Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

Key message: In the present study, we identified 10 significant single nucleotide polymorphisms (SNPs), 3 stable quantitative trait loci (QTLs) and 3 potential candidate genes associated with peanut kernel resistance to Aspergillus flavus stress. Peanut ( Arachis hypogaea L.) is highly susceptible to A. flavus infection, producing highly carcinogenic aflatoxins. Breeding resistant varieties is an effective and sustainable approach to address this issue, and identifying novel genetic sources and loci underlying resistance is crucial. In this study, 353 A. hypogaea accessions were evaluated for resistance to A. flavus infection and aflatoxin production across three environments, leading to the identification of 13 accessions with stable resistance to both infection and aflatoxin production. A genome-wide association study (GWAS) was performed by integrating phenotypic data from multiple environments with 935,231 SNP markers, resulting in the detection of 10 significant marker-trait associations (MTAs). Of these, one showed consistent association with infection resistance in at least two out of three environments, and two were associated with resistance to both aflatoxin B ₁ (AFB ₁ ) and aflatoxin B ₂ (AFB ₂ ) production. Based on linkage disequilibrium (LD) decay estimates (70 kb upstream and downstream of significant SNPs), three novel stable QTLs were identified on chromosomes A03 and A05, designated qII_A05 , qAF_A03 , and qAF_A05 , and 13 candidate genes were initially selected within these QTL regions. Haplotype analysis further validated 3 key candidate genes: Arahy.7046BI.1 (encoding a PH/START domain-containing protein), Arahy.TX2FLU.1 (encoding a receptor-like kinase), and Arahy.ASR4JM.1 (encoding a TIR-NBS-LRR class disease resistance protein). These genes exhibited significant haplotype-trait associations, with favorable haplotypes showing high frequencies (85.43%–94.12%) in the germplasm pool, facilitating their direct utilization in breeding. Overall, this study uncovers key genetic loci and candidate genes governing peanut resistance to A. flavus infection and aflatoxin production, provides elite resistant germplasm resources, and offers practical insights for marker-assisted selection. The findings lay a solid foundation for accelerating the development of A. flavus -resistant peanut varieties and provide valuable genetic information for peanut breeding programs.