Identification of <i>HSSP1</i> as a regulator of soybean protein content through <scp>QTL</scp> analysis and Soy‐<scp>SPCC</scp> network

Soybeans (Glycine max L. Merr.) are a major source of plant-based protein for human nutrition and livestock feed. Enhancing the protein content of soybean seeds is vital for meeting growing dietary needs and promoting sustainable agricultural practices. In this study, we first performed QTL (Quantitative Trait Loci) mapping analysis and constructed a Soybean Seed Protein Content Co-expression (Soy-SPCC) network to identify key genes associated with soybean seed protein accumulation. Next, we investigated the role of High Seed Storage Protein1 (HSSP1) in regulating soybean seed protein content through a comprehensive analysis. Functional validation through overexpression and gene knockout experiments demonstrated that HSSP1, a key component of the Soy-SPCC network, significantly influences seed storage protein levels. Particularly, HSSP1 enhances the expression of GmCG1 by binding directly to its cis-acting element, leading to increased protein content in soybean seeds. Furthermore, we performed a molecular module stacking breeding analysis of 120 candidate genes identified from the Soy-SPCC network, including HSSP1, to identify genetic variations associated with protein content. This study provides a novel perspective on soybean protein regulation. The identification of HSSP1 as a critical regulator offers valuable insights for developing high-protein soybean varieties and advancing breeding strategies aimed at improving soybean seed quality.