Genome-wide Association Study Of Nitrogen Use Efficiency-Related Traits And Gene Fuctional Validation In Rice

Nitrogen fertilizer is an indispensable fertilizer in agricultural production,and excessive application of nitrogen fertilizer will have adverse effects on the environment and crops like water eutrophication,soil acidification,crop lodging,seed quality decline and so on.Therefore,it is very important for the sustainable development of agriculture to improve the nitrogen use efficiency(NUE)of crops and reduce the application of nitrogen fertilizer.Nitrogen use efficiency is a complex quantitative trait including absorption,transport,assimilation and recombination.The nitrate transporter(NRT)family genes provide basic nitrate transport and signal transduction functions throughout the process.Previous studies focused on concentrate protein mutation in NRT and fewer studies in promoter elements.At the same time,previous studies on rice nitrogen use efficiency focused on bi-parental QTL,but did not use the advantage of genome-wide association analysis(GWAS)to study NUE.In this study,two nitrogen-related genes were cloned from rice landraces by GWAS of NUE-related traits for three successive years and dominant haplotypes were found.Mutants and transgenic materials were used to make intensive study,clarify their functions and mechanisms,and provide theoretical basis for breeding materials and gene utilization.The main contents are as follows:1.GWAS of NUE-related traitsA total of 1,531,224 SNPs were obtained by GBS(Genotyping By Sequencing)combined with Imputation from 117 varieties with low nitrogen tolerance and nitrogen sensitivity.Nitrogen use efficiency-related traits(Plant Height Ratio,PHR,Effective Panicle Number Ratio,EPNR,Yield Per Plant Ratio,YPPR)were investigated for three consecutive years.Tassel was used for association analysis to obtain multiple significant loci.Seven QTL loci which were repetitive at least two years were found through screening including reported OsNPF2.4?OsNRT1.1B?OsNiR?OsGS1;1 and TOND1.And two novel genes OsNPF6.1 and OsNAC42,may be obtained by association analysis.2.OsNPF6.1 affects nitrate uptake rate,expression difference affects nitrogenrelated characteristics,and higher expression can increase yield and NUE.A QTL locus at the physical position 0.1M of chr1 was identified by association analysis.OsNPF6.1 was a candidate gene in this region by SNP annotation,gene annotation,expression analysis.Then through subsequent gene complementation and knockout validation,we confirmed that OsNPF6.1 was the gene that caused the phenotype of the QTL.Further sequencing haplotype analysis showed that OsNPF6.HapB was a dominant haplotype with different amino acids.Using subcellular localization,OsNPF6.1 was found to be a membrane protein with 12 transmembrane domains located on the plasma membrane.Different haplotypes had different field phenotypes and high and low nitrate uptake rates at seedling stage in NIL lines.It was also found that the expression level of OsNPF6.1 was related to nitrogen-related traits.The expression level of OsNPF6.1 was very important for nitrogen-related traits in different haplotype overexpression families.Further detection of differences in promoters of different haplotypes revealed that there were 73 SNPs and 14 Indels,which predicted differences in binding sites that might contain transcription factors.Because OsNPF6.1 affects nitrate uptake rate,and then detects nitrogen concentration in leaves and seeds,it is found that OsNPF6.1 affects nitrogen concentration in leaves,but has no effect on nitrogen concentration in seeds.The plot yield and NUE calculation of different materials of OsNPF6.1 showed that OsNPF6.1 could significantly improve rice NUE.Comparing the genotypes of OsNPF6.1 in different species and different wild rice,we found that OsNPF6.1HapBwas specific in Oryza stiva,and only 9.7%of OsNPF6.1HapB existed in Rice3K database,which was a rare haplotype.3.OsNAC42 encodes a NAC transcription factor.The expression patterns of OsNAC42 and OsNPF6.1 are similar,and the transcriptional regulation levels of different haplotypes of OsNPF6.1 are different.In order to find a possible regulatory gene upstream of OsNPF6.1,we found that the candidate gene of QTL may be a transcription factor.So we found that a QTL locus in chr9 in GWAS results.Using SNP annotation,gene annotation,expression analysis and subsequent mutant validation,OsNAC42 is the gene in this region.It was predicted that OsNPF6.1 might be regulated by OsNAC42 by co-expression analysis of two genes and expression of OsNPF6.1 in Tilling mutant osnac42.Furthermore,luciferase reporting system was used to verify that OsNPF6.1 was regulated by OsNAC42 and had higher transcriptional regulation level for dominant haplotype OsNPF6.1HapB.Maybe this is the reason for the difference in expression of OsNPF6.1 haplotypes.By detecting haplotypes of OsNPF6.1 and OsNAC42,dominant haplotypes containing OsNPF6.1 and OsNAC42,such as Suwon264?Kexuan 13 and IR3 6,were found,which provided material basis for breeding.