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SUMMARY: Isoflavone, a secondary metabolite produced by Glycine max (L.) Merr. (soybean), is valuable for human and plant health. The genetic architecture of soybean isoflavone content remains unclear, however, despite several mapping studies. We generated genomic data for 200 soybean cultivars and 150 recombinant inbred lines (RILs) to localize putative loci associated with soybean seed isoflavone content. Using a genome‐wide association study (GWAS), we identified 87 single‐nucleotide polymorphisms (SNPs) that were significantly associated with isoflavone concentration. Using linkage mapping, we identified 37 quantitative trait loci (QTLs) underlying the content of four isoflavones found in the RILs. A major locus on chromosome 8 (qISO8‐1) was co‐located by both the GWAS and linkage mapping. qISO8‐1 was fine mapped to a 99.5‐kb region, flanked by SSR_08_1651 and SSR_08_1656, in a BC2F5 population. GmMPK1, encoding a mitogen‐activated protein kinase, was identified as the causal gene in qISO8‐1, and two natural GmMPK1 polymorphisms were significantly associated with isoflavone content. Overexpression of GmMPK1 in soybean hairy roots resulted in increased isoflavone concentrations. Overexpressing GmMPK1 in transgenic soybeans had greater resistance to Phytophthora root rot, suggesting that GmMPK1 might increase soybean resistance to biotic stress by influencing isoflavone content. Our results not only increase our understanding of the genetic architecture of soybean seed isoflavone content, but also provide a framework for the future marker‐assisted breeding of high isoflavone content in soybean cultivars. Significance Statement: We reported novel loci that control isoflavone content in soybean seeds by genome‐wide association mapping and linkage analysis using a high‐density haplotype map and genetic linkage map. Of these loci, a major QTL qISO8‐1 and its causal gene GmMPK1, underlying the isoflavone content of soybean, was fine mapped. The novel gene GmMPK1 could influence the isoflavone content in soybean and could also be involved in the soybean response to Phytophthora root rot. [ABSTRACT FROM AUTHOR]

• Screening for excellent soybean germplasms with high tocopherol concentration. • The genetic structure of the Toc concentration of the test sample was identified. • Haplotypes significantly associated with tocopherol concentration were detected. • Glyma.17G061900 may regulate tocopherol concentration in soybean seeds. Tocopherol (Toc) in soybean seeds has value for foods, medicines, cosmetics, and animal husbandry. Soybean breeders and soybean-derived food industries have thus focused on breeding new soybean cultivars with higher seed Toc content. In this study, an association panel consisting of 180 diverse soybean accessions were used to detect quantitative trait nucleotides (QTNs) and scan possible candidate genes based on genome-wide association analysis (GWAS) and high-throughput single-nucleotide polymorphisms (SNP). Approximately 22,611 SNPs were identified using the specific locus amplified fragment sequencing (SLAF-seq) approach and based on minor allele frequencies (MAFs) > 5 %, which spanned 84.10 % of the soybean whole genome. For the three individuals including α-tocopherol (α-Toc), γ- tocopherol (γ-Toc), δ- tocopherol (δ -Toc), and total tocopherol (total Toc) levels, a total of 18 quantitative trait nucleotides (QTNs) distributed on the 18 genomic regions of 10 chromosomes were verified via GWAS. Seven QTNs (rs55811336, rs7975863, rs37094897, rs7097743, rs17125409, rs330000, and rs4730199) could be identified in more than three environments, and the other eleven could be identified in least two environments. A total of 6, 8, 4, and 10 QTNs were novel loci for α-Toc, γ-Toc, δ-Toc, and total Toc, respectively. Nine QTNs (rs37505279, rs37686178, rs36359429, rs6898494, rs7097743, rs17125409, rs10200201, rs21080726, and rs5680781) were associated with more than one Toc component. A total of 216 candidate genes near the 200-kb flanking region of these identified QTNs were identified as potential candidate genes associated with soybean seed Toc levels. Of these genes, Glyma.17G061900 (located near QTN rs4730199 on Chr.17), a homogentisate phytyltransferase 1 that catalytic homogentisate to 2-methyl-6-phyl-1,4-benzoquinol in Toc biosynthesis, was detected through association analysis, haplotype analysis, and expression pattern analysis. The identified QTNs with beneficial alleles and the candidate genes would be valuable for improving Toc levels in soybean seeds and improving the nutritional value of soybean oil. [ABSTRACT FROM AUTHOR]