GWAS combined with transcriptome and metabolome to excavate key candidate genes for rice anaerobic germination

Abstract

Abstract In this study, a total of 317 SNP loci were detected based on dynamic genome-wide association study (GWAS) of the length (CL), surface area (CSA), volume (CV) and diameter (CD) of the coleoptile growing in 2d, 3d and 4d under anaerobic conditions in 591 natural rice populations. Integrated dynamic widely targeted metabolomic technology combined with liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) and data analysis, we found that xanthine, L-alanine and GABA may be key biomarkers that are sensitive and respond strongly to hypoxic stress perception. By combining targeted metabolomics and transcriptomics for WGCNA, we found that OsAlaAT1, OsGAD4, OsAAH and Os09g0424600 are the key structural genes that best characterize the metabolic processes of L-alanine, GABA and xanthine. Among them, OsAlaAT1 (Os10g0390500), located in Chr10-12877840, which is within the GWAS location range of CVAN3d, is considered to be a more reliable candidate gene. Through phenotype observation of OsAlaAT1 transgenic plants and qRT‒PCR analysis, it was found that the coleoptile length (CL) and relative expression level of the ko-alaat1 mutant were significantly decreased compared with the control (ZH11). GUS staining showed that OsAlaAT1 was strongly expressed in the coleoptile, embryo and radicle during seed germination under anaerobic conditions. Overall, in addition to providing new insight into the metabolic regulation of L-alanine, GABA and xanthine during hypoxic germination of rice, the outcome of our study establishes a foundation for anaerobic germination (AG) improvement in rice.