Bioscience Method 2024, Vol.15, No.1, 1-8 http://bioscipublisher.com/index.php/bm 5 Figure 5 Identification of rice fragrant gene Badh2-E7by RPA amplification Note: M: DL2000 Marker; 1:Shenfan1; 2:Shenfan2; 3:Shenfan3; 4:Shenfan4; 5:Shenfan6; 6:Shenfan7; 7:Shenfan9; 8:Shenfan10; 9:Shenfan11; 10:Shenfan12; 11:Shenfan13; 12:Shenfan14; 13:Shenfan16; 14:Shenfan17; 15:Shenfan18; 16:Shenfan19; 17:Shenfan21; 18:Shenfan22; 19:Shenfan23; 20:Shenfan24; 21:Shenfan25; 22:Shenhui26; 23:Shenfan27; 24:Shenfan28; 25:Shenfan29; 26:Shenfan30; 27:Shenfan31; 28:Shenfan32; 29:Shenfan33; 30: Shenfan34; 31:Shenfan35; 32:Shenfan36; 33: Shenfan37; 34:Shenfan38; 35:Shenfan43; 36:ShenCR1; 37:Shenyou R1 2 Discussion The cultivation and breeding of fragrant rice has a long history. From ancient times to the present, many high-quality rice varieties with unique aromas have been cultivated both domestically and internationally, such as India's "Basmati type fragrant rice", Thailand's "jasmine fragrance type", Japan's "Gongxiang", the United States' "Jasmine85" and "Della", China's "Northeast rice flower fragrance", "Yunnan crab valley", and "Guangxi Jingxi fragrant glutinous rice" (Jain et al., 2004; Pachauri et al., 2010; Zheng et al., 2012). However, these varieties have geographical characteristics, or low yields and weak resistance (Qi et al., 2020). In recent years, with the improvement of people's living standards, the demand for high-quality fragrant rice has also increased sharply. Therefore, the breeding of more and better fragrant rice varieties has attracted increasing attention. Accurately and quickly identifying aroma traits is an important step in the breeding process of fragrant rice. Chewing method (Berner and Hoff, 1986) and KOH soaking method (Sood and Siddiq, 1978) are commonly used methods for traditional breeding to identify aroma, but the aroma characteristics themselves are greatly influenced by external environmental conditions, and subjective differences among different appraisers lead to low accuracy in aroma identification. In recent years, with the development of molecular biology and sequencing technology, molecular marker assisted selection has been widely applied in the genetic breeding of fragrant rice. At present, the main method for detecting aroma genes using molecular markers is conventional PCR. However, conventional PCR technology requires the use of expensive PCR instruments, and the amplification time is relatively long (about 2 hours), which requires high technical requirements from experimental personnel. These to some extent limit the application of this technology in breeding (Yang and Yu, 2019). RPA isothermal amplification technology, as an emerging isothermal nucleic acid amplification technology, has higher sensitivity and specificity compared to traditional methods. It does not require special equipment and can be performed at lower temperatures (35 °C~40 °C) or even room temperature (Zhang et al., 2022; Banerjee et al., 2023). It is a molecular detection method that is expected to replace PCR. Banerjee et al. (2023) designed a detection method for the Badh2-E7 allele using RPA technology, which can quickly and sensitively detect the presence of the Badh2-E7 gene, but cannot distinguish between homozygous and heterozygous strains of the Badh2-E7 gene. Unlike the study by Banerjee et al. (2023), this study designed an RPA-badh2-E7 detection method based on the deletion of the 8 bp mutation allele in the 7th exon of the aroma gene Badh2. The amplification reaction can be completed in as little as 5 minutes, which is much lower than Banerjee et al.'s (2023) RPA amplification time of 30 minutes and eliminates the need for RPA enzyme inactivation at 65 °C for 10 minutes. And this study explored the temperature of RPA amplification, and the results showed that RPA amplification reaction can be carried out under temperature conditions of 25 °C~45 °C. The RPA-badh2-E7 method created in this study can effectively distinguish genotypes as homozygous Badh2, homozygous Badh2-E7, and heterozygous strains. Compared with
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