Genetic Variation for Seed Metabolite Levels in Brachypodium distachyon

Abstract

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

Keywords: Brachypodium distachyon; QTL analysis; chrysanthemin; metabolome; recombinant inbred line; vitamin B6.

Figure 1

Genotype and metabolotype variation across forty B. distachyon inbred lines. (A) PCA plot of the forty natural B. distachyon lines based on the biallelic SNP dataset generated using publicly available GBS and whole-genome sequencing data. (B) PLS-DA plot of the forty B. distachyon lines based on accumulation profiles of the 183 metabolites in mature seeds. The colors for individuals are the same as in (A). (C) The signature metabolites based on the variable importance of projection (VIP) score (>1.5) of PLS-DA analysis. (D) Accumulation patterns of the signature metabolites across the forty B. distachyon lines. The heatmap represents hierarchically clustered accumulation patterns of the signature metabolites based on the normalized binary logarithm (log2) values of their quantitative data.

Figure 2

Differentially accumulating metabolites in Bd21 and Bd3-1 seeds. (A) Volcano plot representing metabolites in seeds between Bd21 and Bd3-1 (p < 0.05). (B) Accumulation patterns of thirty-seven differentially accumulating metabolites in Bd21 and Bd3-1 seeds as well as their fold change.

Figure 3

Results of mQTL analysis using an RIL population from the cross Bd3-1 × Bd21. Heat map representing the distribution of LOD scores for 17 metabolites across five chromosomes (Bd1, Bd2, Bd3, Bd4 and Bd5) in B. distachyon. The heat map for each metabolite is created using three replicates.

Figure 4

Genetic mapping of QTLs for 4-pyridoxate accumulation in the Bd3-1 × Bd21 RILs. (A) A QTL likelihood (LOD) map of the genomic region that covers the QTL region of interest, with a LOD significance threshold of p < 0.05. The colored LOD curves correspond to three replicates of metabolome measurements. (B) Average relative abundance of 4-pyridoxate in seeds for the genotypes nearest to the closest marker, BD1742_2, in the RILs.

Figure 5

Interaction of QTLs in the accumulation of chrysanthemin in the Bd3-1 × Bd21 RILs. (A) LOD scores corresponding to additive or full (additive and interaction) models for each combination of chromosomes 1 and 4 QTLs for the accumulation of chrysanthemin. The upper left and lower right triangles represent the results of the full and additive models, respectively. The color scale indicates separate LOD score scales for the full and additive models. LOD scores computed using five models are represented on the two-dimensional QTL scan. (B) Combined effects of the two QTLs on the accumulation of chrysanthemin in the RILs. The histogram represents the phenotypic distribution of chrysanthemin accumulation in the RILs for the four genotype combinations corresponding to the two SNP markers Bd3534_1 and BD1893_1, located on chromosomes 1 and 4, with the maximum LOD score.