Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain

Abstract

Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of ∼1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including five that had not been previously associated with maize grain tocochromanols: four biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for co-localized eQTL and GWAS association signals. Our multi-omics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

Leveraging prior biological knowledge improves prediction of tocochromanols in maize grain

“…The first data set of tocochromanol phenotypes was generated by Wu et al (2022). Briefly, physiologically mature self-pollinated ears were harvested from 1,815 inbred lines of the maize North Central Regional Plant Introduction Station panel (hereinafter the Ames panel, Romay et al, 2013) that was grown in an augmented complete block design during the 2015 and 2017 field seasons at Iowa State University Agricultural Engineering and Agronomy Research Farm in Boone, IA.…”

“…Next, read counts were generated using the htseq-count function within HTSeq version 0.11.2 (Anders et al, 2015) based on the B73 version 4.59 annotation and then normalized using the rlog function in the DESeq2 package (Love et al, 2014). After the implementation of several additional stringent quality control measures at the sample and gene levels as described in Wu et al (2022), a total of 741 high quality samples from 13 check and noncheck lines with rlog-transformed read counts for 22,136 genes were retained for generating BLUE values of each gene by fitting a mixed linear model. To coincide with both tocochromanol phenotype data sets, 545 lines were retained after removing 104 lines classified as sweet corn, popcorn, or an endosperm mutant.…”

“…Unfortunately, the predominant maize varieties grown around the world are inadequate in providing grain that meets the recommended dietary intake of vitamin E for healthy individuals (Fitzpatrick et al, 2012). Considerable natural variation, however, exists for levels of tocopherols and tocotrienols in maize grain (Li et al, 2012; Lipka et al, 2013; Baseggio et al, 2019; Wu et al, 2022), thus there is tremendous potential for maize biofortification breeding efforts centered on the elevation of vitamin E and antioxidants levels in maize grain (Diepenbrock & Gore, 2015; Mène-Saffrané & Pellaud, 2017). To identify causal genes controlling grain tocochromanol levels, most genome-wide association studies (GWAS) used moderately-sized maize mapping panels with low genotyping densities that limited identification to a few large-effect loci involved in the genetic control of these nutritionally important grain phenotypes (Li et al, 2012; Lipka et al, 2013; Wang et al, 2018; Baseggio et al, 2019).…”

“…In a transcriptome-wide association study (TWAS), mRNA expression levels are correlated with terminal phenotypes, providing a high-resolution, gene-based association test that is less influenced by LD patterns, as opposed to DNA genetic markers in GWAS (Hirsch et al, 2014; Pasaniuc & Price, 2016; Lin et al, 2017; Kremling et al, 2019; Li et al, 2021; Hershberger et al, 2022). Through combining GWAS and TWAS with the Fisher’s combined test in the maize Ames panel of nearly 1,500 inbred lines, Wu et al (2022) further elucidated the genetic architecture of grain tocochromanol phenotypes that had not been fully resolved in the NAM panel, identifying novel associations with an arodeH2 paralog (Zm00001d014737), dxs1 , vte5 (phytol kinase), vte7 (α-/β-hydrolase family protein), and samt1 (S-adenosylmethionine transporter), along with the re-identification of por1 , por2 , dxs2 , arodeH2 Zm00001d014734, hppd1 , vte1 , vte4 , and hggt1 . Of these 13 genes, seven were detected by TWAS ( por1 , por2 , dxs1 , dxs2 , vte4 , hggt1 , and samt1 ), thus further demonstrating that regulatory variation plays an important role in influencing tocochromanol levels in maize grain.…”

Marker-assisted pyramiding of γ-tocopherol methyltransferase and glutamate formiminotransferase genes for development of biofortified sweet corn hybrids

“…The wide variability for micronutrient content in sweet corn unveils the great prospect of developing biofortified sweet corn varieties. Many quantitative trait loci (QTL) associated with micronutrients content have been identified ( Baseggio et al, 2019 ; Baseggio et al, 2020 ; Diepenbrock et al, 2017 ; Hershberger et al, 2021 ; Lone et al, 2021 ; Simic et al, 2012 ; Wu et al, 2022 ). For example, ZmVTE4 , encoding γ-tocopherol methyltransferase, is capable of catalyzing γ-tocopherol to α-tocopherol.…”