The cultivated soybean (Glycine max (L.) Merrill) is domesticated from wild soybean (Glycine soja) and has heavier seeds with a higher oil content than the wild soybean. In this study, we identified a novel candidate gene associated with SW using a genome-wide association study (GWAS). The candidate gene GmWRI14-like was detected by GWAS analysis in three consecutive years. By constructing transgenic soybeans overexpressing the GmWRI14-like gene and gmwri14-like soybean mutants, we found that overexpression of GmWRI14-like increased the SW and increased total fatty acid content. We then used RNA-seq and qRT-PCR to identify the target genes directly or indirectly regulated by GmWRI14-like. Transgenic soyabeans overexpressing GmWRI14-like showed increased accumulation of GmCYP78A50 and GmCYP78A69 than non-transgenic soybean lines. Interestingly, we also found that GmWRI14-like proteins could interact with GmCYP78A69/GmCYP78A50 using yeast two-hybrid and bimolecular fluorescence complementation. Our results not only shed light on the genetic architecture of cultivated soybean SW, but also lays a theoretical foundation for improving the SW and oil content of soybeans.

Linolenic acid (LA) has poor oxidative stability since it is a polyunsaturated fatty acid. Soybean oil has a high LA content and thus has poor oxidative stability. To identify candidate genes that affect the linolenic acid (LA) content in soybean seeds, a genome-wide association study (GWAS) was performed with 1,060 soybean cultivars collected in China between 2019–2021 and which LA content was measured using matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS). A candidate gene, GmWRI14, encoding an APETALA2 (AP2)-type transcription factor, was detected by GWAS in cultivars from all three study years. Multiple sequence alignments showed that GmWRI14 belongs to the plant WRI1 family. The fatty acid contents of different soybean lines were evaluated in transgenic lines with a copy of GmWRI14, control lines without GmWRI14, and the gmwri14 mutant. MALDI-TOF IMS revealed that GmWRI14 transgenic soybeans had a lower LA content with a significant effect on seed size and shape, whereas gmwri14 mutants had a higher LA content. compared to control. The RNA-seq results showed that GmWRI14 suppresses GmFAD3s (GmFAD3B and GmFAD3C) and GmbZIP54 expression in soybean seeds, leading to decreased LA content. Based on the RNA-seq data, yeast one-hybrid (Y1H) and qRT-PCR were performed to confirm the transcriptional regulation of FAD3s by GmWRI14. Our results suggest that FAD3 is indirectly regulated by GmWRI14, representing a new molecular mechanism of fatty acid biosynthesis, in which GmWRI14 regulates LA content in soybean seeds.