Xu Wu

Carboxylesterases (CXEs) participate in diverse plant metabolic processes, including isoflavone biosynthesis. However, the soybean GmCXE subfamily remains poorly characterized, especially in relation to root isoflavone accumulation and the response to Fusarium oxysporum. Here, fifty-six putative GmCXE genes were identified in the soybean genome and classified into three major phylogenetic clades. Analyses of gene structure, conserved motifs, protein domains, and promoter cis-elements revealed conserved features as well as potential functional divergence among subfamily members. Collinearity and duplication analyses indicated that segmental duplication was the main driver of GmCXE subfamily expansion. Tissue-specific expression profiling and RT-qPCR validation selected five root-expressed genes as candidates associated with isoflavone accumulation. SNP variation analysis and allelic group analysis of 209 soybean accessions further prioritized GmCXE54 as a candidate gene for root isoflavone accumulation. Allelic groups defined by a putative promoter SNP, Chr.20-rs39215413, showed significant differences in root daidzein and total isoflavone contents, with accessions carrying the C allele exhibiting higher levels of both traits than those carrying the T allele. Functional analysis in soybean hairy roots showed that GmCXE54 overexpression increased daidzein and total isoflavone accumulation. At 3 h after F. oxysporum inoculation, GmCXE2, GmCXE39, and GmCXE54 were induced, with GmCXE54 showing the strongest response in the resistant accession ZD27. These findings clarify GmCXE subfamily evolution and identify GmCXE54 as a candidate gene associated with root isoflavone accumulation and early F. oxysporum response, offering new perspectives for improving soybean isoflavone-related traits and investigating root response mechanisms.

Soybean root rot caused by Fusarium graminearum is an important soil-borne disease. It hinders seedling establishment and ultimately reduces soybean yield. Resistant germplasm and reliable molecular markers are therefore needed for resistance breeding. In this study, 336 soybean accessions were evaluated for resistance to F. graminearum root rot using the disease severity index (DSI), which ranged from 5.71 to 100.00 across the association panel. Genome-wide association analysis was performed using resequencing-based single nucleotide polymorphism (SNP) data with mixed linear model (MLM) and Fixed and random model Circulating Probability Unification (FarmCPU) models, which detected 117 and 113 candidate resistance-associated SNPs, respectively. Among these, 105 shared SNPs were used to define candidate genomic intervals containing 247 annotated genes. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, functional annotation, and allelic-effect analysis, six candidate genes and their associated exonic SNPs were prioritized. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed infection-responsive expression patterns for all six candidate genes, with Glyma.17g202500 and Glyma.18g266700 showing stronger induction in the resistant accession. Two SNPs in these genes were converted into Kompetitive allele-specific PCR (KASP) assays. KASP-S17_32244510 and KASP-S18_55105706 were successfully developed for genotype screening, with screening efficiencies of 73.08% and 74.29%, respectively. These findings identify useful genetic targets and molecular markers for improving soybean resistance to root rot caused by F. graminearum.

Fusarium root rot, primarily caused by Fusarium solani, is a damaging soil-borne disease that restricts soybean growth and reduces yield. In the present study, a panel of 330 soybean germplasm accessions was inoculated with F. solani. Disease responses were evaluated using the disease severity index (DSI). The soybean accessions displayed substantial variation in their susceptibility to F. solani. Based on DSI, 39 accessions were classified as highly resistant, 94 as resistant, 125 as susceptible, and 72 as highly susceptible, accounting for 11.82%, 28.48%, 37.88%, and 21.82% of the panel, respectively. The genome-wide association study (GWAS) was performed using a genotyping dataset of 627,436 high-quality single nucleotide polymorphisms (SNPs) and two models, Fixed and random model Circulating Probability Unification (FarmCPU) and mixed linear model (MLM). Both GWAS models detected putative SNP associations across seven chromosomes. Based on SNP allelic-effect analysis and gene function annotation, eight genes were prioritized and subsequently evaluated by quantitative reverse transcription PCR (qRT-PCR) for their responses to F. solani infection. Two Kompetitive allele-specific PCR (KASP) markers, KASP-S13_37431242 and KASP-S13_37529208, were developed from resistance-associated SNPs on chromosome 13 and evaluated across the diverse soybean association panel used in this study. Accessions carrying the favorable genotypes of these markers were enriched for resistant germplasm, with positive predictive values of 65.52% and 66.67%, respectively, indicating their potential value for preliminary favorable-allele tracking and germplasm prioritization. Collectively, these results improve our understanding of the genomic components underlying soybean responses to F. solani. The identified loci, candidate genes, and KASP assays provide a basis for further functional validation and the development of multi-locus strategies for improving soybean resistance to Fusarium root rot.

Isoflavones belong to the class of flavonoid compounds, which are important secondary metabolites that play a crucial role in plant development and defense. Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyzes the conversion of Acetyl-CoA into Malonyl-CoA in plants. It is a key enzyme in fatty acid synthesis and also catalyzes the production of various secondary metabolites. However, information on the ACC gene family in the soybean (Glycine max L. Merr.) genome and the specific members involved in isoflavone biosynthesis is still lacking. In this study, we identified 20 ACC family genes (GmACCs) from the soybean genome and further characterized their evolutionary relationships and expression patterns. Phylogenetic analysis showed that the GmACCs could be divided into five groups, and the gene structures within the same groups were highly conserved, indicating that they had similar functions. The GmACCs were randomly distributed across 12 chromosomes, and collinearity analysis suggested that many GmACCs originated from tandem and segmental duplications, with these genes being under purifying selection. In addition, gene expression pattern analysis indicated that there was functional divergence among GmACCs in different tissues. The GmACCs reached their peak expression levels during the early or middle stages of seed development. Based on the transcriptome and isoflavone content data, a weighted gene co-expression network was constructed, and three candidate genes (Glyma.06G105900, Glyma.13G363500, and Glyma.13G057400) that may positively regulate isoflavone content were identified. These results provide valuable information for the further functional characterization and application of GmACCs in isoflavone biosynthesis in soybean.