTAB extraction buffer and 10 μL of β-mercaptoethanol, mix well, and incubate at 65°C for 60 minutes, with inversion and shaking every 10–15 minutes. (4) Centrifuge at 12,000 rpm at room temperature for 5 minutes, and transfer the supernatant to a new 2.0 mL centrifuge tube. (5) Add an equal volume of chloroform/isopropanol, mix well for 5 minutes, and centrifuge at 12,000 rpm at room temperature for 10 minutes. (6) Transfer the supernatant to a new 2.0 mL centrifuge tube and repeat the extraction process once. (7) Centrifuge, transfer the supernatant to a new centrifuge tube, and add an equal volume of isopropanol to mix well. Incubate at -20°C