Bioscience Method 2024, Vol.15, No.1, 1-8 http://bioscipublisher.com/index.php/bm 2 Figure 1 Design of RPA amplification primer for rice fragrance gene badh2-E7 In the traditional breeding process of new fragrant rice varieties, researchers often use methods such as hot water method (Hu et al., 2006; Yang et al., 2010), KOH method (Sood and Siddiq, 1978), chewing method (Berner and Hoff, 1986), and instrument measurement method (Li J.H., 2008, China Rice, (2): 8-12.) for aroma identification. These methods are either subjectively influenced, or have low accuracy, poor repeatability, and low efficiency. How to simply, accurately, and quickly identify the aroma in fragrant rice is the mainstream of contemporary research (Yan et al., 2015). With the in-depth study of aroma genes, molecular markers have gradually become the main means of aroma detection. At present, the main method for detecting aroma genes is PCR. Recombinase polymerase amplification (RPA) technology is an emerging and rapidly developing isothermal nucleic acid amplification technology. Compared with traditional PCR technology, RPA technology has the advantages of simple operation, short amplification reaction time, and no need for specific instruments (Piepenburg et al., 2006; Zhang et al., 2022; Banerjee et al., 2023). Recently, Banerjee et al. (2023) designed a detection method for the Badh2-E7 allele using RPA technology. However, the directional primers they designed contained an 8 bp missing part, which could only identify whether rice contained the Badh2-E7 gene, but could not distinguish between homozygous and heterozygous lines of the Badh2-E7 gene. This study designed a co dominant marker RPA-badh2-E7 for the Badh2-E7 allele based on RPA technology. This method has four major advantages: (1) time-saving, amplification can be completed in 5 minutes, while conventional PCR requires 1.5 hours; (2) High sensitivity, 100 times higher than conventional PCR; (3) No strict amplification conditions are required, amplification can be performed at 25 ℃~45 ℃; (4) No PCR instrument required, only one constant temperature incubator is needed. Using RPA-badh2-E7, 36 core restoring lines of hybrid japonica rice and 1 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 genotypes as homozygous Badh2, homozygous Badh2-E7, and heterozygous lines. RPA-badh2-E7 can limited identify badh2-E7, which greatly improves the efficiency of molecular marker assisted selection of rice aroma genes and the breeding of fragrant rice varieties in the future. 1 Results and Analysis 1.1 Design and quality validation of RPA amplification primers In order to improve the molecular marker assisted selection efficiency of the aroma gene Badh2 and accelerate the cultivation process of new aroma rice varieties, this study compared the functional variation site of the 7th exon of Badh2 (Figure 1) and designed three pairs of primers based on the principle of RPA amplification primer design (Figure 1; Table 1).
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