The soybean cropping system involves its distribution across the country, the lighting time, accumulated temperature and cropping system of the varieties, the rotation system, as well as the monocropping, intercropping and relay intercropping methods, serves as the foundation for soybean production, breeding, introduction, and technology innovation. Optimizing the soybean cropping system is of decisive significance for enhancing the comprehensive production capacity and benefits of soybeans in China. Since the founding of the People's Republic of China (PRC) 70 years ago, the area planted with soybeans in regions with one crop per year system has expanded, while the area in regions that have shifted from triple crops per two years system to double crops per year system has decreased. In areas that have transitioned from double crops per year and then to triple crops per year, the area planted with soybeans has remained stable with a slight increase. From a national perspective, the soybean cultivation region has expanded to the northern part of Northeast China, and the soybean cultivation region in the South and Southwest has remained stable with a slight increase. The Northwest region has performed a new high-yield area for soybeans. Historically, the division of soybean cultivation regions was based on the basic data, investigations and experiments of the planting system at that time. In the recent 30 years, there have been significant advancements in soybean production, breeding and cultivation techniques, especially in the changes of soybean cultivation areas. The division of ecological cultivation region is a fundamental task closely related to soybean cultivation, resource utilization, introduction and breeding for cultivars. Based on the review of the changes in soybean cultivation region in China since the PRC establishment, including the northward expansion and southward shift of cultivation region, the renewal and upgrading of varieties, the improvement of mechanization levels, the comprehensive progress of cultivation techniques, and the promotion of intercropping system, especially the emphasis on developing the soybean industry as a national policy in China since 2000, this review comprehensively analyzed the dynamic characteristics of the soybean cropping system and technical system in PRC and thus proposed suggestions for adjusting the ecological cultivation region divisions of soybeans. From which a new soybean ecological cultivation region system is proposed. The main results comprise the changes in soybean cropping regions and the advances in cropping system, the environmental cultivation regions and changes of soybeans, the ecology of modern soybeans in China, and discussion and prospect on ecological cultivation region of soybeans in China. Influenced by updates of soybean cultivars, advancements in cultivation and farming technology, and requirements on food security, the soybean cropping system has undergone significant changes. The new six ecological cultivation regions were suggested as Northeast Spring Planting Soybean Ecological Cultivation Region, Northwest Spring Planting Soybean Ecological Cultivation Region, Huang-Huai-Hai Summer Planting Soybean Ecological Cultivation Region, Changjiang Valleys Spring-Summer-Autumn Planting Soybean Ecological Cultivation Region, Southwest Plateau Spring-Summer Planting Soybean Ecological Cultivation Region, and South China All Season Planting Soybean Ecological Cultivation Region. This division and naming system is considered as consistent as that of the national crop cultivation system, and also pays attention to the connection with previous ecological cultivation region division systems in soybean.

Genomic selection (GS) has provided a comprehensive framework for efficient breeding by linking phenotypes to genome-wide markers. However, research has predominantly focused on improving genotype-to-phenotype prediction models, often overlooking optimal cross design, which determines the potential of progeny selection and plays a critical role in crop breeding. In this study, an efficient GS framework, EMLGP (ensemble machine-learning for genomic prediction), was proposed for optimal cross design in crop breeding. EMLGP first employs machine-learning algorithms to train precise genotype-to-phenotype prediction models in a germplasm population and then integrates with genome simulations to predict optimal crosses in a breeding population. GS model training of 14 soybean traits demonstrated that EMLGP achieved superior performance, with the highest prediction accuracy (correlation coefficient) reaching 0.92. The prediction accuracy showed a maximum improvement of 35.85% over the classical GBLUP method. Further simulation studies confirmed that EMLGP exhibited robust performance under conditions of small-to-moderate sample sizes (300–5000), low-to-moderate trait heritabilities (0.4–0.6), and complex genetic architectures (100 causal loci). Validation using real data of rice, maize, cotton, sorghum, and switchgrass consistently affirmed EMLGP’s superiority, outperforming GBLUP and deep learning methods. Among the 14 soybean traits analyzed, 13 traits exhibited transgressive segregation potential in the progeny. Specifically, seed linolenic acid content in the northern China showed the highest recombination potential, exceeding the maximum parental value by 16.89%. In conclusion, EMLGP optimizes parental selection and phenotypic prediction, offering a robust framework for efficient, intelligence-driven crop breeding.

Annual wild soybean, the ancestor of cultivated soybean, underwent a significant increase in seed oil content during domestication. To elucidate the genetic basis of this change, a chromosome segment substitution line population (177 lines) constructed with cultivated soybean NN1138-2 as recipient and wild soybean N24852 as donor was used in this study. Phenotypic evaluation across three distinct environments led to the identification of two major QTL/segments, qOC14 on chromosome 14 and qOC20 on chromosome 20, which collectively explained 39.46% of the phenotypic variation, with individual contributions of 17.87% and 21.59%, respectively. Both wild alleles exhibited negative additive effects, with values of -0.35% and -0.42%, respectively, consistent with the inherently low oil content of wild soybeans. Leveraging transcriptome and genome data from the two parents, two candidate genes were predicted. Notably, Glyma.14G179800 is a novel candidate gene encoding a PHD-type zinc finger domain-containing protein, and the hap-A haplotype exhibits a positive effect on oil content. In contrast, Glyma.20G085100 is a reported POWR1 gene, known to regulate protein and oil content. Our findings not only validate the role of known gene but, more importantly, unveil a new candidate gene, offering valuable genetic resources and theoretical targets for molecular breeding of high-oil soybean.

Phosphoglycerate kinase (PGK) is a vital glycolytic enzyme that provides energy and carbon skeletons to support fatty acid synthesis. However, the PGK gene family has not been characterized in soybean (Glycine max), and its role in soybean oil accumulation remains unclear. Here, we identified six GmPGK genes in soybean, all of which encode proteins containing conserved PGK domains. Phylogenetic analysis clustered soybean PGK proteins into three groups. Analysis of GmPGK promoters revealed relatively abundant cis-elements related to plant growth, development, and phytohormone response. Expression profiling showed that GmPGK5 transcript abundance increases progressively with oil accumulation during seed development, and is significantly higher in the high-oil variety NN1138-2. Overexpression of GmPGK5 significantly increased total fatty acid content in soybean hairy roots. A single nucleotide polymorphism (SNP) located at Chr15:49447855 within the GmPGK5 promoter was significantly associated with both seed oil content and seed weight in natural soybean accessions. Based on this SNP, a derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to facilitate soybean molecular breeding. Our findings suggest that GmPGK5 may positively regulate fatty acid accumulation in soybean. The identified natural variation and dCAPS marker provide potential valuable tools for marker-assisted selection to improve soybean oil content and seed weight.

The subgenus Soja, including annual wild (Glycine soja) and cultivated soybean (Glycine max), is the primary germplasm source of soybeans. We analyzed the genome constitution of 750 wild and cultivated accessions from the Chinese Soybean Germplasm Population (CSGP), covering ~20.42% genic and ~79.58% intergenic regions. Most previous genomic studies focused on gene compositions and functions, with intergenic regions being non-emphasized yet. Our results showed: (i) We defined 48,465 gene blocks (2-23 alleles/gene block) in the genic region. For intergenic region partitioning, the linkage-disequilibrium (LD) confidence interval (CI) method performed the best, identifying 137,104 SNP LD blocks (SNPLDBs, 2-24 haplotypes/SNPLDB). (ii) Wild and cultivated accessions shared 98.5%/80.9% genes/alleles and 88.0%/80.8% SNPLDBs/haplotypes, indicating high wild genomic contribution to the cultivated genome; genic and intergenic regions exhibit distinct allele/haplotype dynamics during domestication. (iii) We proposed the three-case restricted two-stage multi-locus multi-allele genome-wide association study (three-case RTM-GWAS), identifying 82 day-to-flowering (DTF) main-effect genes and 47 intergenic-SNPLDB-impacted genes (by 34 SNPLDBs) (total 129 genes, 746 alleles). These explained 98.87% of phenotypic variance (PV), with main-effect and SNPLDB-impacted genes accounting for 68.61% and 30.26% PV, respectively; gene-allele(s) impacted by SNPLDB-haplotype (one/both sides) were also identified. (iv) Domestication process excluded more large-effect positive alleles that shorten DTF in cultivated accessions, enhancing DTF's transgressive recombination potential in earliness. This study provides insights into genic/intergenic genome regions, offering a novel understanding of soybean functional genomics.

Salinity is a significant factor limiting the cultivation of soybean, a globally important cash crop. However, efficient assessment and genetic dissection of soybean response to salt stress remain challenging. This study leveraged high-throughput phenotyping (HTP) and traditional physiological methods for comprehensive phenotyping of salt tolerance using 261 diverse soybean germplasms and dissected the genetic basis through GWAS. A highly efficient rail-based HTP system with depth-sensing and RGB cameras was developed to collect horizontal and vertical growth and leaf health information. Machine learning pipeline facilitated canopy detection, segmentation, and phenotype extraction processes. Three HTP traits and five traditional physiological traits related to salt tolerance were collected. Divergence between growth status and chlorophyll content was observed, indicating the importance of HTP and the genetic complexity of salt tolerance in soybean. A stepwise regression analysis indicated that "Vegetation color index" (VEG), "Anthocyanin Reflectance Index" (ARI), and "Cyan, Magenta, Yellow" (CMY_Yellow) are the most informative indices of soybean foliar health under salt tolerance. GWAS identified 46 loci for salt tolerance-related traits. Fifteen potential candidate genes were proposed, including Glyma.18g238700 which is known to be involved in salt tolerance mechanisms. Field test indicated that two of the top five tolerant accessions at seedling stage are salt tolerant at full growth stages with high yield potential. Additionally, best crosses were predicted from random mating of the association panel by using linkage and independent assortment models for salt tolerance improvement breeding. This study provided tolerant genotypes, promising candidates and optimized crosses for further exploration.

Phytophthora sojae-induced root rot poses a major threat to soybean production. While the molecular mechanisms underlying soybean-P. sojae interactions have been extensively studied, their biochemical basis remains largely unexplored. Previous research has identified key metabolic modules involved in pathogen defense, but structural diversity has largely been constrained by studies on single soybean accessions. Here, we broadened the chemical search space to a diverse soybean germplasm collection using high-throughput metabolomics as a powerful tool for comprehensive metabolic profiling. Chemical classes of lipids and phenylpropanoids again retrieved the most pronounced responses upon P. sojae infection in general. A two-layer analytical strategy further finely resolved metabolites into pathogenesis-, resistance-, and tolerance-type accumulation patterns, leading to the identification of cinnamaldehyde and coumestrol as potent defense metabolites. Bioassays validated cinnamaldehyde directly and strongly inhibited cyst germination and mycelial growth, and coumestrol, a benzofuran-type metabolite, exhibited broad-spectrum activity against spore germination as an identified phytoalexin. Multiomics analyses nailed down the candidate of coumestrol biosynthesis genes, and genetically overexpression of regulatory genes (Dir2a/4a/4b) in hairy root systems increased coumestrol accumulation thus positively correlating with improved host resistance. Interestingly, tolerance-type compounds may serve distinct ecological roles, as exemplified by daidzein, which, despite being classified as a tolerance-type metabolite, recruits more zoospores facilitating secondary infection in fact. This study highlights a systematic approach for population-level investigations and emphasizes the necessity of integrating bioinformatics with experimental validation to accurately predict metabolite or gene ecological functions.

Previous studies on population evolution relied primarily on allele frequency analysis using molecular markers or genome sequence segments, like selective sweeps. With the sequencing technique developed, we suggest the genome‐wide locus–allele comparison to detect the genomic structure variation among populations. Its key point lies in taking SNP linkage disequilibrium block as uniform genomic marker for genome‐wide gene and inter‐gene regions to meet the requirement of multiple alleles in natural populations. A sample composed of 750 annual wild accessions (WAs), landraces (LRs), and released cultivars (RCs) of soybean from southern, northern, and northeastern China eco‐regions (SC, NC, and NEC, respectively) were analyzed for their evolution dynamics involving four evolutionary processes (WA→LR→RC, WA SC →WA NC →WA NEC , LR SC →LR NC →LR NEC , and LR SC →RC SC /LR NC →RC NC /LR NEC →RC NEC ). Our major finding was the discovery of allele and locus zero/one variation between/among ancestor‐filial populations involving a large part of the whole population alleles and loci, 25.10% and 18.62% in domestication and modern breeding stages, respectively, which was not detected by selective sweeps. The essence of population evolution is the allele zero/one changes based on ordinary allele frequency changes, which causes the locus zero/one changes. The allele/locus zero/one variation happened more often when their frequency was at 0.0–0.3 and 0.8–0.99 in the previous stage generation, respectively. The WA and LR geographic evolution are different processes due to different combination of allele/locus zero/one changes by natural versus artificial selection pressures. Compared to per‐year allele exclusion, the rate of per‐year allele emergence is relatively stable in domestication and modern breeding (2.75E‐5 vs. 1.34E‐5 and 1.42E‐3 vs. 1.10E‐5), respectively.

A wild-allele GsPP2C-51-a1 of Glyma.14g162100 was identified in SojaCSSLP5, back to wild soybean, conferring drought tolerance. Its functions were verified in transgenic hairy root soybeans and Arabidopsis under water deficit and ABA treatment. A population of wild soybean chromosome segment substitution lines (CSSLs), SojaCSSLP5, with NN1138-2 as the cultivated recurrent parent and N24852 as the wild donor parent, was used to identify drought-tolerant loci/segments from the donor. Relative shoot dry weight, a tolerance indicator, varied significantly among the parents and CSSLs. Six drought tolerance loci/segments were detected in SojaCSSLP5, including Gm14_LDB_21 with GsPP2C-51 (Glyma.14g162100) as one of the four possible genes. This gene belongs to the F1 clade of protein phosphatase 2C based on gene ontology annotation, qPCR, and previous research results. Glyma.14g162100 was traced back to the Chinese germplasm population, in which four alleles existed on the locus, with soja holding all four, and max holding only two without any new alleles emerging. N24852 and NN1138-2 hold a1 and a2, respectively. The GsPP2C-51 protein was located inside the nucleus. In transgenic hairy root composite soybean, the GsPP2C-51-a1 overexpressed plants maintained a higher leaf fresh weight (tolerance) under 15

Annual wild soybean is characterized by a high protein content. To elucidate the genetic basis, this study utilized a chromosome segment substitution line population (177 lines) constructed with cultivated soybean NN1138-2 as the recipient and wild soybean N24852 as the donor. Phenotypic analyses across three environments revealed significant variation in protein content ranging from 42.86% to 49.08%, with a high heritability of 0.70, indicating strong genetic control. Through high-throughput sequencing, six wild segments associated with high protein content were detected on chromosomes 3, 6, 9, 15, and 20, with phenotypic variation explained (PVE) by individual segments ranged from 3.58% to 22.46%, with segments on chromosomes 9, 15, and 20 as large-effect segments with PVE > 10%. All wild segments exhibited positive additive effects (0.42–1.09%), consistent with the characteristic of a high protein content in wild soybean. Compared with previous studies, five segments overlapped with reported loci, while qPro6.1 on chromosome 6 was a novel discovery. Integration of genomic and transcriptomic data identified 10 genes involved in nucleic acid binding, transmembrane protein transport, and amino acid synthesis pathway, with homologs validated in soybean, rice, and rapeseed. This research deepens the understanding of wild soybean’s high protein and offers new gene resources for breeding high-protein cultivated soybean.

Soybean, an economically valuable oil and protein crop, is vulnerable to numerous biotic stresses throughout its growth period. Soybean mosaic virus (SMV), a destructive plant pathogen, induces substantial yield reduction and seed quality deterioration globally. In China, a total of 22 distinct SMV strains have been documented, with SMV-SC4 being a widely spread strain. The Chinese cultivar Kefeng-1 (KF) is resistant to this strain. To investigate the resistance mechanism, transcriptional analysis was performed at 0, 6, 24, and 48 h post-inoculation of SC4 in KF (Resistant) and NN1138-2 (NN) (Susceptible). A total of 1201 core differentially expressed genes (DEGs) were identified as active ones against SC4 infection, with most originating from the resistant cultivar at the early infection stages. Gene ontology enrichment analysis indicated that the DEGs directly involved in signal transduction and those related to plant stress response contributed to KF resistance indirectly, including protein phosphorylation, protein kinase activity, oxidation–reduction, oxidoreductase activity, catalytic activity, metal ion transport, and response to auxin. A total of 27 genes in “Signal transduction” with most of them were disease resistance conserved domains, 52 genes active in oxidoreductase activity involving in removing ROS from SMV attack, and 8 genes in “Response to auxin”, a phytohormone that plays a role in biotic stress response in addition to growth and development. These genes expressed more differentially in the resistant versus susceptible cultivar. Our findings provide insights into the molecular networks related to soybean response to SMV, which may be relevant in understanding soybean resistance against the viral infections.

The utilization of heterosis is one of the important ways to improve crop yield, which has been widely applied in crops such as rice, rapeseed, corn, and other crops, and has achieved remarkable economic and social benefits. As early as the last century, both domestic and international researchers-initiated explorations into soybean heterosis, and research results have shown that soybeans have significant heterosis. Across different decades, scientists employing diverse hybrid combinations consistently observed substantial over-parent heterosis in soybean hybrids, particularly in yield traits. The over-parent heterosis rate has been documented to exceed 15.6%, with some high heterosis combinations demonstrating over 50% over-parent heterosis and over 30% superiority compared to control varieties, indicating that soybeans have obvious heterosis in yield traits. Male sterile lines serve as pivotal materials for hybrid seed production in crops. Among the methodologies employed, the cytoplasmic-nuclear male sterility-based "three-line method" and the genic male sterility-based "two-line method" are the two most widely adopted strategies for hybrid breeding in crops. Currently, soybean hybrid breeding predominantly relies on the "three-line method". At present, the reported soybean sterile cytoplasm types that achieve the "three-line system" in China mainly include RN type, ZD type, N8855 type, N21566 type, and N23661 type. These sterile cytoplasms originate from five distinct cultivated soybean varieties. Importantly, China has established a comprehensive hybrid seed production technology system that integrates soybeans, insects, and the environment, enabling male sterile lines with high outcrossing rates to achieve over 90% outcrossing efficiency. So far, 46 hybrid soybean varieties have been examined and approved using the "three-line system" with an average yield increase of nearly 13% compared with the control. Among them, 13 hybrid soybean varieties increased by more than 15%. Since the "Twelfth Five-Year Plan" period, 39 hybrid soybean varieties have been approved, reflecting both the expanding scale of hybrid soybean breeding and continuous advancements in heterosis utilization technologies in China. With the application of gene editing and transgenic breeding technology, fertility regulation genes such as fertility restorer genes, genic male sterility genes, and high-temperature tolerance genes have gradually been discovered and applied to the utilization of soybean heterosis. It can be seen that the utilization of heterosis can serve as a breakthrough technology for the improvement of soybean yield in China, which has significant strategic significance for enhancing soybean production capacity and ensuring food security. This review article focuses on the research progress of genetic composition and utilization ways of soybean heterosis, the cytoplasmic-nuclear male sterility and "three line" breeding of soybean, the genic male sterility and utilization of soybean, the examination and approval of soybean hybrid variety and the production technology of hybrid seeds. After decades of systematic research and development, significant progress has been made in the utilization of soybean heterosis, but there is still a certain gap in commercial application. Some key issues still need to be further overcome, mainly including efficient sterile cytoplasm needs to be explored, high outcrossing rate male sterile lines and strong restorer lines need to be selected, hybrid seed production efficiency needs to be further improved, strong advantage combinations need to be further screened, the utilization ways of heterosis need to be expanded, and fertility genes with significant breeding value need to be discovered. With the continuous exploration and utilization of excellent soybean germplasm resources and fertility genes with significant breeding value, the continuous improvement of commercial production technology systems for soybean hybrid seeds, and the continuous development of biotechnology, the utilization of soybean heterosis will show broad application prospects and significant economic and social benefits.

Soybean Mosaic Virus (SMV) poses a serious threat to soybean production, often resulting in considerable yield losses or complete crop failure, particularly if infection occurs during early growth stages. While several SMV resistance genes have been identified, the genetic basis of resistance to certain strains remains poorly understood. Among the 22 SMV strains, SC4 and SC20 are considered pathogenic in Central China. Dominant genes resistant to SC4 (Rsc4) on Chr.14 in Dabaima and to SC20 (Rsc20) on Chr.13 in Qihuang-1 have been identified. Kefeng-1 is resistant to SC4 and SC20. This study aimed to determine whether the resistance to SC4 and SC20 in Kefeng-1 was identical and whether Rsc4 and Rsc20 in Dabaima and Qihuang-1 are also present in Kefeng-1 due to translocation. Mendelian experiments using F1, F2, and recombinant inbred lines (RIL3:8) of Kefeng-1 (resistant) and NN1138-2 (susceptible) indicated a single dominant gene inheritance pattern in SC4 and SC20, respectively. Linkage mapping showed two loci for SC4 and SC20 in neighboring single nucleotide polymorphism linkage disequilibrium blocks (SNPLDB) marker regions of 253 kb and 375 kb, respectively, in Kefeng-1. Association between SNPs in possible gene regions of Kefeng-1 and resistance data showed SNP11692903 jointly as the most significant SNP, exhibiting the highest χ2 value. By comparing SNP11692903 to possible gene sequences in the coding region, Glyma02g13380 was identified as a joint candidate gene. The results were validated using qRT-PCR, virus induced gene silencing (VIGS), and gene-sequence. Therefore, the two Mendelian genes on chromosome 2 in Kefeng-1 responsible for SC4 and SC20 resistance are unique genes, different from Rsc4 in Dabaima and Rsc20 in Qihuang-1. Hence, one gene is involved in resistance toward two SMV strains resistance. This result challenged our previous hypothesis of a single dominant gene responsible for resistance against a single strain and underscored the potential for using multiple resistance sources aimed at enhancing SMV resistance in breeding practices.

Soybean mosaic virus(SMV)poses a substantial threat to the yield and quality of soybean(Glycine max(L.)Merr.),leading to significant economic losses in soybean production.However,the mining of SMV-resistance loci and the exploration of the underlying disease resistance mechanisms remain relatively limited.MicroRNAs(miRNAs)are a class of post-transcriptional regulators that play a pivotal role in mod-ulating plant growth,development and responding to various stresses.In this study,we demonstrated the function of the"miR398c/d-GmCSDs"module between soybean resistant and susceptible varieties,focusing on its differential regulatory roles in SMV infection.Specifically,SMV infection downregulated gma-miR398c/d expression in the resistant variety(Qihuang 1,QH),while upregulated them in the sus-ceptible variety(Nannong 1138-2,NN).Transient expression assay in N.benthamiana confirmed that gma-miR398c/d can target six superoxide dismutase(SOD)family genes,which responded to SMV infec-tion in both varieties.Stable overexpression of Gma-MIR398c/d in soybean or inhibition of the corre-sponding target genes' expression via Bean pod mottle virus(BPMV)-induced gene silencing(VIGS)led to reduced H2O2 content and thereby promoted SMV infection.Conversely,plants overexpressing the tar-get genes exhibited the opposite phenotypes.The functions of gma-miR398c/d and their target genes were further validated in N.benthamiana through transient co-expression with SMV infectious clone(pSC7-GFP),indicating that gma-miR398c/d negatively regulated soybean resistance to SMV,while the target genes positively contributed to disease resistance.Collectively,our findings provide novel insights into the regulatory mechanisms underlying soybean resistance to SMV.

Shade tolerance is a key trait for cultivars in inter/relay-cropped soybeans in maize fields. Our previous genome-wide association study (GWAS) results on southern China soybean germplasm revealed that the shade tolerance was conferred by a complex of genes with multiple alleles. To complete our understanding of the shade tolerance gene system, GWAS with gene-allele sequences as markers (designated GASM-RTM-GWAS) was conducted in a recombinant inbred line (RIL) population between two extreme parents using the shade tolerance index (STI) and relative pith cell length (RCL) as indicators. Altogether, 211 genes, comprising 99 and 119 genes (seven shared) for STI and RCL, respectively, were identified and then annotated into a similar set of five biological categories. Furthermore, transcriptome analysis detected 7837 differentially expressed genes (DEGs), indicating plentiful DEGs involved in the expression of regulatory/causal GWAS genes. Protein-protein interaction (PPI) analysis and gene functional analysis for both GWAS genes and DEGs showed a group of interrelated causal genes and a group of interrelated DEGs; the former were included in the latter and their functions were interconnected as a gene network. For further understanding of the response of soybean to shade stress in a sequential connection, six chronological gene modules were grouped as signal activation and transport, signal-transduction, signal amplification, gene expression, regulated metabolites, and material transport. From the modules, 12 key genes were selected as entry points for further analysis. Our study provides an overview of the shade tolerance gene network as a new insight into a complex-trait genetic system, rather than the usual way of starting from a hand-picked single gene.

Abstract Chloride (Cl − ) ions cause major damage to crops in saline soils. Understanding the key factors that influence Cl − uptake and translocation will aid the breeding of more salt-tolerant crops. Here, using genome-wide association study and transcriptomic analysis, we identified a NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER family (NPF) protein, GmNPF7.5, as the dominant gene locus influencing Cl − homeostasis in soybean (Glycine max). A natural SNP variation resulted in two haplotypes (GmNPF7.5 HapA and GmNPF7.5 HapB ), which was associated with Cl − content. GmNPF7.5 HapA mediated Cl − or nitrate (NO 3 − ) uptake in a pH-dependent manner and exhibited higher permeability for Cl − over NO 3 − . The suppression of GmNPF7.5 HapA expression decreased Cl − accumulation and salt damage in plants, whereas its overexpression showed the opposite effects. The elite haplotype GmNPF7.5 HapB diminished Cl − transport activity independently from NO 3 − permeability, thus enhancing soybean salt tolerance. Furthermore, the protein kinase GmPI4Kγ4 could phosphorylate GmNPF7.5, which repressed Cl − uptake without affecting NO 3 − permeability. Our findings define a regulatory mechanism for Cl − control under NaCl stress, providing a strategy for the improvement of salt tolerance in soybean plants.

Soybean is an important source of oil, protein, and feed. However, its yield is far below that of major cereal crops. The green revolution increased the yield of cereal crops partially through high-density planting of lodging-resistant semi-dwarf varieties, but required more nitrogen fertilizers, posing an environmental threat. Genes that can improve nitrogen use efficiency need to be integrated into semi-dwarf varieties to avoid the overuse of fertilizers without the loss of dwarfism. Unlike cereal crops, soybean can assimilate atmospheric nitrogen through symbiotic bacteria. Here, we created new alleles of GmGID1-2 (Glycine max GIBBERELLIN INSENSITIVE DWARF 1-2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) editing, which improved soybean architecture, yield, seed oil content, and nitrogen fixation, by regulation of important pathways and known genes related to branching, lipid metabolism, and nodule symbiosis. GmGID1-2 knockout reduced plant height, and increased stem diameter and strength, number of branches, nodes on the primary stem, pods, and seeds per plant, leading to an increase in seed weight per plant and yield in soybean. The nodule number, nodule weight, nitrogenase activity, and nitrogen content were also improved in GmGID1-2 knockout soybean lines, which is novel compared with the semi-dwarf genes in cereal crops. No loss-of-function allele for GmGID1-2 was identified in soybean germplasm and the edited GmGID1-2s are superior to the natural alleles, suggesting the GmGID1-2 knockout mutants generated in this study are valuable genetic resources to further improve soybean yield and seed oil content in future breeding programs. This study illustrates the pleiotropic functions of the GID1 knockout alleles with positive effects on plant architecture, yield, and nitrogen fixation in soybean, which provides a promising strategy toward sustainable agriculture.

Shade tolerance is essential for soybeans in inter/relay cropping systems. A genome-wide association study (GWAS) integrated with transcriptome sequencing was performed to identify genes and construct a genetic network governing the trait in a set of recombinant inbred lines derived from two soybean parents with contrasting shade tolerance. An improved GWAS procedure, restricted two-stage multi-locus genome-wide association study based on gene/allele sequence markers (GASM-RTM-GWAS), identified 140 genes and their alleles associated with shade-tolerance index (STI), 146 with relative pith cell length (RCL), and nine with both. Annotation of these genes by biological categories allowed the construction of a protein–protein interaction network by 187 genes, of which half were differentially expressed under shading and non-shading conditions as well as at different growth stages. From the identified genes, three ones jointly identified for both traits by both GWAS and transcriptome and two genes with maximum links were chosen as beginners for entrance into the network. Altogether, both STI and RCL gene systems worked for shade-tolerance with genes interacted each other, this confirmed that shade-tolerance is regulated by more than single group of interacted genes, involving multiple biological functions as a gene network.

Clanis bilineata tsingtauica Mell (Lepidoptera: Sphingidae, CBT), as a traditional edible insect, is becoming popular in China due to its high nutritional value, but production needs to be improved to meet the expanding market. In the present study, CBT eggs were artificially inoculated on soybean leaves to evaluate the effects of leaf position, growth stage, sowing season, and soybean variety on CBT larval growth, respectively. The results showed that (1) the larval weight and survival rate were poorly correlated, so they could represent two different larval growth and development indicators for CBT. The 21-day-old larval weight was significantly different between the sowing seasons and between soybean growth stages, which was suitable as a key indicator for evaluating CBT larval rearing factors. (2) Compared with autumn-sown soybeans, the weight of 21-day-old larvae feeding on V6 stage (sixth trifoliolate) leaves of summer-sown soybeans was significantly higher, with an average increase of 44.7%. (3) Under autumn sowing conditions, the weight of 21-day-old larvae feeding on soybeans in the V6 stage was significantly higher than those fed on soybeans in the R3 stage (beginning pod), increasing by 33.9%. (4) Under summer sowing conditions, the weight of 21-day-old larvae feeding on the third-top leaf (the third leaf from the top of the soybeans’ main stem) was significantly higher than those feeding on the third-bottom leaf (the third leaf from the bottom of the soybeans’ main stem) at V6 stage by 35.7%. Similar results also appeared in autumn sowing; the average weights of 21-day-old larvae feeding on the third-top leaf increased significantly by 29.9% compared to those feeding on the third-bottom leaf. Moreover, the survival rate of larvae fed with the third-top leaf was significantly higher than that of those fed with the third-bottom leaf at the V6 stage in autumn sowing. Leaf position is the main factor affecting the survival rate of larvae. (5) Under summer sowing conditions, the weights of larvae fed with the third-top leaf of the susceptible-soybean varieties NN89-29 and NN1138-2 were significantly higher than that of those fed with the third-bottom leaf of these varieties. This difference was significantly reduced with autumn sowing. In conclusion, the CBT eggs inoculated on the third-top leaf of NN89-29 and NN1138-2 at the V6 stage in summer sowing could achieve maximum larval yield.