Genome-wide Association Studies And Linkage Mapping Of Amino Acids In Maize & Comparative Analysis Of Maize And Rice Metabolomes

Maize(Zea mays L.),as one of the most important crops widely grown all over the world,is prevalently processed into a range of food and industry products.As a kind of human food and a main energy ingredient in livestock feed,maize kernel is not fully used accounting for its low content of protein and amino acids.In order to characterize the genetic architecture of amino acides in maize kernel,one diverse association panel of 513 inbred lines and three recombinant inbred line(RIL)populations were used to quantify the total amino acids of mature maize kernel;a set of high density SNPs across the whole genome were applied to perform genome-wide association study and QTL mapping.By the means of expression QTL mapping,QTL fine mapping,bioinformatics analysis and genetic transformation,three candidate genes were well validated.The main results are as follows:1.Natural variation of amino acids in maize kernel: The content of each amino acid-related trait varied broadly within the association panel under two environments(AM1 and AM2)and three RIL populations(B73/By804: BB,Kui3/B77: KB and Zong3/Yu87-1: ZY).There was 2.04-11.49 fold change and 2.62-13.06 fold change of phenotypic variation detected within the asscociation panel under AM1 and AM2 enviroments,respectively.In BB,KB and ZY RIL populations,there was 1.51-3.86 fold change,1.79-4.81 fold change and 1.98-5.50 fold change of phenotypic variation identified,respectively.2.Genome-wide association study: A set of 1.25 million high quality SNPs were used to perform genome-wide association study on the diverse panel of 513 inbred lines.At a significant level of p ? 2.04×10-6,247 and 281 genetic loci were identified respectively at AM1 and AM2 environments.Within each study,each locus explained 7.44% and 7.90% of phenotypic variations on average.3.Linkage mapping: A set of high density SNP array was applied to conduct QTL mapping in three populations(BB,KB and ZY).There were 404 QTLs identified for amino acid traits across all three populations.Within BB population,89 QTLs were identified and each QTL explained 9.03% of phenotyptic viraition on avaergae;150 QTLs were detected in KB population,each of which explained 9.39% of phenotypic variartion;within ZY population,there were 165 QTLs identified and each QTL averagely explaind 10.15% of phenotypic variation.The average mapping resolution of BB,KB and ZY populations were 8.6,11.3 and 7.2Mb,and the number of QTL with the confidence interval less than 5Mb were 57,95 and 99 respectively.4.Candidate genes and co-expression network: 308 unique candidate genes corresponding to 528 trait-loci associations were identified at AM1 and AM2 environments.Based on the current database,27% of these candidates encode enzymes directly or indirectly affecting amino acid metabolism;29% of these genes involve in other biological process;7% of them are transcription factors;2% encode storage proteins in kernel.These genes are enriched in cellular nitrogen metabolism,amine metabolism,the metabolism of amino acids and derivative metabolism,organic acids,and other processes.By the investiagetion of the previous RNA-sequencing data of immature maize kernels,it was found that 133 out of these candidate genes possessing e QTLs.23 candidate genes involving in amino acid anabolism and catabolism were identified by GWAS.The co-expression analysis was performed between these 23 candidate genes and 28,769 genes obtained by RNA-sequencing from immature kernels.The co-expression genes contained 4,670 target genes.Among these 4,670 genes,49 genes were identified by GWAS,another 140 annotated genes were identified to be directly or indirectly associated with amino acid metabolism.GO term analysis of the 4,670 co-expressed genes revealed significant enrichment in terms relating to amine metabolism,cellular processes,developmental processes and biological regulation.5.Validation of candidate genes: By means of genetic approaches,e QTL analysis,bioinformatics,gene transformation and co-expression analysis,three candidate genes(O2,GRMZM2G138727 and GRMZM2G143008)were soundly investigated in this study.These results provided clues to understanding the metabolism and genetic variation and basis of amino acids in maize mature kernel,and provided the relevant candidate genes for research of maize amino acids,meanwhile,provided the materials for high Lys maize breeding as well.Another area in this research was the comparative investigation of maize and rice metabolomes.Based on the published metabolomes data of maize kernel,rice grain and rice leaf,an investigation of the metabolites distribution,variance coefficient and fold change were processed among maize and rice.It was noted that the metabolites distribution were significantly different between maize and rice,particularly,the flavonoids.According to the different level of comparisons,it was noted that different metabolites obviously differing between maize and rice and even within each subgroup.The fold change of secondary metabolite classes were greater than primary metabolite classes both in maize and rice.There were pretty more variations of metabolites observed in TST(tropical/subtropical)maize accessions than in TEM(temperate maize)group.As for the metabolites in rice,much more variations were identified in indica varities than in japonica varites;in addition,the fold change of mebolites was significantly different between grain and leaf.According the enrichment analysis of difference metabolites,the flavonoid was significantly enriched in maize kernel,rice leaf and rice grain.The most enriched metabolites in TEM accessions were flavonoids;within TST inbred lines,there were relative higher level of phenolamides.The most enriched metabolites in indica varieties were flavonoids;within japonica accessions,there were relative higher level of phenolamides.The correlations between metabolites of the same class are relative higher than those between metabolites of the different classes in TEM,TST,japonica and indica subgroups.While,the correlations among metabolites of the different classes were considerable different between japonica and indica subspecies,but not in TEM and TST.Compared with TEM and indica,there were less positive correlations but more negative correlations in TST and japonica.The PCA and cluster analysis based on the metabolites or flavonoids and phenolamides,shown the TEM and TST or japonica and indica could be separate.