Mining Functional Loci Of Nitrate Reductase Gene ZmNR2 Based On Association Analysis In Maize

Nitrogen metabolism is one of the basic substance metabolism in the growth and development of higher plants.Nitrate reductase(NR)is a key enzyme of nitrogen metabolism and responsible for the initial reduction step of nitrogen cycling.Nitrate reductase activity(NRA)is closely related to nitrate accumulation and directly affects the nitrogen efficiency of plants.Therefore,analyzing the inheritance of nitrate reductase genes in maize core inbred lines at the molecular level would help accelerate the breeding process and improve the breeding efficiency.In this study,we constructed an association mapping population consisting of 126 maize core inbred lines as experimental material.We identified and evaluated 8 related phenotypic traits such as nitrate reductase activity in maize seedling stage.Combining the population structure analysis of the 126 inbred lines and the sequence polymorphism of nitrate reductase gene ZmNR2,candidate gene association analysis was performed.The aim of this study was to explore superior allelic variations associated with nitrogen-efficient traits and nitrogen-efficient haplotypes.The main findings are as follows:1.This study identified eight nitrogen efficiency-related traits in the associated group: nitrate reductase activity,chlorophyll SPAD value,root dry weight,shoot dry weight,total dry weight,root-shoot ratio,total plant length and main root length.It was found that all traits in this population showed continuous normal distribution and varying degrees of variation,with heritability ranging from 60.03% to 82.21%.Among them,nitrate reductase activity has a large variation range,low heritability and is greatly affected by the environment.The results of correlation analysis showed that there was a significant positive correlation between NRA and main root length,root dry weight and total dry weight in E1 and E2 environments.2.Sequence polymorphism analysis of ZmNR2 gene revealed that 58 polymorphic loci were detected in 113 maize inbred lines,including 50 SNPs(one per 62bp)and 8 Indels(one per 389bp).The SNPs of the gene were mainly distributed in the exon region,and the Indels were mainly distributed in the intron region.Linkage disequilibrium analysis showed that there were some obvious LD structures on the ZmNR2 gene sequence.The linkage disequilibrium of the gene began to decline rapidly at around 250 bp with the increase of distance and declined to below 0.1 level at 1000 bp.3.Association analysis between all the related traits and polymorphic loci used three statistical models in TASSEL software,including general linear model(GLM),general linear model considering population structure Q matrix(GLM+Q)and mixed linear model(MLM(Q+K)).A total of 38 significant polymorphic loci are detected in ZmNR2 gene sequence in the three models,including 30 SNPs and 8 Indels.A total of 8 polymorphic loci were significantly associated with corresponding traits in all three statistical models.Six polymorphic loci were significantly associated with corresponding traits under P ? 0.01.4.Four haplotypes(MAF?0.05)were divided based on 6 important significant associated SNPs/Indels.The results showed that Hap1,Hap2,Hap3 and Hap4 had 3,3,1 and 1 excellent allelic variations,respectively.The T-test results of nitrate reductase activity of each haplotype showed that the average values of Hap1 and Hap4 were significantly different(P < 0.05).Therefore,this study concluded that Hap1 is a N-efficient haplotype and Hap4 is a N-inefficient haplotype.5.The N-efficient haplotype Hap1 and N-inefficient haplotype Hap4 were further verified by field yield phenotypic identification test and ZmNR2 gene expression analysis.The results showed that the yield traits of N-efficient haplotype Hap1 were significantly higher than those of N-inefficient haplotype Hap4;the gene expression of Hap1 was significantly higher than that of Hap4.It is further confirmed that there are abundant synergistic allele variations in Hap 1 inbred lines,and S633-T and S1337-1 may be an important excellent allele combination.These variations have common responses to multiple regulatory pathways of nitrogen nutrition utilization,and can be applied to molecular marker-assisted selection to improve the efficiency of nitrogen efficient use of maize.