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Background  Soybean is a globally important legume crop that provides a primary source of high-quality vegetable protein and oil. Seed protein content (SPC) is a valuable quality trait controlled by multiple genes in soybean.    Results  In this study, we performed quantitative trait loci (QTL) mapping, QTL-seq, and RNA sequencing (RNA-seq) to reveal the genes controlling protein content in the soybean by using the high protein content variety Nanxiadou 25. A total of 50 QTL for SPC distributed on 14 chromosomes except chromosomes 4, 12, 14, 17, 18, and 19 were identified by QTL mapping using 178 recombinant inbred lines (RILs). Among these QTL, the major QTL q SPC _20–1 and q SPC _20–2 on chromosome 20 were repeatedly detected across six tested environments, corresponding to the location of the major QTL detected using whole-genome sequencing-based QTL-seq. 329 candidate DEGs were obtained within the QTL region of q SPC _20–1 and q SPC _20–2 via gene expression profile analysis. Nine of which were associated with SPC, potentially representing candidate genes. Clone sequencing results showed that different single nucleotide polymorphisms (SNPs) and indels between high and low protein genotypes in  Glyma.20G088000  and  Glyma.16G066600  may be the cause of changes in this trait.    Conclusions  These results provide the basis for research on candidate genes and marker-assisted selection (MAS) in soybean breeding for seed protein content.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-021-03176-2.

Genetic mapping high protein content QTL from soybean ‘Nanxiadou 25’ and candidate gene analysis