Peanut kernels rich in oil, particularly those with oleic acid as their primary fatty acid, are in high demand among consumers, the food industry, and farmers due to their superior nutritional content, extended shelf life, and health benefits. The oil content and fatty acid composition are governed by multiple genetic factors. Identifying the quantitative trait loci (QTLs) related to these attributes will facilitate marker-assisted selection and genomic selection, thus enhancing quality-focused peanut breeding programs. For this purpose, we developed a population of 521 recombinant inbred lines (RILs) and tested their kernel quality traits across five different environments. We identified two major and stable QTLs for oil content, qOCAh12.1 and qOCAh16.1. The markers linked to these QTLs were designed by Kompetitive allele-specific PCR (KASP) and subsequently validated. Moreover, we found that the superior haplotype of oil content in the qOCAh16.1 region was conserved within the plant introduction (PI) germplasm cluster, as evidenced by a diverse peanut accession panel. In addition, we determined that qAh09 and qAh19.1, which harbor the key gene encoding fatty acid desaturase 2 (FAD2), influence all seven fatty acids, palmitic, stearic, oleic, linoleic, arachidic, gadoleic, and behenic acids. Regarding the protein content and the long-chain saturated fatty acid behenic acid, qAh07 emerged as the major and stable QTL, accounting for over 10% of the phenotypic variation explained (PVE). These findings can enhance marker-assisted selection in peanut breeding, with the aim of improving the oil content, and deepen our understanding of the genetic mechanisms that shape fatty acid composition.

Oligo probe staining is a low-cost and efficient chromosome identification technique. In this study, oligo genomic in situ hybridization (Oligo-GISH) technology was established in peanut. Peanut A and B subgenome-specific interspersed repeat (IR) oligo probe sets were developed based on clustering and electronic localization of tandem repeat sequences in the reference genome of Tifrunner. The Oligo-GISH kit was then used to perform staining of 15 Arachis species. The A-subgenome probe set stained the chromosomes of A- and E-genome Arachis species, the B-subgenome probe set stained those of B-, F-, K-, and E-genome species, and neither set stained those of H-genome species. These results indicate the relationships among the genomes of these Arachis species. The Oligo-GISH kit was also used for batch staining of the chromosomes of 389 seedlings from the irradiated M₁ generation, allowing 67 translocation and deletion lines to be identified. Subsequent Oligo-FISH karyotyping, FISH using single-copy probe libraries, and trait investigation identified seven homozygous chromosomal variants from the M₃ generation and suggested that there may be genes on chromosome 4B controlling seed number per pod. These findings demonstrate that the IR probe sets and method developed in this study can facilitate research on distant hybridization and genetic improvement in peanut.

Oil and protein content and fatty acid composition are quality traits in peanut. Elucidating the genetic mechanisms underlying these traits may help researchers to obtain improved cultivars by molecular breeding. Whole-genome resequencing of a recombinant inbred population of 318 lines was performed to construct a high-density linkage map and identify QTL for peanut quality. The map, containing 4561 bin markers, covered 2032 cM with a mean marker density of 0.45 cM. A total of 110 QTL for oil and protein content, and fatty acid composition were mapped on the 18 peanut chromosomes. The QTL qA05.1 was detected in four environments and showed a major phenotypic effect on the contents of oil, protein, and six fatty acids. The genomic region spanned by qA05.1, corresponding to a physical interval of approximately 1.5 Mb, contains two SNPs polymorphic between the parents that could cause missense mutations. The two SNP sites were employed as KASP markers and validated using lines with extremely high and low oil contents. These sites may be useful in the marker-assisted breeding of peanut cultivars with high oil contents.