Peanut (Arachis hypogaea L.) is a great plant protein source for human diet since it has high protein content in the kernel. Therefore, seed protein content (SPC) is considered a major agronomic and quality trait in peanut breeding. However, few genetic loci underlying SPC have been identified in peanuts, and the underlying regulatory mechanisms remain unknown, limiting the effectiveness of breeding for high-SPC peanut varieties. In this study, a major QTL (qSPCB10.1) controlling peanut SPC was identified within a 2.3 Mb interval in chromosome B10 by QTL-seq using a recombinant inbred line population derived from parental lines with high and low SPCs, respectively. Sequence comparison, transcriptomic analysis, and annotation analysis of the qSPCB10.1 locus were performed. Six differentially expressed genes with sequence variations between two parents were identified as candidate genes underlying qSPCB10.1. Further locus interaction analysis revealed that qSPCB10.1 could not affect the seed oil accumulation unless qOCA08.1_XH13 was present, a high seed oil content (SOC) allele for a major QTL underlying SOC. In summary, our study provides a basis for future investigation of the genetic basis of seed protein accumulation and facilitates marker-assisted selection for developing high-SPC peanut genotypes.

Peanut is susceptible to Aspergillus flavus infection, and the consequent aflatoxin contamination has been recognized as an important risk factor affecting food safety and industry development. Planting peanut varieties with resistance to aflatoxin contamination is regarded as an ideal approach to decrease the risk in food safety, but most of the available resistant varieties have not been extensively used in production because of their low yield potential mostly due to possessing small pods and seeds. Hence, it is highly necessary to integrate resistance to aflatoxin and large seed weight. In this study, an RIL population derived from a cross between Zhonghua 16 with high yield and J 11 with resistance to infection of A. flavus and aflatoxin production, was used to identify quantitative trait locus (QTL) for aflatoxin production (AP) resistance and hundred-seed weight (HSW). From combined analysis using a high-density genetic linkage map constructed, 11 QTLs for AP resistance with 4.61–11.42% phenotypic variation explanation (PVE) and six QTLs for HSW with 3.20–28.48% PVE were identified, including three major QTLs for AP resistance (qAFTA05.1, qAFTB05.2 and qAFTB06.3) and three for HSW (qHSWA05, qHSWA08 and qHSWB06). In addition, qAFTA05.1, qAFTB06.3, qHSWA05, qHSWA08 and qHSWB06 were detected in multiple environments. The aflatoxin contents under artificial inoculation were decreased by 34.77–47.67% in those segregated lines harboring qAFTA05.1, qAFTB05.2 and qAFTB06.3, while the HSWs were increased by 47.56–49.46 g in other lines harboring qHSWA05, qHSWA08 and qHSWB06. Conditional QTL mapping indicated that HSW and percent seed infection index (PSII) had no significant influence on aflatoxin content. Interestingly, the QT 1059 simultaneously harboring alleles of aflatoxin content including qAFTA05.1 and qAFTB05.2, alleles of PSII including qPSIIB03.1, qPSIIB03.2, and qPSIIB10 and alleles of HSW including qHSWA05, qHSWB06, qHSWA08 had better resistance to A. flavus infection and to toxin production and higher yield potential compared with the two parents of the RIL. The above identified major loci for AP resistance and HWS would be helpful for marker-assisted selection in peanut breeding.

Peanut ( Arachis hypogaea ) is an allotetraploid legume crop of agricultural and economic importance worldwide. Despite advances in peanut genomics, the integration of genetic diversity with multi‐omics landscapes remains limited, and a comprehensive understanding of subgenome asymmetry is still lacking. Here, we present a high‐quality genome assembly of an elite peanut cultivar Zhonghua 5 (ZH05; 2,622.85 Mb with 73,718 predicted genes) and construct a species‐specific pan‐genome based on 1,286 accessions, uncovering 849.82 Mb of non‐reference sequences and 18,484 novel genes. Over 23 million genetic variants and extensive gene PAVs substantially expand the landscape of genetic diversity in peanut. Integrative multi‐omics (transcriptome, DNA methylation, chromatin accessibility, and three‐dimensional genome architecture), combined with population genomics analyses, reveal pronounced and multi‐layered subgenome asymmetry. Specifically, SubA shows higher chromatin accessibility and overall gene expression, whereas SubB displays more transposable elements, elevated DNA methylation, more genes under selection during domestication, and stronger three‐dimensional chromatin interactions with more stable topologically associating domains. Furthermore, genetic dissection using a recombinant inbred line population identified several major stable QTLs for seed size, whose pyramiding contributes to ZH05's superior yield performance. Collectively, this study establishes an integrated genomic framework for peanut, illuminates asymmetric subgenome evolution across sequence, epigenetic, and chromatin architecture, and provides valuable resources for accelerating molecular breeding and crop improvement.

Aflatoxin contamination caused by Aspergillus flavus threatens the development of peanut industry, breeding aflatoxin-resistant peanut varieties are highly needed. In this study, a recombinant inbred line (RIL) population was developed from a cross between Zhonghua 16 and Kainong H03-3—a high oleic acid peanut line exhibiting resistance to both A. flavus infection and aflatoxin production. Using this RIL population, a high-density genetic map was constructed and employed to identify quantitative trait loci (QTLs) associated with resistance to A. flavus infection (described as percentage seed infection index, PSII) and aflatoxin production (described as aflatoxin content, ATC) across four environments. Three QTLs for PSII and ATC were detected, among which novel QTLs with major effect for both traits were co-localized in chromosome B06, explaining 20.71% and 22.73% of phenotypic variance for PSII and ATC, respectively. Within this co-localized genomic region, 13 candidate genes exhibited strong co-expression patterns linked to both PSII and ATC. Among them, Chr16g3738, harboring a non-synonymous variant in its exon, was used to develop a diagnostic KASP marker KASP-Chr16g3738. Validation of KASP-Chr16g3738 in a peanut germplasm panel and a breeding population (Jihua 6×Kaixuan 01–6) demonstrated that the favorable allele of the candidate gene was associated with a reduction of 15.27–56.67% in PSII and 27.59–72.76% in ATC. This study was the first report in identification of a genomic interval co-localized with QTLs conferring resistance to both A. flavus infection and aflatoxin production, and provides an efficient marker-assisted selection approach for accelerating improvement of resistance to aflatoxin in peanut.