Genetic Dissection Of Tocopherol Content In Maize Kernels By Combining Association And Linkage Analyses And Fine Mapping Of A Major QTL

Tocopherols play an important role in plants and in human health.Although the biosynthetic pathway of tocopherols in model plants as Synechocystis and Arabidopsis thaliana has been studied comprehensively,the genetic architecture of tocopherol content in maize(Zea mays L.)kernels has remained largely unknown.In this study,linkage and association analyses were conducted to examine the genetic architecture of tocopherol content in maize kernels.?-,?-,?-,total tocopherol content and ?/? ratio in maize kernels were measured as the target traits in this study.The main results are as follows:Six populations of recombinant inbred lines(RILs)with diverse genetic and phenotypic variation were developed for linkage analysis.High-density genetic maps had been constructed previously.Forty-one unique quantitative trait loci(QTLs)were identified by linkage mapping,representing 89 QTLs in total.Among the 89 QTLs,44 were detected in multiple RIL populations while 45 were only detected in single populations.Among the 41 unique QTLs,10 QTLs were major ones each explaining more than 10% of phenotypic variation,and 11 QTLs could influence more than one tocopherol traits.A comparison with previous studies showed 10 unique QTLs have not been detected before,revealing the advantage of using multiple populations from diverse genetic backgrounds.An epistatic analysis showed only a minor role of epistasis between QTL pairs in contributing to phenotypic variation of tocopherols.A diverse association panel composed of 508 inbred lines genotyped with 0.56 million SNPs derived from RNA deep sequencing was used for genome-wide association study(GWAS).282 significant SNPs corresponding to 32 independent significant loci were detected via GWAS.Unexpectedly,the significant loci identified via GWAS scarcely overlapped with previous known pathway homologs and revealed a role for non-tocopherol pathway genes typically with genes involved in fatty acid metabolism,chlorophyll metabolism and plastid function in the modulation of natural tocopherol variation in maize kernels.These findings were validated through an example of mutant analysis of LACS which functions in the fatty acid pathway.Among the 32 loci from GWAS,18 co-localized with the QTLs identified by linkage mapping.By utilizing the transcriptomic data of the association panel,a putative regulation network was constructed centred with key homologs in tocopherol biosynthetic pathway.Besides,it was discovered that most favorable alleles identified via GWAS were rare alleles with a frequency of less than 0.1.The variation of tocopherols could be mainly explained by the accumulation of favorable alleles.A major QTL conferring multiple tocopherol traits was validated and fine-mapped on the short arm of chromosome 8.By utilizing heterogeneous inbred family(HIF)derived from RIL lines,recombinant screening and progeny test were conducted.Eventually,the QTL were confined to an 860-kb interval.This result also suggests a role for non-pathway genes since candidate gene analysis of the only pathway homolog in the QTL interval(DXR2)did not result in any possible causative polymorphisms.Moreover,a significant locus from GWAS fell into the fine-mapping interval,located in an intergenic region.In this way,the fine-mapping validated the power and accuracy of GWAS and QTL mapping results.Together,this study revealed that the tocopherol variation in maize kernels are controlled by a few QTL with major effects and a number of QTLs with minor effects.Many novel genes beyond the tocopherol biosynthetic pathways were found to involve in the variation of tocopherol content in maize kernels.The favorable alleles for many of the significant SNPs/QTLs represented rare alleles in natural populations.Pyramiding of the favorable alleles of the newly elucidated genes would greatly improve tocopherol content in maize.