Bioscience Method 2024, Vol.15, No.1, 1-8 http://bioscipublisher.com/index.php/bm 1 Research Article Open Access Rapid Detection of Rice Fragrance Allele badh2-E7 by Recombinant Polymerase Amplification (RPA) Jihua Zhou 1*, Anpeng Zhang 1*, Wenke Xu 2, Can Cheng 1, Fuan Niu 1, Bin Sun 1, Liming Cao 1, Jianming Zhang 1 , Huangwei Chu1 1 Institute of Crop Breeding and Cultivation, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China 2 Shanghai Chongming Sanxing Town Agricultural Comprehensive Technology Extension Service Center, Shanghai, 202152, China * These authors contributed equally to this work Corresponding authors: zhangjianming@saas.sh.cn; chuhuangwei@saas.sh.cn Bioscience Method, 2024, Vol.15, No.1 doi: 10.5376/bm.2024.15.0001 Received: 23 Jan., 2024 Accepted: 26 Jan., 2024 Published: 31 Jan., 2024 Copyright © 2024 Zhou et al., This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Zhou J.H., Zhang A.P., Xu W.K., Cheng C., Niu F.A., Sun B., Cao L.M., Zhang J.M., and Chu H.W., 2024, Rapid detection of rice fragrance allele badh2-E7 by recombinant polymerase amplification (RPA), Bioscience Method, 15(1): 1-8 (doi: 10.5376/bm.2024.15.0001) Abstract Fragrant rice is favored deeply by consumers due to the strong fragrance. Fragrant rice is mainly caused by the loss-of-function mutation of the Betaine aldehyde dehydrogenase 2 (Badh2) gene in rice. Badh2-E7, with 8 bp deletion and 3 bp substitution in exon 7 of Badh2, is the main mutation allele used in fragrant rice breeding. In this study, a genotyping method named RPA-badh2-E7 for Badh2-E7 allele was designed. This method has the characteristics of rapid (completed amplification in 5 min), sensitivity (100-fold than conventional PCR), no strict amplification conditions required (25 °C~45 °C), and independent of PCR amplifier (only one thermostatic incubator is enough for amplification). This method greatly improved the efficiency of molecular marker-assisted selection of rice fragrant genes and breeding of fragrant rice varieties. Keywords Rice; Fragrant gene; badh2-E7; RPA Rice (Oryza sativa L.) is the most important grain crop in China. With the development of the times and social progress, people's requirements for the quality of rice are also increasing (Zhang et al., 2020). Aroma is an important characteristic of rice quality. Rice with aroma not only has a fragrant aroma, but also is rich in nutrients such as vitamins and aromatic amino acids, which are deeply loved by consumers (Peng et al., 2018). The molecular regulation mechanism of aroma traits and the breeding of new varieties of fragrant rice are receiving increasing attention from scientists. As early as the 1980s, scientists conducted research on the components, formation mechanisms, and genetic characteristics of rice aroma, and found that mutations in the Badh2 (Betaine aldehyde dehydrogenase2) gene encoding betaine aldehyde dehydrogenase were related to the formation of fragrant rice. The functional deficiency of Badh2 leads to the accumulation of 2-acetyl-1-pyrrolin (2-AP) (Yang et al., 2008; Fukuda et al., 2014), a substance directly related to the aroma of rice, resulting in the rich aroma of rice (Chen et al., 2008; Kovach et al., 2009). The Badh2 gene is located on chromosome 8 and consists of 15 exons and 14 introns. Up to now, multiple different allele variations of Badh2 have been reported, including mutation types occurring in different regions such as exons 1, 2, 4, 5, 7, 10, 12, 13, and 14 of Badh2, the cleavage site of intron 1, and the 5'UTR region (Amarawathi et al., 2008; Shi et al., 2008; Shao et al., 2011; Shao et al., 2013; Ootsuka et al., 2014; Shi et al., 2014; He and Park, 2015; Cheng et al., 2018). The deletion of 8 bp and substitution of 3 bp in exon 7 of Badh2 are the main types of variation (Figure 1), which have been applied in the breeding of many fragrant rice varieties (Sun et al., 2021).
RkJQdWJsaXNoZXIy MjQ4ODYzMg==