Dynamic QTL Analysis And Gene Identification Of Wheat Kernel Metabolism At Different Developmental Stages

Wheat(Triticum aestivum L.)is an important crop,probing the dynamic changes of its kernels during seed maturation and germination is essential for wheat breeding.Metabolomics has long been regarded as a bridge between genotypes and phenotypes.Therefore,a comprehensive understanding of the composition content changes of metabolites during wheat kernel maturity and germination is helpful to explore the underlying molecular mechanisms.To achieve this goal,the combination of metabolic profiling methodologies and genetic designs has been proved effective in identifying candidate genes that affect the content of metabolites,which has been universally utilized in model crops such as rice,maize,tomato.Recently,similar studies were also applied in wheat.However,only a few cases subjected to such experimental designs that including several sequential developmental stages are reported.In current study,the widely-targeted metabolomics approach was harnessed to profile the kernels of 159 recombinant inbred lines(RILs)that derived from Aikang 58(AK58)and Jingdong 8(JD8).A total of three sequential seed developmental stages were included,namely filling stage(abbreviated as FK,three weeks after flowering),mature stage(MK,five weeks after flowering)and germination stage(GK,from germination to exposure).Subsequently,numerous reliable mQTL(metabolic quantitative trait loci)were obtained by combining the metabolite contents and the high-density molecular markers.Moreover,we have proved that mQTL obtained from the three seed developmental stages could better facilitate pathway elucidation and metabolic network construction.The detailed results are as follows:1.Detection and analysis of metabolites:The previously established widely-targeted metabolomics method was applied to the three stages(i.e.,FK,MK and GK)for each of the 159 RILs.As a result,a total of 625 known metabolites that tentatively classified as amino acids,nucleotides,lipids,phytohormones,vitamins,flavonoids and phenolamides were detected.The coefficients of variation(CV)and the broad-sense heritability(H~2)of the 625 metabolites fitted the normal distribution,which respectively peaked at 20%-30%and 0.4-0.6.The heritability of flavonoids in kernels at filling stage was the highest,wherein the H~2for 67%of flavonoids was>0.6.2.mQTL mapping and the verification of candidate genes:Based on the high-density genetic map generated by 50k SNP chip in wheat,a total of 3173,3143and 2644 mQTL were detected at FK,MK and GK stages,respectively,among which52 mQTL were jointly mapped among the three stages.The mapped mQTL were unevenly distributed on the wheat chromosomes,wherein the mostly located ones are2B,3D and 4B,with 50,40 and 44 mQTL were respectively mapped.A total of 177candidate genes that regulate metabolite contents were identified by data mining.Traes CS3A01G296900(Tyrosinedecarboxylase)and Traes CS4B01G055300/Traes CS4B01G055400(Acyltransferase)were verified by enzyme activity in vitro.The Traes CS7A01G068600(O-methyltransferase)and Traes CS2A01G070100(Glycosyltransferase)were further verified in transgenic wheat.3.Analysis of dynamic mQTL:It is found that there is obvious co-mapped and specifically mappedmQTL among the three stages,indicating that different metabolites have common and specific biological functions as well as the regulatory mechanisms.Metabolic pathways can be analyzed more comprehensively by integrating mQTL at different stages.4.Constructing metabolic network of wheat kernels:By integrating the results obtained from three stages of mQTL and the verified candidate genes,the metabolic network in wheat kernels was constructed.It is mainly the flavonoid metabolic pathway downstream of phenylpropane and the biosynthesis and decomposition pathway of several important amino acids,including the tryptophan,tyrosine and the phenolamide downstream of arginine,which improves our cognition of the complex metabolic network.5.Study on Agronomic traits by metabolomics:Through correlation analysis and QTL co-location analysis of 625 known metabolites and 7 agronomic traits,it was found that 44 agronomic traits QTL(phenotypic QTL,p QTL)were co-located with249 mQTL,involving 195 metabolites.Among them,a candidate gene Traes CS4D01G038200(encoding a sugar transporter)which affects both sugar accumulation and kilo-grain weight was predicted in the co-location interval of chromosome 4D,which promoted our research on the mechanism of complex agronomic trait of wheat by metabolomics.