Mining And Functional Analysis Of The Candidate Gene Involved In Maize Flavonoids Metabolic Pathway

Flavonoids, as a a group of representative polyphenols secondary metabolites, ditribute extensively in the plants kingdom. Flavonoids can help the plants producing these compounds improve the resistance to the biotic or abiotic stresses. While Flavonoids exhibit distinctive bioactivity to animals, which significantly contributes to the exploitation of new medicine for humanity. Maize is one of the critical crops for food and fuel. Since flavonoids are important metabolic product of maize kernel, it is of great significance to comprehensively study the pathway of flavonoids to improve maize production and accumulation of nutrition. In my study, I detected 39 flavonoids in maturity kernel and measured their contents that were regarded as phenoytpes for genetic analyisis. The goal of this study was to identify the candidate genes controlling maize kernel flavonoids content and validate them by combining genome wide association study, linkage analysis and transgenic anlayisis. The results will provide insights into the genetic architecture of the metabolic pathway of flavonoids and high nutrinal maize breeding. The major results are summarized as following:1. A maize QTL mapping population( B73 × By804) consisting of 173 maize recombinant inbred lines was used as the materials. We detected 39 flavonoids in maturity kernel and measured their contents that were regarded as phenoytpes for QTL mapping. A total of 235 quantitative trait loci(QTLs) were detected in two environments(BBR1, Hainan, 2010; BBR2, Henan, 2011). 110 QTLs affecting 31 traits were detected in the BBR1, with LOD value ranging from 2.50 to 56.30. Single QTL explaining the phenotypic variation ranged between 2.15% and 76.78%. In BBR2, 125 QTLs affecting 30 traits were detected with LOD value ranged from 2.50 to 53.84. Single QTL explaining the phenotypic variation ranged between 2.04% and 73.51%.2. A recombinant inbred population of maize(Zong3×Y8701) consisting of 161 lines was used for QTL mapping. A total of 265 quantitative trait loci(QTLs) were detected in two environments(ZYR1, Yunnan, 2010; ZYR2, Henan, 2011). 138 QTLs affecting 31 traits were detected in the ZYR1, with LOD value ranging from 2.50 to 12.70. Single QTL explaining the phenotypic variation ranged between 4.16% and 23.17%. 127 QTLs affecting 31 traits were detected in the ZYR2, with LOD value ranging from 2.50 to 9.77. Single QTL explaining the phenotypic variation ranged between 2.85% and 18.84%.3. Genome-wide association studies for 39 traits in three environments were perfomed with 560 K high quality SNPs(minor allele frequency ≥ 0.05) in an association mapping panel containing 368 diverse lines. In total, 114 distinct locus–trait associations were identified at a genome-wide significance level of p≤1/N(calculated by mixed linear model controlling Q and K) across three environments. In E1(Hainan, 2010), 7 significant associated loci for 5 traits were detected. Each locus explained 7.17%- 15.39% of the observed flavonoids content variance. A total of 68 loci were associated with 29 traits in E2, which could explain phenotypic variation ranging from 6.53% to 19.77%. The numbers of loci associated with 23 traits in E3 were 39, and each locus explained 6.70%-19.48% of the observed flavonoids content variance. In total, 13 loci were detected in more than one environment.4. Eight candidate genes were identified to play important roles in maize flavonoids metabolic pathway by combining linkage analysis, genome-wide association analysis and transgenic analysis. These candidate genes were: GRMZM2G059590(Regulator of Vps4 activity in the MVB pathway), GRMZM2G156145(ABC transporter group), GRMZM5G843555(2OG-Fe(II) oxygenase superfamily), GRMZM2G084799(Myb-like DNA-binding domain, p1), GRMZM2G104710(O-methyltransferase), GRMZM2G119175(Pyruvate kinase), GRMZM2G383404(UDP-glucoronosyl and UDP-glucosyl transferase), GRMZM2G110233(Putative sno RNA binding domain). Two candidate genes(GRMZM2G383404 and GRMZM5G843555) were overexpressed in rice, 42 and 24 positive transgenic seedlings were obtained respectively. These results provide a foundation for further functional and mechanism study of these important genes in maize flavonoids metabolic pathway.