Untargeted Metabolomics Reveals Regional and Cultivar-Associated Metabolomic Variation in Soybeans Across Northeast China.

Soybean (Glycine max) quality and metabolic composition are shaped by complex interactions between genetic background and agro-ecological conditions, making metabolic characterization across major production regions valuable for germplasm evaluation and quality-oriented breeding. In this study, an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach was applied to compare the mature seed metabolomes of nine representative commercial soybean cultivars sampled from two major production regions in Northeast China: Heilongjiang (HLJ, four cultivars from Harbin) and Liaoning (LN, five cultivars from Shenyang). Because region-specific cultivars were collected from a single site and seed lot per province without direct site-level meteorological or soil measurements, genotype and environment are confounded in this observational design, and the observed differences reflect the combined variation in the sampled cultivar-location cohorts. After preprocessing, 8283 features were putatively annotated (MSI Level 2/3). Multivariate statistical analyses (PCA, PLS-DA, and OPLS-DA) combined with cultivar-grouped cross-validation revealed clear metabolic separation between the sampled HLJ and LN cohorts. A total of 1485 putatively annotated metabolites (VIP > 1.0, subsample-level FDR < 0.05, fold change HLJ/LN > 2.0 or <0.5) were screened as exploratory candidate differential metabolites, with 953 exhibiting higher abundance (FC>2.0) and 532 exhibiting lower abundance (FC<0.5, i.e., higher abundance in LN) in HLJ soybeans relative to LN soybeans. Exploratory KEGG pathway mapping (based on nominal p-values, as no pathway reached FDR-adjusted q<0.05) indicated nominal enrichment in isoflavonoid biosynthesis (p=0.0043, q=0.261) and tryptophan metabolism (p=0.0093, q=0.283); specifically, sampled LN cultivar-location combinations exhibited higher steady-state abundances of isoflavones and their glycosylated derivatives, whereas sampled HLJ soybeans exhibited higher steady-state abundances of specific lipid species (unsaturated fatty acyls, sphingolipids) and tryptophan-derived indoleamines. In the absence of direct environmental measurements, potential links between these steady-state metabolic signatures and regional macroclimatic differences (such as thermal regimes or high-latitude cold stress) are proposed strictly as exploratory, literature-informed hypotheses. Overall, these findings provide preliminary candidate metabolic markers for characterizing the sampled regional soybean germplasm; however, independent validation across additional harvest years, multiple geographic sites, independent seed lots, and broader cultivar panels will be required before these candidate metabolites can be considered validated biomarkers for geographic authentication.