The oil content of maize kernels is essential to determine its nutritional and economic value. A multiparent population (MPP) consisting of five recombinant inbred line (RIL) subpopulations was developed to elucidate the genetic basis of the total oil content (TOC) in maize. The MPP used the subtropical maize inbred lines CML312 and CML384, along with the tropical maize inbred lines CML395, YML46, and YML32 as the female parents, and Ye107 as the male parent. A genome-wide association study (GWAS) was performed using 429 RILs of the multiparent population across three environments, employing 584,847 high-quality single nucleotide polymorphisms (SNPs). Furthermore, linkage analysis was performed in the five subpopulations to identify quantitative trait loci (QTL) linked to TOC in maize. Through QTL mapping and GWAS, 18 QTLs and 60 SNPs that were significantly associated with TOC were identified. Two novel candidate genes, Zm00001d029550 and Zm00001d029551, related to TOC in maize and located on chromosome 1 were reported, which have not been previously reported. These genes are involved in biosynthesis, lipid signal transduction, plant development and metabolism, and stress responses, potentially influencing maize TOC. Haplotype analysis of Zm00001d029550 and Zm00001d029551 revealed that Hap3 could be considered a superior haplotype for increasing TOC in maize. A co-located SNP (SNP-75791466) on chromosome 1, located 5648 bp and 11,951 bp downstream of the candidate genes Zm00001d029550 and Zm00001d029551, respectively, was found to be expressed in various maize tissues. The highest expression was observed in embryos after pollination, indicating that embryos are the main tissue for oil accumulation in maize. This study provides a theoretical basis for understanding the genetic mechanisms underlying maize TOC and developing high-quality, high-oil maize varieties.
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