Fusarium wilt, caused by the soil-borne Fusarium oxysporum f.sp. niveum (FON), is a devastating disease constraining global watermelon production. While three physiological races (0, 1 and 2) are established, resistance mechanisms to the foundational race remain largely unexplored. This study first evaluated seedling resistance to FON race 0 across 41 diverse watermelon accessions revealing that wild germplasm (Citrullus amarus) possessed superior resistance with 24.4% classified as highly resistant, whereas cultivated accessions were predominantly susceptible. The resistant accession WM216 and susceptible G42 were selected for in-depth analysis. Physiological assessment demonstrated that WM216 mounted a robust and systemic defence response, characterized by a significant post-inoculation induction of ROS-scavenging enzymes like catalase and peroxidase. Integrated transcriptome and BSA-seq analysis identified a major resistance QTL on chromosome 8 (15-22 Mb). Cross-referencing these datasets pinpointed a single key candidate gene, ClG42_08g0072500, encoding a two-component response regulator (ARR family) implicated in cytokinin signalling. Expression profiling suggested this gene may act as a negative regulator of resistance. Our findings underscore the invaluable resistance alleles present in wild watermelon germplasm, provide a foundational genetic map for FON-0 resistance and deliver critical molecular resources for marker-assisted breeding of durable Fusarium wilt-resistant cultivars.

Soybean is crucial to food processing and agricultural output. However, pests and diseases can easily impact soybeans, reducing their production. Soybean cyst nematode (SCN) is a soilborne pathogen that has a large geographic range, a long lifespan, and the potential to inflict substantial harm to the soybean industry. Persistent use of major resistance genes leads to a progressive loss of resistance; therefore, continuous identification of new soybean strains and genes is essential for continued sustainable soybean production. In this research, the SCN-resistant and SCN-sensitive germplasm DN-L10 and Heinong 37 were inoculated with SCN 3. After stress treatment, the stressed roots were collected for RNA-Seq analysis. The sequencing results screened out the differentially expressed gene GmbHLH18. The GmbHLH18 gene was cloned, and the overexpression vector pCAMBIA3300-GmbHLH18 was constructed. Agrobacterium infected soybean hairy roots and genetically modified the roots of DN50 soybeans, and transgenic root seedlings were obtained. The transgenically identified root seedlings were transplanted in soil infested with SCN 3, and resistance to root nematodes was determined by magenta staining. The secondary and tertiary structures of the protein, phosphorylation sites, as well as the hydrophilicity related to the GmbHLH18 gene were analyzed. Subsequently, the recombinant subcellular localization vector pCAMBIA1302-GmbHLH18 was employed. Agrobacterium was injected into tobacco leaves, and organelle-specific expression was observed. Finally, stress resistance-related indexes of the roots of overexpressing plants and WT plants under SCN 3 stress were measured. The results showed that overexpression and subcellular localization vectors were successfully constructed and transformed into Agrobacterium K599 and GV3101, respectively. The encoded protein had 1149 amino acids, a molecular weight of 95.76 kDa, an isoelectric point of 5.04, 60 phosphorylation sites, a tertiary structure of a-helix (36.39%), random coil (53.40%), extended chain (8.64%), and corner (1.57%), and was hydrophilic. The protein that the gene encoded was a nuclear-localized protein, according to the results of subcellular localization analysis. Moreover, the Agrobacterium-induced hairy root test revealed that the number of overexpressed pCAMBIA3300-GmbHLH18 transgenic roots in the unit area of DN50 was substantially lower than in the control group, which at first suggested that the gene had partial resistance to SCN 3. Stress resistance-related indexes suggest that the contents of POD, SOD, and proline in the overexpressing root significantly increase after SCN 3 stress, demonstrating that this gene can enhance the plant’s resistance to the SCN 3 pathogen. Future research could focus on further elucidating the molecular mechanism underlying the gene’s resistance to SCN 3 and exploring its potential application in breeding soybean varieties with enhanced resistance.

Soybean (Glycine max) is a major crop for grain and oil production worldwide. However, soybean cyst nematode (SCN) disease is a serious threat to soybean cultivation, causing major challenges for agriculture in China and globally. In this study, 306 soybean germplasms were evaluated for resistance to SCN HG type 0 using the female index (FI), a metric quantifying nematode reproduction on soybean roots. Furthermore, we performed a genome-wide association analysis study using 1,332,548 high-density single nucleotide polymorphism (SNP) markers. Through this analysis, the study identified quantitative trait nucleotides (QTN) and pinpointed candidate genes that are linked to resistance against SCN HG type 0. A total of 77 SNPs and 117 candidate genes associated with soybean resistance to SCN HG type 0 were identified. Subsequently, two kompetitive allele-specific PCR (KASP) markers, S19_rs8522772 and S19_rs8384176, along with four cleaved amplified polymorphic sequence (CAPS) markers (S19_rs838271, S19_rs8522589, S19_rs8466511 and S19_rs8481473) were developed. These markers were found to be closely linked to soybean resistance to SCN based on the results of beneficial allele analysis of candidate genes. These markers not only expanded the soybean germplasm resource, but also provided a solid foundation for molecular breeding of soybean varieties resistant to SCN.

The plant stress response mechanism is activated by biotic and abiotic stresses, but its continuous activation typically affects growth. The role of ferritin in regulating biomass accumulation has been extensively characterized in diverse plant species; however, the underlying mechanisms through which it contributes to salt stress tolerance and Fusarium resistance remain poorly understood. Here, we confirm that overexpression of ferritin leads to iron accumulation and Fe3+ sequestration in both aboveground and roots, activating the iron uptake and transport system. More importantly, GmFER1 enhances salt stress tolerance and Fusarium resistance. First, GmFER1 is localized in chloroplasts and significantly induced by salt stress and Fusarium infection. Overexpression of GmFER1 increases soybean yield per plant by enhancing net photosynthetic rate and Rubisco enzyme activity, without activating the reactive oxygen scavenging mechanism. Under salt stress, GmFER1 enhances resistance by improving the activities of SOD and CAT enzymes, as well as Na+ efflux capacity. Under Fusarium infection, GmFER1 enhances resistance to the pathogen by boosting antioxidant capacity. Moreover, iron-deficiency tests revealed that increased CAT and SOD activities under salt stress are linked to iron ions accumulation. Lastly, we analysed the effects of GmFER1 gene variation on salt tolerance, disease resistance and 23 agronomic traits related to yield and quality. Further analysis of GmFER1 gene variation revealed that the Hap2 haplotypes could potentially enhance salt resistance, disease resistance, pod number and oil content in soybean. Our research offers a new way to reduce growth penalties while boosting plant resistance to salt stress and Fusarium infection.

Rhizoctonia solani root rot (RSRR) is a major disease that significantly reduces soybean yields, causing substantial economic losses to global soybean production. To elucidate the genetic basis of RSRR resistance, 310 soybean germplasm accessions were evaluated using the disease severity index (DSI) following inoculation with R. solani. Among these accessions, 46.13% were susceptible, and only 2.26% exhibited high resistance. Utilizing resequencing data consisting of 738,561 Single Nucleotide Polymorphism (SNP) loci, a genome-wide association study (GWAS) was performed by integrating both general linear model (GLM) and mixed linear model (MLM) approaches, resulting in the identification of 21 SNPs significantly associated with resistance on chromosomes 3, 13, 15, 16, 17, and 18, and six candidate genes. RT-qPCR expression analysis revealed that four genes, including Glyma.03G166300, Glyma.03G168100, Glyma.13G212700, and Glyma.13G212300, were significantly upregulated in resistant genotypes after inoculation. Furthermore, Cleaved Amplified Polymorphic Sequences (CAPS) and Kompetitive Allele Specific PCR (KASP) molecular markers were successfully developed based on the RSRR-associated SNPs S3_38086892, S3_38247290, and S13_32595026, providing effective tools for marker-assisted selection (MAS). The findings strengthen our genetic knowledge concerning RSRR resistance and contribute to the molecular breeding of resistant soybean cultivars.

Using the cotyledonary node method, four traits related to callus induction rate were identified in 185 soybean germplasm resources. Cultivation of callus tissue is crucial for soybean (Glycine max (L.) Merr.) genetic transformation and functional genomics studies. Identifying genes associated with the induction rate of soybean callus tissue is therefore essential for biotechnological breeding and for understanding the molecular genetic mechanisms of soybean regeneration. The efficiency of genetic transformation impacts the breeding rate of soybeans, with its success rate dependent on the soybean regeneration system. Subsequently, whole genome association analysis (GWAS) and multidimensional functional validation were conducted. GWAS identified 66 significantly associated SNP loci corresponding to the four traits. Expression analysis in extreme phenotypes highlighted four candidate genes: Glyma.12G164100 (GmARF1), Glyma.12G164700 (GmPPR), Glyma.02G006200 (GmERF1), and Glyma.19G128800 (GmAECC1), which positively regulate callus formation. Overexpression and gene-editing assays in hairy roots confirmed that these genes significantly enhanced callus formation rate and density, with GmARF1 exerting the most prominent effect. Hormone profiling revealed elevated levels of gibberellin (GA), auxin (IAA), cytokinin (CTK), and other phytohormones in transgenic lines, consistent with enhanced responsiveness to exogenous GA. Overall, the results suggest that these four candidate genes may promote soybean regeneration, with GmARF1 showing the most pronounced effect. These results provide valuable genetic resources for improving soybean regeneration efficiency and accelerating genetic transformation-based breeding.

Fusarium root rot, caused by Fusarium equiseti, poses a significant threat to soybean production. This study aimed to explore the genetic basis of resistance to Fusarium equiseti root rot (FERR) by evaluating the resistance phenotype of 346 soybean germplasms and conducting a genome-wide association study (GWAS) using 698,949 SNP markers obtained from soybean germplasm resequencing data. GWAS analysis identified 101 SNPs significantly associated with FERR resistance, distributed across nine chromosomes, with the highest number of SNPs on chromosomes 13 and 20. Further gene-based association and allele variation analyses identified candidate genes whose mutations are closely related to FERR resistance. To accelerate soybean FERR resistance breeding screening, we developed CAPS markers S13_14464319-CAPS1 and S15_9215524-CAPS2, targeting these SNP sites, and KASP markers based on the S15_9205620-G/A, providing an effective tool for marker-assisted selection (MAS). This study offers a valuable theoretical foundation and molecular marker resources for the functional validation of FERR resistance genes and soybean disease resistance breeding.

The ABI3 transcription factor is a key regulator in plant growth and development. Through transcriptome analysis of the resistant soybean cultivar ‘Dongnong L10′ and the susceptible cultivar ‘Heinong 37′ exposed to soybean cyst nematode race 3 (SCN 3) stress, the differentially expressed gene GmABI3VP1 was identified. The GmABI3VP1 gene was then cloned and analyzed through bioinformatics, subcellular localization, and qRT-PCR analysis of resistant and susceptible soybean germplasms, as well as overexpression and gene editing of soybean hairy roots followed by SCN 3 identification analysis. It was found that the protein encoded by GmABI3VP1 is an acidic and hydrophilic protein with transmembrane domains. It has a collinear relationship with Arabidopsis and is widely distributed in plants. Through the analysis of promoter elements, it was shown that this gene contains multiple hormone-responsive promoter elements like ABRE/ABRE3a/ABRE/4a/as-1 and stress-responsive elements such as Myb/MYC/MYc. Transient expression in tobacco indicated that the GmABI3VP1 gene is located in the nucleus. The transcription of GmABI3VP1 responds to the stress of SCN, and its transcriptional level is relatively high in the roots of resistant materials. Genetic transformation mediated by Agrobacterium rhizogenes was used to obtain GmABI3VP1 gene overexpressed and CRISPR-Cas9 gene-edited soybean hairy roots. In comparison to the wild type (WT), the density of nematodes per area was notably lower in hairy roots overexpressing (OX) the gene, whereas the density of SCN per unit area (per cm of lateral root length) significantly increased in gene-edited (KO) soybean hairy roots. Through SCN phenotyping, GmABI3VP1 was identified as a contributor to SCN 3 resistance. This study provides initial insights into the role of the GmABI3VP1 gene in SCN resistance, establishing a robust basis for future research on the mechanisms underlying SCN disease resistance and offering valuable genetic reservoirs for SCN 3 resistance.

Soybean cyst nematode (SCN, Heterodera glycines) is one of the major pathogens of soybean worldwide. We utilized the CHIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) data from the transgenic GmMYB29 strain (Glycine Max roots). We then performed enrichment analysis using KEGG and GO to identify potential candidate genes within the promoter-binding region. A targeted regulatory relationship between the GmMYB29 and GmPP2C-37like genes was further identified using the dual-luciferase Assay (Luciferase, LUC) and yeast one-hybrid Assay (Y1H). Hairy roots with target gene overexpression and gene-edited hairy roots were generated, and their resistance to soybean cyst nematode (SCN) was evaluated. Meanwhile, the presence of reciprocal genes with GmPP2C-37like was determined by the yeast two-hybrid library screening method. The targeting relationship between GmMYB29 and GmPP2C-37like genes was further validated through the Y1H assay and LUC assay. Based on phenotypic assessments of SCN, transgenic soybean roots overexpressing GmPP2C-37like exhibited significantly enhanced resistance to SCN 3 compared to wild-type. Further analysis revealed that GmPP2C-37like collaborates with other regulatory factors to modulate soybean resistance against SCN. Yeast two-hybrid library (Y2H) screening identified 18 interacting proteins. These findings not only illuminate the functional role of GmPP2C-37like but also provide a foundation for dissecting its molecular network. Moreover, the results offer promising candidate genes for enhancing SCN resistance and optimizing soybean resilience through targeted genetic strategies.

Soybean (Glycine max) is a globally important grain and oil crop, but its yield and quality are severely limited by soybean cyst nematode (SCN, Heterodera glycines Ichinohe), a devastating soil-borne pathogen. Here, we evaluated SCN race 3 resistance in 306 soybean germplasms and combined a genome-wide association study (GWAS) with transcriptome analysis to identify key resistance-related genes. GWAS using 30× resequencing data (632,540 SNPs) revealed 77 significant quantitative trait loci (QTLs) associated with SCN resistance, while transcriptome comparison between the extreme resistant accession Dongnong L10 and susceptible Heinong 37 identified 4185 upregulated and 3195 downregulated genes. Integrating these results, we characterized the GmRF2-like gene as a candidate resistance gene. Subcellular localization showed GmRF2-like encodes a nuclear-localized protein. Functional validation via soybean hairy root transformation demonstrated that overexpression of GmRF2-like significantly inhibits SCN race 3 infection. Collectively, our findings confirm that GmRF2-like plays a positive role in soybean resistance to SCN race 3, providing critical insights for dissecting the molecular mechanism of SCN resistance and facilitating the development of resistant soybean varieties.

Soybean cyst nematode (SCN, Heterodera glycines) is a major pathogen harmful to soybean all over the world, causing huge yield loss every year. Soybean resistance to SCN is a complex quantitative trait controlled by a small number of major genes (rhg1 and Rhg4) and multiple micro-effect genes. Therefore, the continuous identification of new resistant lines and genes is needed for the sustainable development of global soybean production. Here, a novel disease-resistance quantitative trait locus Rscn-16 was identified and fine mapped to an 8.4-kb interval on chromosome 16 using an F2 population. According to transcriptome and metabolome analysis, a UDP-glucosyltransferase encoding gene, GmUGT88A1, was identified as the most likely gene of Rscn-16. Soybean lines overexpressing GmUGT88A1 exhibited increased resistance to SCN, higher isoflavone glycosides and larger seed size while the phenotype of RNA-interference and knockout soybean lines showed sensitivity to SCN and decreased in seed size compared to wild-type plants. GmMYB29 gene could bind to the promoter of GmUGT88A1 and coordinate with GmUGT88A1 to regulate soybean resistance to SCN and isoflavone accumulation. Under SCN infection, GmUGT88A1 participated in the reorientation of isoflavone biosynthetic metabolic flow and the accumulation of isoflavone glycosides, thus protecting soybean from SCN stress. GmUGT88A1 was found to control soybean seed size by affecting transcription abundance of GmSWEET10b and GmFAD3C, which are known to control soybean seed weight. Our findings provide insights into the regulation of SCN resistance, isoflavone content and seed size through metabolic flux redirection, and offer a potential means for soybean improvement.

Vitamin E, comprising tocopherols and tocotrienols, is a crucial fat-soluble antioxidant that helps maintain intracellular redox homeostasis in plants when they are under stress. Soybeans are a significant source of natural vitamin E. GGDR catalyzes the formation of phytyl diphosphate (PDP), a key vitamin E precursor, and it is involved in chlorophyll degradation. The GmGGDR gene, identified via RNA-seq in soybean germplasms with high and low vitamin E contents, encodes GGDR, a key enzyme involved in both vitamin E synthesis and chlorophyll degradation. This study shows that the GmGGDR-encoded protein is hydrophilic and stable, predominantly expressed in leaves, and markedly responsive to gibberellins. The GmGGDR gene enhances the tolerance of transgenic Arabidopsis and soybean plants to salt and drought stresses; transgenic soybeans overexpressing GmGGDR exhibited an approximately 8-fold increase in POD activity, with no significant changes in SOD and CAT activities. Moreover, the GmGGDR gene enhances the levels of α-, γ-, δ-, and total tocopherol content in transgenic soybean and Arabidopsis plants and also increases the chlorophyll a levels in the leaves of these transgenic plants. The increases in α-tocopherol, γ-tocopherol, and δ-tocopherol and total tocopherol in transgenic Arabidopsis seeds ranged from 177.8% to 600.0%, 42.9% to 90.0%, 17.6% to 292.9%, and 71.4% to 127.3% over the control, respectively. Similarly, transgenic soybeans exhibited a minimum increase of 42.9%, 27.8%, 7.1%, and 25.0% in these tocopherol fractions. Overexpression of GmGGDR also significantly elevated chlorophyll a levels in the leaves of these transgenic plants by 33.3–112.5%. This study preliminarily elucidated the function of the GmGGDR gene. It provides a theoretical foundation for further research. It presents a novel strategy for the genetic enhancement of soybean vitamin E content.

Fusarium oxysporum root rot (FORR) is an important disease threatening soybean production. The development of marker-assisted selection (MAS) molecular markers will help accelerate the disease resistance breeding process and achieve the breeding goal of improving soybean disease resistance. This study evaluated the FORR disease resistance of 356 soybean germplasm accessions (SGAs) and screened resistance-related loci using genome-wide association analysis (GWAS) to develop molecular markers for MAS. A total of 1,355,930 high-quality SNPs were analyzed, 150 SNP sites significantly associated with FORR resistance were identified, and these sites were distributed within 41 QTLs. Additionally, 240 candidate genes were screened near these QTL regions, involving multiple functions such as hormone metabolism, signal transduction, stress defense, and growth regulation. Cleaved amplified polymorphic sequence (CAPS) and Kompetitive Allele-Specific PCR (KASP) molecular markers were developed based on candidate genes with significant SNP loci and beneficial haplotypes. The CAPS markers, S15_50486939-CAPS1 and S15_50452626-CAPS2, can effectively distinguish resistant and sensitive genotypes through enzyme digestion. The KASP marker is based on S07_19078765-G/T and exhibits a genotype clustering pattern consistent with disease resistance, demonstrating its application value in breeding. The CAPS and KASP markers developed in this study can provide reliable tools for MAS in FORR disease-resistant varieties. The research results will help reveal the genetic structure of FORR disease resistance and provide support for efficient breeding.

Protein content (PC) is crucial to the nutritional quality of soybean [Glycine max (L.) Merrill]. In this study, a total of 266 accessions were used to perform a genome-wide association study (GWAS) in three tested environments. A total of 23,131 high-quality SNP markers (MAF ≥ 0.02, missing data ≤ 10%) were identified. A total of 40 association signals were significantly associated with PC. Among them, five novel quantitative trait nucleotides (QTNs) were discovered, and another 32 QTNs were found to be overlapping with the genomic regions of known quantitative trait loci (QTL) related to soybean PC. Combined with GWAS, metabolome and transcriptome sequencing, 59 differentially expressed genes (DEGs) that might control the change in protein content were identified. Meantime, four commonly upregulated differentially abundant metabolites (DAMs) and 29 commonly downregulated DAMs were found. Remarkably, the soybean gene Glyma.08G136900, which is homologous with Arabidopsis hydroxyproline-rich glycoproteins (HRGPs), may play an important role in improving the PC. Additionally, Glyma.08G136900 was divided into two main haplotype in the tested accessions. The PC of haplotype 1 was significantly lower than that of haplotype 2. The results of this study provided insights into the genetic mechanisms regulating protein content in soybean.

Context Sclerotinia stem rot (SSR) is one of the diseases that seriously affect soybean yield, leading to heavy losses all over the world. A well-known SSR resistant variety is ‘Maple Arrow’. Aims In this study, transcriptome sequencing analysis of resistant variety ‘Maple Arrow’ and susceptible variety ‘Hefeng25’ was conducted to understand the resistance mechanism of resistant and susceptible soybean varieties to SSR and to look for candidate genes. Methods RNA sequencing of Maple Arrow and Hefeng25 generated 75.09 GB and 64.97 GB clean readings, respectively. In total, 417 differentially expressed genes (DEGs) were found among the different comparable groups. Gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes analysis and haplotype analysis were performed for genes with different expression levels in Maple Arrow and Hefeng25. Key results It was found that DEGs from Maple Arrow and Hefeng25 were involved in the regulation of ‘oxidation–reduction process’, ‘regulation of transcription’, ‘amino acid metabolism’, ‘methylation’ and ‘membrane’, ‘integral component of membrane’ and ‘epidermal growth-factor receptor substrate 15’. In total, 31 haplotypes of 12 genes were screened out with significant or extremely significant differences among soybeans with different levels of SSR resistance. Conclusions These genes may be involved in the relevant pathways of soybean sclerotiniose. Implications To provide excellent gene resources for further disease-resistance breeding.

Soybean vegetable oil is an important source of the human diet. However, the analysis of the genetic mechanism leading to changes in soybean oil content is still incomplete. In this study, a total of 227 soybean materials were applied and analyzed by a genome-wide association study (GWAS). There are 44 quantitative trait nucleotides (QTNs) that were identified as associated with oil content. A total of six, four, and 34 significant QTN loci were identified in Xiangyang, Hulan, and Acheng, respectively. Of those, 26 QTNs overlapped with or were near the known oil content quantitative trait locus (QTL), and 18 new QTNs related to oil content were identified. A total of 594 genes were located near the peak single nucleotide polymorphism (SNP) from three tested environments. These candidate genes exhibited significant enrichment in tropane, piperidine, and pyridine alkaloid biosynthesiss (ko00960), ABC transporters (ko02010), photosynthesis-antenna proteins (ko00196), and betalain biosynthesis (ko00965). Combined with the GWAS and weighted gene co-expression network analysis (WGCNA), four candidate genes (Glyma.18G300100, Glyma.11G221100, Glyma.13G343300, and Glyma.02G166100) that may regulate oil content were identified. In addition, Glyma.18G300100 was divided into two main haplotypes in the studied accessions. The oil content of haplotype 1 is significantly lower than that of haplotype 2. Our research findings provide a theoretical basis for improving the regulatory mechanism of soybean oil content.

Soybean (Glycine max [L.] Merr.) is a major oil-producing crop worldwide. Although several related proteins regulating soybean oil accumulation have been reported, little is known about the regulatory mechanisms. In this study, we characterized vascular plant one-zinc-finger 1A (GmVOZ1A) that interacts with WRINKLED 1a (GmWRI1a) using yeast 2-hybrid library screening. The GmVOZ1A-GmWRI1a interaction was further verified by protein-protein interaction assays in vivo and in vitro. GmVOZ1A enhanced the seed fatty acid and oil contents by regulating genes involved in lipid biosynthesis. Conversely, a loss-of-function mutation in GmVOZ1A resulted in a reduction in triacylglycerol (TAG) content in soybean. Protein-DNA interaction assays revealed that GmVOZ1A and GmWRI1a cooperate to upregulate the expression level of acyl-coenzyme A-binding protein 6a (GmACBP6a) and promote the accumulation of TAG. In addition, GmACBP6a overexpression promoted seed fatty acid and oil contents, as well as increased seed size and 100-seed weight. Taken together, these findings indicate that the transcription factor GmVOZ1A regulates soybean oil synthesis and cooperates with GmWRI1a to upregulate GmACBP6a expression and oil biosynthesis in soybean. The results lay a foundation for a comprehensive understanding of the regulatory mechanisms underlying soybean oil biosynthesis and will contribute to improving soybean oil production through molecular breeding approaches. Cooperation between 2 transcription factors upregulates expression of a gene encoding an acyl-coenzyme A-binding protein and increases oil biosynthesis in soybean.

The utilization of saline land is a global challenge, and cultivating salt-tolerant soybean varieties is beneficial for improving the efficiency of saline land utilization. Exploring the genetic basis of salt-tolerant soybean varieties and developing salt-tolerant molecular markers can effectively promote the process of soybean salt-tolerant breeding. In the study, the membership function method was used to evaluate seven traits related to salt tolerance and comprehensive salt tolerance at the soybean seedling stage; genome-wide association analysis (GWAS) was performed in a natural population containing 200 soybean materials; and linkage analysis was performed in 112 recombinant inbred lines (RIL) population to detect quantitative trait loci (QTLs) of salt tolerance. In the GWAS, 147 SNPs were mapped, explaining 5.28-17.16% of phenotypic variation. In the linkage analysis, 10 QTLs were identified, which could explain 6.9-16.16% of phenotypic variation. And it was found that there were two co-located regions between the natural population and the RIL population, containing seven candidate genes of salt tolerance in soybean. In addition, one colocalization interval was found to contain qZJS-15-1, rs47665107, and rs4793412, all of which could explain more than 10% of phenotypic variation rates, making it suitable for molecular marker development. The physical positions of rs47665107 and rs47934112 were included in qZJS-15-1. Therefore, a KASP marker was designed and developed using Chr. 15:47907445, which was closely linked to the qZJS-15-1. This marker could accurately and clearly cluster the materials of salt-tolerant genotypes in the heterozygous population tested. The QTLs and KASP markers found in the study provide a theoretical and technical basis for accelerating the salt-tolerant breeding of soybean.

The dQTG.seq model was utilized to investigate the genetic underpinnings of phenotypic plasticity in soybean isoflavone content, leading to the identification of 100 marker sites associated with phenotypic plasticity, including 27 transcription factors. Overexpression of Glyma.18G091600 (GmERF7) hairy roots under low temperature, salt, and drought stress confirmed the regulatory role of GmERF7 in the phenotypic plasticity of soybean isoflavone content. Phenotypic plasticity is characteristic of organisms that undergo phenotypic changes in response to environmental fluctuations. This phenomenon is pivotal in evolutionary processes and the emergence of new traits. Isoflavones, significant secondary metabolites found in soybeans, have garnered considerable attention owing to their beneficial physiological effects on human health. The variation in isoflavone content among different soybean varieties is influenced by diverse environmental factors, thereby influencing the evaluation of high and low isoflavone varieties. In this study, we measured the phenotypic plasticity of isoflavone content in recombinant inbred lines Hefeng 25 and L-28 in three different environments over two years. Utilizing the dQTG.seq model, 100 statistically significant markers were identified, and 101 potential genes, including 27 transcription factors, were screened. Through qRT-PCR analysis, elevated expression levels of Glyma.18G091600, Glyma.09G196200, and Glyma.05G229500 were observed in various parts of soybean plants. Under low temperature, drought or salt stress conditions, the related enzymes involved in the isoflavone synthesis pathway were notably upregulated in Glyma.18G091600 (GmERF7) overexpressed hairy roots compared to wild-type controls, resulting to higher phenotypic plasticity values for DZ, GC, GT, and TI. These results suggest that GmERF7 influences the phenotypic plasticity of soybean isoflavone content, enhancing adaptation to adverse environments, while also promoting the synthesis and accumulation of soybean isoflavones.

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.