Bioscience Method 2024, Vol.15, No.1, 1-8 http://bioscipublisher.com/index.php/bm 6 conventional PCR, the method significantly shortens the amplification time and has a sensitivity 100 times higher than conventional PCR. Additionally, this method does not require an expensive PCR instrument and can be amplified at temperatures ranging from 25 °C to 45 °C. Using RPA-badh2-E7, 36 core restoring lines of hybrid japonica rice and one new hybrid japonica rice variety Shenyou R1 from the Rice Center Resource Library of Shanghai Academy of Agricultural Sciences were identified. It was found that this method can effectively distinguish homozygous Badh2, homozygous Badh2-E7, and heterozygous genotypes, greatly improving the efficiency of molecular marker assisted selection of rice aroma genes and the breeding of fragrant rice varieties. 3 Materials and Methods 3.1 Research materials All rice samples used in this study are the backbone parents or varieties of hybrid japonica rice selected by the Rice Center Heterosis Utilization Research Group of the Crop Breeding and Cultivation Research Institute of Shanghai Academy of Agricultural Sciences, including 36 restoring line resources and 1 hybrid japonica rice variety. The RPA isothermal amplification kit (TABAS03KIT) was purchased from TwistDx Co., Ltd. The RPA amplification primers were synthesized by Biotechnology (Shanghai) Co., Ltd. 3.2 Extraction of rice DNA The DNA extraction from rice leaves was carried out using an improved CTAB method (Murray and Thompson, 1980), and the extracted DNA was measured and quantified using a NanoDropTM 2000 micro spectrophotometer. Take a leaf with a length of about 1.5 cm and place it in a 2 mL centrifuge tube; Join 750 μL 1.5×CTAB solution (1.5% CTAB, 75 mmol/L Tris-HCl, 15 mmol/L EDTA, 1.05 mol/L NaCl, ph 8.0) and a steel ball with a diameter of 6 mm; Set the frequency parameter of the plant tissue rapid grinder to 65 Hz and oscillate for 90 seconds; After grinding, the sample is incubated in a 65 °C water bath for 45 minutes, with 500 μL chloroform added, vigorously shaken, centrifuged at 12 000 r/min for 8 minutes; Transfer 500 μL of supernatant to a new 1.5 mL centrifuge tube, add an equal volume of anhydrous ethanol, mix up and down, place the centrifuge tube in a -20 °C freezer for 1 hour, centrifuge at 12 000 r/min for 8 minutes, discard the supernatant, air dry or dry at 37 °C, then add 500 μL of ddH2O to dissolve DNA. Store the dissolved DNA in a -20 ℃ freezer for later use. 3.3 RPA reaction system The RPA amplification system includes 29.5 μL Primer free rehydrogenation buffer, 2.4 μL each of 10 μM forward and reverse primers, 1 L DNA template, 12.2 μL sterile ddH2O, and a total volume of 50 μL. Gently mix with a pipette and transfer the mixture to a TwistAmp reaction tube containing freeze-dried enzyme powder. Then add 2.5 μL of magnesium acetate with a concentration of 280 mmol/L, mix well, and perform constant temperature amplification. Unless otherwise specified, the amplification conditions are 39 °C and incubate for 20 minutes. Authors’ contributions ZJH was the experimental designer and executor of this study; ZJH and ZAP completed data analysis and wrote the first draft of the paper; XWK, CC, NFA, SB, ZJM participated in experimental design and analysis of experimental results; CLM and CHW are the project conceptualizers and leaders, guiding experimental design, data analysis, paper writing, and revision. All authors read and approved the final manuscript. Acknowledgements This study was jointly funded by the Shanghai Rice Industry Technology System Construction Project (Hu Nong Ke Chan Zi (2023) No. 3), the Shanghai Science and Technology Innovation Action Plan Agricultural Science and Technology Field Project (23N61900100), and the Shanghai Science and Technology Innovation Action Plan Natural Science Foundation Project (23ZR1455600).
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