Understanding the genetic and regulatory mechanisms underlying wood traits and secondary cell wall (SCW) development in Ginkgo biloba is crucial for improving wood quality. We identified key genes related to wood traits and SCW development through integrated genome-wide association studies (GWAS), transcriptome-wide association studies (TWAS), and weighted gene co-expression network analysis (WGCNA). Cellulose biosynthesis in the SCW is catalyzed by the CesA4–CesA7–CesA8 complex encoded by GbCesA4, GbCesA7, and GbCesA8A/8B. These CesA genes form a co-expression network with TUBA/TUBB and EG, indicating coordination among cellulose synthesis, cytoskeletal guidance, and cell wall remodeling. Additionally, loss of function of GbCesA8B caused only a slight reduction in cellulose content, supporting potential functional redundancy between GbCesA8A and GbCesA8B. For hemicellulose biosynthesis, GbCSLA9A/9B and IRX9/IRX14 were major contributors to mannan/glucomannan and xylan synthesis, respectively, and formed a co-expression network with UXS, UXE, IRX7, GXMT, and URGT, spanning nucleotide sugar supply, transport, and polymer elongation and modification. Moreover, MYB46 may regulate mannan/glucomannan biosynthesis in the SCW by activating CSLA9 transcription. For lignin biosynthesis, TWAS identified multiple genes involved in phenylalanine biosynthesis, phenylpropanoid metabolism, and lignin monomer polymerization, including ADT/PDT, PAL, and PER, as well as MYB91 and several bHLH genes that may positively regulate lignin accumulation. Furthermore, several transcription factors potentially involved in SCW development were identified, including GATA9 as a putative positive regulator, WRKY12 and HB15 as potential negative regulators, and ELF6, which may facilitate tracheid expansion. Our findings provide valuable insights into the genetic regulation of wood traits and SCW development in Ginkgo.

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