Rapid and Visual Detection of High Amylose Gene Wxa in Rice (Oryza sativa) Using LAMP Method
DAI An-Qi1,LIANG Xiang-Shuai1,YE Wen-Wei2,WANG He-Fei1,WU Xian-Jun2*, TIAN Yong-Hang1*
1 College of Food Science and Engineering, Hainan Tropic Ocean Univercity, Sanya 572022, China; 2 Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, China
Abstract：Mining and utilizing the allelic variation Wxa of high amylose gene particle bound starch synthase (waxy, Wx) is one of the hot spots in rice (Oryza sativa) breeding, but the key is to screen the gene Wxa conveniently. In this study, the visual loop-mediated isothermal amplification (LAMP) of rice high amylose gene Wxa was designed to explore a convenient screening method. Firstly, the Ex10-115(T/C) variant site and upstream and downstream consistent sequences of Wxa gene were identified as target sequences by sequence alignment. Then, using Primer3 Input, NEBcutter V2.0 and Primerexplore V5 software, cleaved amplified polymorphic sequences (CAPs) and LAMP markers were designed for genotyping. Finally, the visibility of the functional markers were tested. The results showed that there were conserved sequences near the Ex10-115(T/ C) variant site, from which CAPs markers (Wxa-2F and Wxa-2R) and LAMP markers (F3, B3, FIP and BIP) were designed. Using the CAPs marker to detect the Ex10-115(T/C) variant site in 10 rice cultivars, the gene Wxa was found in 'D62B', 'G46B', 'II-32B', 'ZS97B' and 'GLA4' those cultivars with higher amylose contents, while the other 5 cultivars did not carry Wxa that there's concordance between genotype and phenotype. The Wxa gene in rice was detected by LAMP marker combined with hydroxynaphthol blue (HNB) at 63° C for 60 minutes that the Wxa gene may be detected with the naked eye in rice varieties. The result was consistent with that of CAPs marker. In this study, a convenient method was designed to screen Wxa genes in rice. The results can provide references for the development and application of functional markers for other important genes in rice.
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