Genetic Determinants of the Network of Primary Metabolism and Their Relationships to Plant Performance in a Maize Recombinant Inbred Line Population

Abstract

Deciphering the influence of genetics on primary metabolism in plants will provide insights useful for genetic improvement and enhance our fundamental understanding of plant growth and development. Although maize (Zea mays) is a major crop for food and feed worldwide, the genetic architecture of its primary metabolism is largely unknown. Here, we use high-density linkage mapping to dissect large-scale metabolic traits measured in three different tissues (leaf at seedling stage, leaf at reproductive stage, and kernel at 15 d after pollination [DAP]) of a maize recombinant inbred line population. We identify 297 quantitative trait loci (QTLs) with moderate (86.2% of the mapped QTL, R(2) = 2.4 to 15%) to major effects (13.8% of the mapped QTL, R(2) >15%) for 79 primary metabolites across three tissues. Pairwise epistatic interactions between these identified loci are detected for more than 25.9% metabolites explaining 6.6% of the phenotypic variance on average (ranging between 1.7 and 16.6%), which implies that epistasis may play an important role for some metabolites. Key candidate genes are highlighted and mapped to carbohydrate metabolism, the tricarboxylic acid cycle, and several important amino acid biosynthetic and catabolic pathways, with two of them being further validated using candidate gene association and expression profiling analysis. Our results reveal a metabolite-metabolite-agronomic trait network that, together with the genetic determinants of maize primary metabolism identified herein, promotes efficient utilization of metabolites in maize improvement.

Figure 1.

Chromosomal Distribution of Metabolic QTLs Identified in This Study. QTL regions (represented by the confidence interval) across the maize genome responsible for metabolite level from the three tissues are shown as green (leaf1, leaf at seedling stage), blue (leaf2, leaf at reproductive stage), and orange (kernel) boxes, respectively. The x axis indicates the genetic positions across the maize genome in cM. Heat map under the x axis illustrates the density of metabolic QTL across the genome. The window size is 10 cM. Detailed information of all detected QTLs is shown in Supplemental Data Set 1C. Metabolites from different chemical groups are marked by distinct colors as shown on the right. m1, alanine; m2, arginine; m3, asparagine; m4, aspartic acid; m5, GABA; m6, glutamic acid; m7, glutamine; m8, glycine; m9, histidine; m10, homoserine; m11, isoleucine; m12, lysine; m13, methionine; m14, ornithine; m15, phenylalanine; m16, proline; m17, serine; m18, threonine; m19, tryptophan; m20, tyrosine; m21, valine; m22, β-alanine; m23, 2-oxo-glutaric acid; m24, ascorbic acid; m25, cinnamic acid, 4-hydroxy-, trans; m26, cis-aconitic acid; m27, cis-caffeic acid; m28, citric acid; m29, dehydroascorbic acid; m30, fumaric acid; m31, galactonic acid; m32, galactonic acid-1,4-lactone; m33, glyceric acid; m34, isocitric acid; m35, lactic acid; m36, malic acid; m37, malic acid, 2-methyl; m38, nicotinic acid; m39, pyruvic acid; m40, quinic acid; m41, quinic acid-3-caffeoyl-, cis; m42, quinic acid-3-caffeoyl-, trans; m43, succinic acid; m44, threonic acid; m45, trans-caffeic acid; m46, putrescine; m47, dopamine; m48, tyramine; m49, fructose; m50, fructose-6-phosphate; m51, fucose; m52, galactinol; m53, glucoheptose; m54, glucose; m55, glucosone-3-deoxy; m56, isomaltose; m57, maltose; m58, mannose; m59, melezitose; m60, myo-inositol; m61, raffinose; m62, rhamnose; m63, squalene, all-trans; m64, sucrose; m65, tagatose; m66, threitol; m67, trehalose; m68, xylose; m69, xylulose; m70, glyceraldehyde-3-phosphate; m71, glycerol; m72, proline-4-hydroxy; m73, N-acetyl-serine; m74, urea.

Figure 2.

Schematic Summary of QTL Identification in This Study. (A) Pie chart showing the proportions of metabolites that have different numbers of QTL. “A” represents the proportion of metabolites that have more than one QTL; “B” represents the proportion of metabolites that have only one QTL; number in the parenthesis represents the proportion of metabolites in “A” for which significant epistatic interactions were detected. (B) Proportion of phenotypic variation explained by all the single QTLs and epistatic interactions. The bars above the metabolite names represent different tissue types: light green, leaf at seedling stage; dark green, leaf at reproductive stage; orange, kernel.

Figure 3.

A Maize Primary Metabolic Network Involving Key Genes and Metabolites Identified in This Study. Metabolites that were not identified in this study are shown in purple. Candidate genes identified in this study are shown in the respective pathway or under the corresponding associated metabolites. ACOX, acyl-CoA oxidase; AcoT, aconitase; ADH, arogenate dehydrogenase; AGD, diaminopimelate aminotransferase; AGT, alanine glyoxylate aminotransferase; AS, anthranilate synthase component II; AK, aspartate kinase; BAM, β-amylase; CesA, cellulose synthase; DHDPR, dihydrodipicolinate reductase; DST, dihydrolipoamide S-acetyltransferase; FRUCT, β-fructofuranosidase; gpa1, glyceraldehyde-3-phosphate dehydrogenase1; GK, glutamate kinase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase; HexK, hexokinase; IVD, isovaleryl-CoA-dehydrogenase; MDH, malate dehydrogenase; O2, Opaque2; PDK2, pyruvate dehydrogenase kinase isoform 2; PEP4, phosphoenolpyruvate carboxylase 4; PGM, phosphoglucomutase; PK, pyruvate kinase; SDH, succinate dehydrogenase; SIT, sugar/inositol transporter; SKDH, shikimate dehydrogenase; TDC, tyrosine/DOPA decarboxylase; TH, thioredoxin H-type; THA, threonine aldolase; THS, threonine synthase; 2OGDH, 2-oxoglutarate dehydrogenase.

Figure 4.

Validation of Candidate Gene AGT within QTL Interval Using Association Analysis. (A) LOD curves of QTL mapping for level of β-alanine in maize leaves at seedling stage on chromosome 1. (B) Scatterplot of association results between polymorphic markers (i.e., SNPs and InDels) in the confidence interval and the level of β-alanine. The −log₁₀-transformed P values from the association analysis are plotted against the genomic physical position. Physical position of candidate gene AGT is indicated by the vertical dashed line. The bigger dot represents the 43-bp InDel marker. Markers shown in blue were in the association analysis for level of β-alanine in maize leaves at seedling stage (leaf 1), while markers in red were in the association analysis for level of β-alanine in maize leaves at reproductive stage (leaf 2). Association analysis was performed using the mixed linear model controlling for the population structure (Q) and kinship (K). (C) Gene structure of AGT and natural variation between alleles from By804 and B73. The star marks the 43-bp insertion in By804. (D) Relative levels of AGT mRNA in B73 and By804. Expression levels were measured by reverse transcription-quantitative PCR and values for three biological replications were averaged (P = 0.04; t test). (E) Box plot for level of β-alanine (gray) and expression of AGT (white) plotted as a function of genotypes at the site InDel_0/43. P value for the expression level was calculated based on ANOVA, and P value for the level of β-alanine was calculated using MLM controlling for population structure (Q) and kinship (K).

Figure 5.

Validation of Candidate Genes TDC1 and TDC2 within QTL Interval Using Association Analysis. (A) Diagram of linkage mapping result for the level of tyramine and dopamine in maize leaves at reproductive stage. LOD values of the bins at the peak of QTL interval are shown as a function of their genetic positions. (B) Scatterplot of association results between polymorphic markers (i.e., SNPs and InDels) in the peak bin and the level of metabolites tyramine and dopamine in maize leaves at reproductive stage. The −log₁₀-transformed P values from the association analysis are plotted against the genomic physical position. Genes located within the 17.8- to ∼17.9-Mb region on chromosome 1 are indicated as green bars. Association analysis was performed using the mixed linear model controlling for the population structure (Q) and kinship (K). (C) Gene structure of TDC1 and TDC2 and sequence variation between alleles from By804 and B73. Stars mark SNPs, while arrowheads mark insertion-deletion polymorphisms (InDels) between the allelic sequence of By804 and B73. The bigger orange arrowhead in the promoter region of TDC2 represents the 83-bp InDel.

Figure 6.

Metabolite-Metabolite-Agronomic Trait Association Network. This illustration represents the union of metabolite-agronomic trait association network with the metabolic relevance networks obtained for each tissue (i.e., leaf at seedling stage, leaf at reproductive stage, and kernel). Nodes that correlate with each other are linked by gray edges. The color of the nodes represents the metabolite classes. The nodes with the star stand for agronomic traits, and the circles, triangles, and squares correspond to the metabolites in leaf at seedling stage, leaf at reproductive stage, and kernel, respectively. The r-square, which measures how well the data fitted to the regression model, was calculated for each model and represented in the network with the size of the corresponding node. The better the model fitted to the data, the bigger the size of the node.

Figure 7.

Dissection of Metabolite and Candidate Genes Associated with Length of Ear Leaf. (A) LOD curves of QTL mapping for length of ear leaf (Len.EL) and level of fucose and xylulose in maize leaves on chromosome 1. QTLs for these three traits overlap at ∼174 cM on chromosome 1. (B) LOD values of the bins at the peak of QTL interval identified in (A) are shown as a function of their genetic positions. (C) Graphical representation of genes within the genomic region spanning the single bin at the peak. Twelve genes represented by boxes were found within the ∼600-kb region. Five genes that have putative function are marked in light blue.