Rapid Visual Detection of High Nitrogen-Use Efficiency Gene OsGRF4 in Rice (Oryza sativa L.) Using Loop-Mediated Isothermal Amplification Method

The GROWTH-REGULATING FACTOR4 (OsGRF4) allele is an important target for the development of new high nitrogen-use efficiency (NUE) rice lines that would require less fertilizers. Detection OsGRF4 through PCR (polymerase chain reaction)-based assay cumbersome and needs advanced laboratory skills facilities. Hence, a method conveniently rapidly detecting on-field key requirement further research applications. In this study, we employed cleaved amplified polymorphic sequences (CAPs) loop-mediated isothermal amplification (LAMP) techniques to develop convenient visual detection NUE gene OsGRF4NM73 (OsGRF4 from line NM73). TC→AA mutation at 1187–1188 bp loci was selected as sequence allele. We identification in 10 varieties carried results revealed one variety (NM73) carries allele, while other did not possess osgrf4 genotype. optimal LAMP reaction using hydroxynaphthol blue (HNB), chromogenic indicator, out 65 °C 60 min, presence confirmed by color changes violet sky blue. study showed can be accurately used detect rice.