GWAS combined with TWAS reveals the QTLs and putative candidate genes associated with leaflet shape-associated traits in soybean (Glycine max (L.) Merr.)

Background Leaf shape critically determines soybean light interception and yield. Given the natural leaflet phenotypic variation across genetic backgrounds and environments, identifying leaflet position-dependent quantitative trait loci (QTLs) is essential for unraveling the genetic basis of leaf development and accelerating the breeding selection of soybean varieties with ideal plant architecture. Method In this study, multi-location association panels were constructed for GWAS and TWAS: 1062 (2019 Jingzhou) and 1161 (2020 Beijing) accessions for GWAS, while 285 (2019 Jingzhou) and 231 (2020 Beijing) accessions for TWAS. Results Six QTLs were detected: an independent, newly identified association signal on Chr3 (40534981–40825558) modulating lateral leaflet width (LW) and leaflet length-to-width ratio (LLWR) at the fourth upper node (4 S); a hotspot on Chr19 (44522754–45100493) modulating LW, LLWR and leaflet area (LA) of lateral leaflets at the fourth (4 S) and fifth (5 S) upper nodes; and an interval on Chr20 (35784232–36559289) containing the characterized gene Ln , which modulates LLWR across lateral leaflets (4 S, 5 S) and terminal leaflets at the fourth and fifth upper nodes (4 M, 5 M). Integrating gene expression profiling and haplotype analysis, four GWAS-derived genes ( Glyma.03G196600 , Glyma.03G196700 , Glyma.19G193700 and Glyma.19G194600 ) were predicted as putative candidate genes modulating leaflet shape. Two additional candidate genes ( Glyma.05G019900 and Glyma.20G119400 ) with suggestive associations to LLWR were further identified via TWAS, integrating transcriptome variation and haplotype analysis. Conclusions This study advances our understanding of the genetic basis governing soybean leaflet shape and supplies valuable gene resources and theoretical insights for molecular breeding targeting ideal soybean plant architecture.