Soybean ZINC FINGER PROTEIN03 targets two SUPEROXIDE DISMUTASE1s and confers resistance to Phytophthora sojae.

Phytophthora sojae causes Phytophthora root and stem rot disease of soybean (Glycine max), leading to huge annual yield loss worldwide, but resistance to Phytophthora sojae (Rps) genes remains elusive. Soybean cultivar "Yudou 29" is resistant to P. sojae strain PsMC1, and this study aimed to clone, identify, and characterize the Rps gene in Yudou 29 (RpsYD29) and clarify its functional mechanism. We map-based cloned RpsYD29 (ZINC FINGER PROTEIN03, GmZFP03) using the families of a cross between Yudou 29 and a P. sojae-susceptible soybean cultivar "Jikedou 2". P. sojae resistance of GmZFP03 was functionally validated by stable soybean genetic transformation and allele-phenotype association analysis. GmZFP03 was identified as a C2H2-type zinc finger protein transcription factor, showing 4 amino acid residue polymorphisms (V79F, G122-, G123-, and D125V) and remarkably different expression patterns between resistant and susceptible soybeans. Notably boosted activity and gene expression of superoxide dismutase (SOD) in resistant-type GmZFP03-expressed transgenic soybean, substantial enhancement of P. sojae resistance of wild-type soybean by exogenous SOD treatment, and GmZFP03 binding to and activation of 2 SOD1 (Glyma.03g242900 and Glyma.19g240400) promoters demonstrated the involvement of SOD1s in GmZFP03-mediated resistance to P. sojae strain PsMC1. Thus, this study cloned the soybean P. sojae-resistant GmZFP03, the product of which specifically targets 2 SOD1 promoters. GmZFP03 can be directly used for precise P. sojae-resistance soybean breeding.

Deep Learning Model for Classifying and Evaluating Soybean Leaf Disease Damage.

Soybean (Glycine max (L.) Merr.) is a major source of oil and protein for human food and animal feed; however, soybean crops face diverse factors causing damage, including pathogen infections, environmental shifts, poor fertilization, and incorrect pesticide use, leading to reduced yields. Identifying the level of leaf damage aids yield projections, pesticide, and fertilizer decisions. Deep learning models (DLMs) and neural networks mastering tasks from abundant data have been used for binary healthy/unhealthy leaf classification. However, no DLM predicts and categorizes soybean leaf damage severity (five levels) for tailored pesticide use and yield forecasts. This paper introduces a novel DLM for accurate damage prediction and classification, trained on 2930 near-field soybean leaf images. The model quantifies damage severity, distinguishing healthy/unhealthy leaves and offering a comprehensive solution. Performance metrics include accuracy, precision, recall, and F1-score. This research presents a robust DLM for soybean damage assessment, supporting informed agricultural decisions based on specific damage levels and enhancing crop management and productivity.

Proteomic, Transcriptomic, Mutational, and Functional Assays Reveal the Involvement of Both THF and PLP Sites at the GmSHMT08 in Resistance to Soybean Cyst Nematode.

The serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) is involved in the interconversion of serine/glycine and tetrahydrofolate (THF)/5,10-methylene THF, playing a key role in one-carbon metabolism, the de novo purine pathway, cellular methylation reactions, redox homeostasis maintenance, and methionine and thymidylate synthesis. GmSHMT08 is the soybean gene underlying soybean cyst nematode (SCN) resistance at the Rhg4 locus. GmSHMT08 protein contains four tetrahydrofolate (THF) cofactor binding sites (L129, L135, F284, N374) and six pyridoxal phosphate (PLP) cofactor binding/catalysis sites (Y59, G106, G107, H134, S190A, H218). In the current study, proteomic analysis of a data set of protein complex immunoprecipitated using GmSHMT08 antibodies under SCN infected soybean roots reveals the presence of enriched pathways that mainly use glycine/serine as a substrate (glyoxylate cycle, redox homeostasis, glycolysis, and heme biosynthesis). Root and leaf transcriptomic analysis of differentially expressed genes under SCN infection supported the proteomic data, pointing directly to the involvement of the interconversion reaction carried out by the serine hydroxymethyltransferase enzyme. Direct site mutagenesis revealed that all mutated THF and PLP sites at the GmSHMT08 resulted in increased SCN resistance. We have shown the involvement of PLP sites in SCN resistance. Specially, the effect of the two Y59 and S190 PLP sites was more drastic than the tested THF sites. This unprecedented finding will help us to identify the biological outcomes of THF and PLP residues at the GmSHMT08 and to understand SCN resistance mechanisms.

Genome-wide identification and analysis of soybean acyl-ACP thioesterase gene family reveals the role of GmFAT to improve fatty acid composition in soybean seed.

KEY MESSAGE: Soybean acyl-ACP thioesterase gene family have been characterized; GmFATA1A mutants were discovered to confer high oleic acid, while GmFATB mutants presented low palmitic and high oleic acid seed content. Soybean oil stability and quality are primarily determined by the relative proportions of saturated versus unsaturated fatty acids. Commodity soybean typically contains 11% palmitic acid, as the primary saturated fatty acids. Reducing palmitic acid content is the principal approach to minimize the levels of saturated fatty acids in soybean. Though high palmitic acid enhances oxidative stability of soybean oil, it is negatively correlated with oil and oleic acid content and can cause coronary heart diseases for humans. For plants, acyl-acyl carrier protein (ACP) thioesterases (TEs) are a group of enzymes to hydrolyze acyl group and release free fatty acid from plastid. Among them, GmFATB1A has become the main target to genetically reduce the palmitic acid content in soybean. However, the role of members in soybean acyl-ACP thioesterase gene family is largely unknown. In this study, we characterized two classes of TEs, GmFATA, and GmFATB in soybean. We also denominated two GmFATA members and discovered six additional members that belong to GmFATB gene family through phylogenetic, syntenic, and in silico analysis. Using TILLING-by-Sequencing+, we identified an allelic series of mutations in five soybean acyl-ACP thioesterase genes, including GmFATA1A, GmFATB1A, GmFATB1B, GmFATB2A, and GmFATB2B. Additionally, we discovered mutations at GmFATA1A to confer high oleic acid (up to 34.5%) content, while mutations at GmFATB presented low palmitic acid (as low as 5.6%) and high oleic acid (up to 36.5%) phenotypes. The obtained soybean mutants with altered fatty acid content can be used in soybean breeding program for improving soybean oil composition traits.

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis.

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing+ (TbyS+), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS+ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS+, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.

A pathogenesis-related protein GmPR08-Bet VI promotes a molecular interaction between the GmSHMT08 and GmSNAP18 in resistance to Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is the most devastating pest affecting soybean production worldwide. SCN resistance requires both the GmSHMT08 and the GmSNAP18 in 'Peking'-type resistance. Here, we describe the molecular interaction between GmSHMT08 and GmSNAP18, which is potentiated by a pathogenesis-related protein GmPR08-Bet VI. Like GmSNAP18 and GmSHMT08, GmPR08-Bet VI expression was induced in response to SCN and its overexpression decreased SCN cysts by 65% in infected transgenic soybean roots. Overexpression of GmPR08-Bet VI did not have an effect on SCN resistance when the two cytokinin-binding sites in GmPR08-Bet VI were mutated, indicating a new role of GmPR08-Bet VI in SCN resistance. GmPR08-Bet VI was mapped to a QTL for resistance to SCN using different mapping populations. GmSHMT08, GmSNAP18 and GmPR08-Bet VI localize to the cytosol and plasma membrane. GmSNAP18 expression and localization hyper-accumulated at the plasma membrane and was specific to the root cells surrounding the nematode in SCN-resistant soybeans. Genes encoding key components of the salicylic acid signalling pathway were induced under SCN infection. GmSNAP18 and GmPR08-Bet VI were also induced under salicylic acid and cytokinin exogenous treatments, while GmSHMT08 was induced only when the resistant GmSNAP18 was present, pointing to the presence of a molecular crosstalk between SCN-resistant genes and defence genes. Expression analysis of GmSHMT08 and GmSNAP18 identified the need of a minimum expression requirement to trigger the SCN resistance reaction. These results provide insight into a new response mechanism towards plant nematode resistance involving haplotype compatibility, gene dosage and hormone signalling.

Identification of introduced and stably inherited DNA methylation variants in soybean associated with soybean cyst nematode parasitism.

DNA methylation is a widespread epigenetic mark that contributes to transcriptome reprogramming during plant-pathogen interactions. However, the distinct role of DNA methylation in establishing resistant and susceptible responses remains largely unexplored. Here, we developed and used a pair of near-isogenic lines (NILs) to characterize DNA methylome landscapes of soybean roots during the susceptible and resistant interactions with soybean cyst nematode (SCN; Heterodera glycines). We also compared the methylomes of the NILs and their parents to identify introduced and stably inherited methylation variants. The genomes of the NILs were substantially differentially methylated under uninfected conditions. This difference was associated with differential gene expression that may prime the NIL responses to SCN infection. In response to SCN infection, the susceptible line exhibited reduced global methylation levels in both protein-coding genes and transposable elements, whereas the resistant line showed the opposite response, increased global methylation levels. Heritable and novel nonparental differentially methylated regions overlapping with genes associated with soybean response to SCN infection were identified and validated using transgenic hairy root system. Our analyses indicate that DNA methylation patterns associated with the susceptible and resistant interactions are highly specific and that novel and stably inherited methylation variants are of biological significance.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating plant-parasitic nematode. Most commercial soybean varieties with SCN resistance are derived from PI88788. Resistance derived from PI88788 is breaking down due to narrow genetic background and SCN population shift. PI88788 requires mainly the rhg1-b locus, while 'Peking' requires rhg1-a and Rhg4 for SCN resistance. In the present study, whole genome re-sequencing of 106 soybean lines was used to define the Rhg haplotypes and investigate their responses to the SCN HG-Types. The analysis showed a comprehensive profile of SNPs and copy number variations (CNV) at these loci. CNV of rhg1 (GmSNAP18) only contributed towards resistance in lines derived from PI88788 and 'Cloud'. At least 5.6 copies of the PI88788-type rhg1 were required to confer SCN resistance, regardless of the Rhg4 (GmSHMT08) haplotype. However, when the GmSNAP18 copies dropped below 5.6, a 'Peking'-type GmSHMT08 haplotype was required to ensure SCN resistance. This points to a novel mechanism of epistasis between GmSNAP18 and GmSHMT08 involving minimum requirements for copy number. The presence of more Rhg4 copies confers resistance to multiple SCN races. Moreover, transcript abundance of the GmSHMT08 in root tissue correlates with more copies of the Rhg4 locus, reinforcing SCN resistance. Finally, haplotype analysis of the GmSHMT08 and GmSNAP18 promoters inferred additional levels of the resistance mechanism. This is the first report revealing the genetic basis of broad-based resistance to SCN and providing new insight into epistasis, haplotype-compatibility, CNV, promoter variation and its impact on broad-based disease resistance in plants.

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean (Glycine max) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure.

Systematic Mutagenesis of Serine Hydroxymethyltransferase Reveals an Essential Role in Nematode Resistance.

Rhg4 is a major genetic locus that contributes to soybean cyst nematode (SCN) resistance in the Peking-type resistance of soybean (Glycine max), which also requires the rhg1 gene. By map-based cloning and functional genomic approaches, we previously showed that the Rhg4 gene encodes a predicted cytosolic serine hydroxymethyltransferase (GmSHMT08); however, the novel gain of function of GmSHMT08 in SCN resistance remains to be characterized. Using a forward genetic screen, we identified an allelic series of GmSHMT08 mutants that shed new light on the mechanistic aspects of GmSHMT08-mediated resistance. The new mutants provide compelling genetic evidence that Peking-type rhg1 resistance in cv Forrest is fully dependent on the GmSHMT08 gene and demonstrates that this resistance is mechanistically different from the PI 88788-type of resistance that only requires rhg1 We also demonstrated that rhg1-a from cv Forrest, although required, does not exert selection pressure on the nematode to shift from HG type 7, which further validates the bigenic nature of this resistance. Mapping of the identified mutations onto the SHMT structural model uncovered key residues for structural stability, ligand binding, enzyme activity, and protein interactions, suggesting that GmSHMT08 has additional functions aside from its main enzymatic role in SCN resistance. Lastly, we demonstrate the functionality of the GmSHMT08 SCN resistance gene in a transgenic soybean plant.

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens.

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

Correlation between the Chemical and Genetic Relationships among Thymus saturejoides Genotypes Cultured under in vitro and in vivo Environments.

In this study, the in vitro and in vivo essential oil (EO) composition and genetic variability in six micropropagated genotypes of Thymus saturejoides Coss., a Mediterranean medicinal and aromatic plant, were analyzed by GC/MS and randomly amplified polymorphic DNA (RAPD). Yield and composition of the EO varied between genotypes. Cluster analysis based on RAPD data and EO grouped the six genotypes in three groups in both culture conditions, thus showing considerable intraspecific genetic and chemical variations. Applying the Mantel test, the result showed a significant correlation between the two proximity matrices RAPD and EO obtained from in vitro genotypes, whereas this correlation was not observed when using the EO obtained from the in vivo genotypes.

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance.

BACKGROUND: Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance. RESULTS: Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F5-derived recombinant inbred lines (RILs) from G00-3213xLG04-6000. CONCLUSIONS: Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

A Summary of Two Decades of QTL and Candidate Genes That Control Seed Tocopherol Contents in Maize (Zea mays L.).

Tocopherols are secondary metabolites synthesized through the shikimate biosynthetic pathway in the plastids of most plants. It is well known that α-Tocopherol (vitamin E) has many health benefits for humans and animals; therefore, it is highly used in human and animal diets. Tocopherols vary considerably in most crop (and plant) species and within cultivars of the same species depending on environmental and growth conditions; tocopherol content is a polygenic, complex traits, and its inheritance is poorly understood. The objective of this review paper was to summarize all identified quantitative trait loci (QTL) that control seed tocopherols and related contents identified in maize (Zea mays) during the past two decades (2002-2022). Candidate genes identified within these QTL regions are also discussed. The QTL described here, and candidate genes identified within these genomic regions could be used in breeding programs to develop maize cultivars with high, beneficial levels of seed tocopherol contents.

No Pairwise Interactions of GmSNAP18, GmSHMT08 and AtPR1 with Suppressed AtPR1 Expression Enhance the Susceptibility of Arabidopsis to Beet Cyst Nematode.

GmSNAP18 and GmSHMT08 are two major genes conferring soybean cyst nematode (SCN) resistance in soybean. Overexpression of either of these two soybean genes would enhance the susceptibility of Arabidopsis to beet cyst nematode (BCN), while overexpression of either of their corresponding orthologs in Arabidopsis, AtSNAP2 and AtSHMT4, would suppress it. However, the mechanism by which these two pairs of orthologous genes boost or inhibit BCN susceptibility of Arabidopsis still remains elusive. In this study, Arabidopsis with simultaneously overexpressed GmSNAP18 and GmSHMT0 suppressed the growth of underground as well as above-ground parts of plants. Furthermore, Arabidopsis that simultaneously overexpressed GmSNAP18 and GmSHMT08 substantially stimulated BCN susceptibility and remarkably suppressed expression of AtPR1 in the salicylic acid signaling pathway. However, simultaneous overexpression of GmSNAP18 and GmSHMT08 did not impact the expression of AtJAR1 and AtHEL1 in the jasmonic acid and ethylene signaling pathways. GmSNAP18, GmSHMT08, and a pathogenesis-related (PR) protein, GmPR08-Bet VI, in soybean, and AtSNAP2, AtSHMT4, and AtPR1 in Arabidopsis could interact pair-wisely for mediating SCN and BCN resistance in soybean and Arabidopsis, respectively. Both AtSNAP2 and AtPR1 were localized on the plasma membrane, and AtSHMT4 was localized both on the plasma membrane and in the nucleus of cells. Nevertheless, after interactions, AtSNAP2 and AtPR1 could partially translocate into the cell nucleus. GmSNAP18 interacted with AtSHMT4, and GmSHMT4 interacted with AtSNAP2. However, neither GmSNAP18 nor GmSHMT08 interacted with AtPR1. Thus, no pairwise interactions among α-SNAPs, SHMTs, and AtPR1 occurred in Arabidopsis overexpressing either GmSNAP18 or GmSHMT08, or both of them. Transgenic Arabidopsis overexpressing either GmSNAP18 or GmSHMT08 substantially suppressed AtPR1 expression, while transgenic Arabidopsis overexpressing either AtSNAP2 or AtSHMT4 remarkably enhanced it. Taken together, no pairwise interactions of GmSNAP18, GmSHMT08, and AtPR1 with suppressed expression of AtPR1 enhanced BCN susceptibility in Arabidopsis. This study may provide a clue that nematode-resistant or -susceptible functions of plant genes likely depend on both hosts and nematode species.

Quantitative Trait Loci and Candidate Genes That Control Seed Sugars Contents in the Soybean 'Forrest' by 'Williams 82' Recombinant Inbred Line Population.

Soybean seed sugars are among the most abundant beneficial compounds for human and animal consumption in soybean seeds. Higher seed sugars such as sucrose are desirable as they contribute to taste and flavor in soy-based food. Therefore, the objectives of this study were to use the 'Forrest' by 'Williams 82' (F × W82) recombinant inbred line (RIL) soybean population (n = 309) to identify quantitative trait loci (QTLs) and candidate genes that control seed sugar (sucrose, stachyose, and raffinose) contents in two environments (North Carolina and Illinois) over two years (2018 and 2020). A total of 26 QTLs that control seed sugar contents were identified and mapped on 16 soybean chromosomes (chrs.). Interestingly, five QTL regions were identified in both locations, Illinois and North Carolina, in this study on chrs. 2, 5, 13, 17, and 20. Amongst 57 candidate genes identified in this study, 16 were located within 10 Megabase (MB) of the identified QTLs. Amongst them, a cluster of four genes involved in the sugars' pathway was collocated within 6 MB of two QTLs that were detected in this study on chr. 17. Further functional validation of the identified genes could be beneficial in breeding programs to produce soybean lines with high beneficial sucrose and low raffinose family oligosaccharides.

Soybean gene co-expression network analysis identifies two co-regulated gene modules associated with nodule formation and development.

Gene co-expression network analysis is an efficient systems biology approach for the discovery of novel gene functions and trait-associated gene modules. To identify clusters of functionally related genes involved in soybean nodule formation and development, we performed a weighted gene co-expression network analysis. Two nodule-specific modules (NSM-1 and NSM-2, containing 304 and 203 genes, respectively) were identified. The NSM-1 gene promoters were significantly enriched in cis-binding elements for ERF, MYB, and C2H2-type zinc transcription factors, whereas NSM-2 gene promoters were enriched in cis-binding elements for TCP, bZIP, and bHLH transcription factors, suggesting a role of these regulatory factors in the transcriptional activation of nodule co-expressed genes. The co-expressed gene modules included genes with potential novel roles in nodulation, including those involved in xylem development, transmembrane transport, the ethylene signalling pathway, cytoskeleton organization, cytokinesis and regulation of the cell cycle, regulation of meristem initiation and growth, transcriptional regulation, DNA methylation, and histone modifications. Functional analysis of two co-expressed genes using TILLING mutants provided novel insight into the involvement of unsaturated fatty acid biosynthesis and folate metabolism in nodule formation and development. The identified gene co-expression modules provide valuable resources for further functional genomics studies to dissect the genetic basis of nodule formation and development in soybean.

Genetic Mapping for QTL Associated with Seed Nickel and Molybdenum Accumulation in the Soybean 'Forrest' by 'Williams 82' RIL Population.

Understanding the genetic basis of seed Ni and Mo is essential. Since soybean is a major crop in the world and a major source for nutrients, including Ni and Mo, the objective of the current research was to map genetic regions (quantitative trait loci, QTL) linked to Ni and Mo concentrations in soybean seed. A recombinant inbred line (RIL) population was derived from a cross between 'Forrest' and 'Williams 82' (F × W82). A total of 306 lines was used for genotyping using 5405 single nucleotides polymorphism (SNP) markers using Infinium SNP6K BeadChips. A two-year experiment was conducted and included the parents and the RIL population. One experiment was conducted in 2018 in North Carolina (NC), and the second experiment was conducted in Illinois in 2020 (IL). Logarithm of the odds (LOD) of ≥2.5 was set as a threshold to report identified QTL using the composite interval mapping (CIM) method. A wide range of Ni and Mo concentrations among RILs was observed. A total of four QTL (qNi-01, qNi-02, and qNi-03 on Chr 2, 8, and 9, respectively, in 2018, and qNi-01 on Chr 20 in 2020) was identified for seed Ni. All these QTL were significantly (LOD threshold > 2.5) associated with seed Ni, with LOD scores ranging between 2.71-3.44, and with phenotypic variance ranging from 4.48-6.97%. A total of three QTL for Mo (qMo-01, qMo-02, and qMo-03 on Chr 1, 3, 17, respectively) was identified in 2018, and four QTL (qMo-01, qMo-02, qMo-03, and qMo-04, on Chr 5, 11, 14, and 16, respectively) were identified in 2020. Some of the current QTL had high LOD and significantly contributed to the phenotypic variance for the trait. For example, in 2018, Mo QTL qMo-01 on Chr 1 had LOD of 7.8, explaining a phenotypic variance of 41.17%, and qMo-03 on Chr 17 had LOD of 5.33, with phenotypic variance explained of 41.49%. In addition, one Mo QTL (qMo-03 on Chr 14) had LOD of 9.77, explaining 51.57% of phenotypic variance related to the trait, and another Mo QTL (qMo-04 on Chr 16) had LOD of 7.62 and explained 49.95% of phenotypic variance. None of the QTL identified here were identified twice across locations/years. Based on a search of the available literature and of SoyBase, the four QTL for Ni, identified on Chr 2, 8, 9, and 20, and the five QTL associated with Mo, identified on Chr 1, 17, 11, 14, and 16, are novel and not previously reported. This research contributes new insights into the genetic mapping of Ni and Mo, and provides valuable QTL and molecular markers that can potentially assist in selecting Ni and Mo levels in soybean seeds.

QTL and Candidate Genes for Seed Tocopherol Content in 'Forrest' by 'Williams 82' Recombinant Inbred Line (RIL) Population of Soybean.

Soybean seeds are rich in secondary metabolites which are beneficial for human health, including tocopherols. Tocopherols play an important role in human and animal nutrition thanks to their antioxidant activity. In this study, the 'Forrest' by 'Williams 82' (F×W82) recombinant inbred line (RIL) population (n = 306) was used to map quantitative trait loci (QTL) for seed α-tocopherol, ß-tocopherol, δ -tocopherol, γ-tocopherol, and total tocopherol contents in Carbondale, IL over two years. Also, the identification of the candidate genes involved in soybean tocopherols biosynthetic pathway was performed. A total of 32 QTL controlling various seed tocopherol contents have been identified and mapped on Chrs. 1, 2, 5, 6, 7, 8, 9, 10, 12, 13, 16, 17, and 20. One major and novel QTL was identified on Chr. 6 with an R2 of 27.8, 9.9, and 6.9 for δ-tocopherol, α-tocopherol, and total tocopherol content, respectively. Reverse BLAST analysis of the genes that were identified in Arabidopsis allowed the identification of 37 genes involved in soybean tocopherol pathway, among which 11 were located close to the identified QTLs. The tocopherol cyclase gene (TC) Glyma.06G084100 is located close to the QTLs controlling δ-tocopherol (R2 = 27.8), α-tocopherol (R2 = 9.96), and total-tocopherol (R2 = 6.95). The geranylgeranyl diphosphate reductase (GGDR) Glyma.05G026200 gene is located close to a QTL controlling total tocopherol content in soybean (R2 = 4.42). The two methylphytylbenzoquinol methyltransferase (MPBQ-MT) candidate genes Glyma.02G002000 and Glyma.02G143700 are located close to a QTL controlling δ-tocopherol content (R2 = 3.57). The two γ-tocopherol methyltransferase (γ-TMT) genes, Glyma.12G014200 and Glyma.12G014300, are located close to QTLs controlling (γ+ß) tocopherol content (R2 = 8.86) and total tocopherol (R2 = 5.94). The identified tocopherol seed QTLs and candidate genes will be beneficial in breeding programs to develop soybean cultivars with high tocopherol contents.