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

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