Genetic regulation of major immunogenic protein accumulation in peanut seeds

Peanut is a major oilseed crop in the U.S., which ranks third globally in production, with South Carolina ranking sixth nationally. Despite their economic and nutritional value, peanuts are unsuitable for 1-2% of the U.S. population due to allergic reaction, including anaphylaxis, making the development of reduced-allergen peanuts a priority. This study aimed to identify peanut lines with reduced Ara h1, h2, h3, and h6 levels and to elucidate their genetic regulation using genome-wide association studies (GWAS). Ninety-two accessions from the U.S. peanut mini-core collection were screened using ELISA and SDS-PAGE, and the resulting data were evaluated for association with 5,532 SNP markers. Substantial phenotypic diversity was observed. Twenty-three lines with extreme protein phenotypes were further evaluated by RP-UPLC, and nine by LC-MS. GWAS identified 165 marker-trait associations (MTAs) across raw and log-transformed datasets, with 13 MTAs common to both analyses. These MTAs were grouped into protein quantitative loci (PQLs), revealing six trans-PQLs and cis-PQLs for two of the five Ara h genes. Seed-expressed candidate transcription factors (TFs), including MYB, MYC, ERF, and bZIP, and pleiotropic PQLs were identified. Promoter analysis (1 kb upstream of transcription start site or TSS) of the corresponding Ara h genes revealed binding sites for TFs underlying these PQLs. We hypothesize that these TFs trans-regulate the Ara h genes either directly or through a regulatory cascade. These findings provide initial insights into the regulatory landscape of Ara h genes and will facilitate breeding for reduced allergen content in peanut.