Combining GWAS,QTT And EQTL Analysis To Identify Key Genes And Molecular Pathways Affecting Lead Accumulation In Maize Seedling Stage

As the world’s largest food and energy crop,maize not only plays an important role in maintaining national food security,but also has great potential in remediation of heavy metals in soil.Lead is a very toxic heavy metal,once absorbed by the human body,it will often cause damage to various organs in the human body,and then endanger human health.Therefore,it is of great theoretical significance to study the stress tolerance mechanism of maize to lead stress,to clarify the mechanism of lead ion absorption and transport in plants,to excavate related genes and to construct related networks.1)To evaluate phenotypic variations among the experimental lines,we investigated root lead content(RLC),shoot lead content(SLC)and transfer coefficient(TC)(SLC/RLC)in 24 h,48 h and 72 h lead treatment for 315 maize lines,which showed abundant phenotypic variations in the populations(range:SLC24=104.39 mg/kg,SLC48=138.74 mg/kg,SLC72=162.27 mg/kg,RLC24=1926.30 mg/kg,RLC48=2380.90 mg/kg,RLC72=2309.50mg/kg,TC14=18.70%,TC48=31.20%,TC=20.29%).SLC is positively correlated with RLC and TC,and its basic performance is significant(P<0.05)or extremely significant(P<0.01).On the contrary,RLC and TC show negative correlation and were basically at the level of significant(P<0.05)or extremely significant(P<0.01).The heritability of Pb content and transport coefficient of shoot and root in three time periods were higher than 70%,suggesting that the phenotypic variance of these traits was mainly controlled by genetic factors.2)RNA-seq was carried out on 315 maize inbred lines,and 80 of 315 materials were selected for resequencing.Combined with the data results of the two,SNP markers were excavated and interpolated through a series of means such as quality control processing.Finally,1,004,950 SNPs were obtained for subsequent data analysis.3)The MLM model in GAPIT was used to analyze the correlation between SNP markers and SLC,RLC and TC in 24 h,48 h and 72 h lead treatment,and a tatol of 68 significant SNPs associated with SLC,RLC and TC were determined to be randomly located on ten chromosomes.In the LD regions of GWAS,we searched 544 candidate genes whose the median expression was greater than 0 in the population,among which Zm00001d015133,Zm00001d017427 and other genes have been proved to be related to the absorption and transport of heavy metals.4)Quantitative trait transcripts(QTT)were analyzed by using linear regression model to identify genes affecting RLC,SLC and TC in three time periods,and the correlation between gene expression and trait phenotype was calculated.At a significant level of P <0.05,a total of xx genes were detected to be significantly associated with three time periods RLC,SLC,and TC.Compared with GWAS results,153 genes were found to overlap with their results.5)We used matrix eQTL software to perform eQTL analysis on 315 maize inbred lines,the eQTL with less than 1Mb away from the target gene was identified as Cis-eQTL and otherwise were considered Trans-eQTL.As a result,we detected a total of 598,317 eQTLs,of which,the cis-eQTL were 99,377,accounting for 16.61%,and the trans-eQTL were498,940,accounting for 83.39%.Combined with gwas results,170 genes were found to be detected by GWAS and QTT.GO enrichment analysis of these genes showed that they were mainly concentrated in the process of protein synthesis and physiological metabolism.6)Combination of the results of GWAS,QTT and eQTL,We thus identified 121 genes that are putatively related to Lead accumulation.Gene function annotations show that most genes play an important role in abiotic stress in plants.The gene Zm00001d012484 on chromosome 8 was annotated as a heavy metal transport/detoxification superfamily protein,which was significantly associated with two trans-eQTLs on chromosome 10.This gene is likely to be a key gene regulating lead accumulation,which is worth doing further research.7)Based on gene annotation and previous studies,six of 121 genes were further verified by candidate gene association analysis.A total of six SNP,which annotated as 3’UTR variant,5’UTR variant and missense mutation,from four candidate genes were significantly associated with corresponding traits.Combined with eQTL analysis,we finally determined that the gene Zm00001d013154 may be a key gene in response to lead stress,and it was constructed for a hypothesis model of its effect on the accumulation and transport of lead ions in maize seedling.