Identification and utilization of a novel major QTL and linked markers for enhancing protein content in peanut

Peanut is a vital source of protein for humans, playing a key role in maintaining a steady protein supply. In this study, the high protein cultivar Zhonghua6 (29.14±1.69%) was crossed with Xuhua13 (24.55±1.84%) to construct a recombinant inbred line (RIL) population. The protein content of the RIL population exhibited significant variation, ranging from 21.05% to 30.28%. To discover genomic regions associated with protein content, four libraries were constructed (two parents and two extreme bulks) for bulked segregant sequencing (BSA-seq). The results revealed significant associations between protein content and the genomic regions on chromosomes A07, A10, B03, and B10. Through linkage analysis, nine QTLs (quantitative trait loci) for protein content were identified, among which the major and stable QTL qPCB03 on chromosomes B03 (125.92-127.94 Mb) explained 11.03%-12.86% of the phenotypic variation. This QTL ( qPCB03 ), simultaneously identified by both BSA-Seq and linkage mapping, had not been documented in prior studies. Within the ~2Mb interval of qPCB03 , a total of 349 genomic variants were discovered, including six single nucleotide polymorphisms (SNPs) that resulted in nonsynonymous mutations in six genes. By assessing allelic effects in peanut germplasm and analyzing transcriptome sequencing data, five candidate genes ( Ah13g469400 , Ah13g473200 , Ah13g475000 , Ah13g477200 and Ah13g477400 ) were identified. According to the marker-assisted selection, favorable genotypes could potentially enhance protein content by 1.23% to 1.57% in the RIL populations. The identification of stable loci and the development of markers facilitate marker-assisted breeding in peanuts, while the discovery of candidate genes lays the groundwork for the fine mapping of key genes regulating protein content.