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

Yonghang Tian; Wenwei Ye; Xiangshuai Liang; Peizhou Xu; Xianjun Wu; Xiangdong Fu; Yaoxian Chin; Yongxiang Liao

doi:10.3390/genes14101850

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

Genes (Basel). 2023 Sep 23;14(10):1850. doi: 10.3390/genes14101850.

Authors

Yonghang Tian^{1

2}, Wenwei Ye³, Xiangshuai Liang¹, Peizhou Xu³, Xianjun Wu³, Xiangdong Fu⁴, Yaoxian Chin^{1

2}, Yongxiang Liao³

Affiliations

¹ College of Food Science and Engineering, Hainan Tropical Ocean University, No. 1 Yucai Road, Sanya 572022, China.
² Marine Food Engineering Technology Research Center of Hainan Province, No. 1 Yucai Road, Sanya 572022, China.
³ Rice Research Institute, Sichuan Agricultural University, No. 211 Huiming Road, Wenjiang District, Chengdu 611130, China.
⁴ Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China.

Abstract

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 of OsGRF4 through PCR (polymerase chain reaction)-based assay is cumbersome and needs advanced laboratory skills and facilities. Hence, a method for conveniently and rapidly detecting OsGRF4 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 OsGRF4^NM73 (OsGRF4 from the rice line NM73). The TC→AA mutation at 1187-1188 bp loci was selected as the target sequence for the OsGRF4^NM73 allele. We further employed this method of identification in 10 rice varieties that carried the OsGRF4 gene and results revealed that one variety (NM73) carries the target OsGRF4^NM73 allele, while other varieties did not possess the osgrf4 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 OsGRF4^NM73 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 OsGRF4^NM73 gene in rice.

Keywords: CAPs; HNB; LAMP; NUE; OsGRF4; rice.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Molecular Diagnostic Techniques
Nucleic Acid Amplification Techniques / methods
Oryza* / genetics
Polymerase Chain Reaction

Supplementary concepts

LAMP assay

Grants and funding

This study was supported by National Natural Science Foundation of China Grant No. 31960399, Natural Science Foundation of Hainan Province Grant No. 321MS050, and funding from the Chengdu Science and Technology Bureau Grant No. 2022-YF09-00036-SN.