Soybean Gene Research Articles

Soybean balanced the growth and defense in response to SMV infection under different light intensities.

Light is essential for the growth and defense of soybean. It is not clear how soybeans adjust their defenses to different light environments with different cropping patterns. The mechanism of soybean response to Soybean mosaic virus (SMV) infection under different light intensities was analyzed by RNA-seq sequencing method. Enrichment analysis illustrated that most defense-related genes were down-regulated in the dark and the shade, and up-regulated under hard light and normal light. Soybean can resist SMV infection mainly by activating salicylic acid signaling pathway. Light is essential for activating salicylic acid defense signaling pathways. With the increase of light intensity, the oxidative damage of soybean leaves was aggravated, which promoted the infection of virus. When light was insufficient, the growth of soybean was weak, and the plant-pathogen interaction pathway, MAPK pathway and hormone defense pathway in infected soybean was inhibited. Under hard light, some defense genes in infected soybean were down-regulated to reduce the degree of oxidative damage. The expression of differentially expressed genes was verified by real-time fluorescence quantitative RT-PCR. In order to adapt to the change of light intensity, soybean balanced allocation of resources between growth and defense through a series regulation of gene expression. The results of this study will provide a theoretical basis for the research of SMV resistance in intercropping soybean.

Genome-Wide Identification and Characterization of the Soybean Snf2 Gene Family and Expression Response to Rhizobia.

Sucrose nonfermenting 2 (Snf2) family proteins are the core component of chromatin remodeling complexes that can alter chromatin structure and nucleosome position by utilizing the energy of ATP, playing a vital role in transcription regulation, DNA replication, and DNA damage repair. Snf2 family proteins have been characterized in various species including plants, and they have been found to regulate development and stress responses in Arabidopsis. Soybean (Glycine max) is an important food and economic crop worldwide, unlike other non-leguminous crops, soybeans can form a symbiotic relationship with rhizobia for biological nitrogen fixation. However, little is known about Snf2 family proteins in soybean. In this study, we identified 66 Snf2 family genes in soybean that could be classified into six groups like Arabidopsis, unevenly distributed on 20 soybean chromosomes. Phylogenetic analysis with Arabidopsis revealed that these 66 Snf2 family genes could be divided into 18 subfamilies. Collinear analysis showed that segmental duplication was the main mechanism for expansion of Snf2 genes rather than tandem repeats. Further evolutionary analysis indicated that the duplicated gene pairs had undergone purifying selection. All Snf2 proteins contained seven domains, and each Snf2 protein had at least one SNF2_N domain and one Helicase_C domain. Promoter analysis revealed that most Snf2 genes had cis-elements associated with jasmonic acid, abscisic acid, and nodule specificity in their promoter regions. Microarray data and real-time quantitative PCR (qPCR) analysis revealed that the expression profiles of most Snf2 family genes were detected in both root and nodule tissues, and some of them were found to be significantly downregulated after rhizobial infection. In this study, we conducted a comprehensive analysis of the soybean Snf2 family genes and demonstrated their responsiveness to Rhizobia infection. This provides insight into the potential roles of Snf2 family genes in soybean symbiotic nodulation.

Functional Characterization of a (E)-β-Ocimene Synthase Gene Contributing to the Defense against Spodoptera litura.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-β-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-β-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-β-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-β-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-β-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

Overexpression of GmNF-YA14 produced multiple phenotypes in soybean

Soybean is an important oil crop, and more and more soybean gene functions have been reported; however, one soybean gene with multiple functions is rarely seen. The NF-YA subfamily plays a broad regulatory role in plant growth and development, and GmNF-YA14 (Glyma.14G010000) is one of them. We found that the expression level of GmNF-YA14 in the root of soybean seedlings was significantly increased after a one-hour treatment with 200 mM salt. The transgenic soybean was obtained by Agrobacterium-mediated method, and all experiments were carried out using transgenic soybean homozygous lines. Under a 200 mM salt treatment, the leaf chlorophyll content of overexpressed GmNF-YA14 transgenic soybean seedlings was significantly decreased compared to the control. Moreover, the first flowering time of overexpressed GmNF-YA14 transgenic soybean was shortened by more than six days compared to the control, and the growth period structure changed significantly. Overexpressed GmNF-YA14 transgenic soybean plants had two or three branches, whereas control soybean plants had eight or nine branches. Compared to the control, the number of main stem nodes and plant height of overexpressed GmNF-YA14 transgenic soybean plants were significantly reduced. The 100-seed weight of GmNF-YA14 transgenic soybean line OE-8 was significantly higher than that of the control. Transcriptome analysis indicated that the multiple phenotypes of the overexpressed GmNF-YA14 transgenic soybean may be related to the mitogen-activated protein kinase (MAPK) signaling pathway. These results indicated that compared to the control, overexpressed GmNF-YA14 transgenic soybeans had changes in salt stress tolerance, growth period, plant type, and 100-seed weight, and these changes may be related to the MAPK signaling pathway.

A genome-wide analysis of the USDA Soybean Isoline Collection.

The USDA Soybean Isoline Collection has been an invaluable resource for the soybean genetics and breeding community. This collection, established in 1972, consists of 611 near-isogenic lines (NILs) carrying one or multiple genes conferring traits that had been determined to exhibit Mendelian inheritance. It has been used in multiple studies on the genetic basis, physiology, and agronomy of these qualitative traits. Here, we used publicly available genotype (SoySNP50K), phenotype, and pedigree data on this collection to characterize the isogenicity of the NILs and identify chromosomal positions of unmapped genes. A total of 368 NILs had at least 80% identity to their recurrent parent and, thus, were useful for what can be called introgression mapping. Both on-target and off-target introgressions were evaluated. The size of on-target introgressions into individual NILs ranged from 61kb to 8.4 Mb, whereas off-target introgressions ranged from 2.6kb to 54.8 Mb. The observed large off-target introgressions indicated that some NILs carry introgressions nearly the size of an entire chromosome. By applying introgression mapping to genes that had never been mapped, we identified the likely chromosomal positions of six such genes: ab, im, lo, Np, pc, and Rpm. The size of mapping intervals was large in some cases (10.28 Mb for im) but small in others (0.21 Mb for Np). The results reported herein will provide future researchers with a resource to help select informative NILs for future studies, and provide a starting point to further fine map, and ultimately clone and functionally characterize these six soybean genes.

Identification of QTNs, QEIs interactions and genes for isoflavones in soybean seeds

Isoflavones are important components of soybean seeds, and the contents affect the nutritional value of soybean products. To further reveal and improve the regulation mechanism of soybean isoflavones content, it is an effective way to construct a large enough germplasm combination and explore the genetic loci related to soybean isoflavone traits. In this study, 179 local soybean germplasms mainly from China were selected to analyze the phenotypic diversity and genetic basis of isoflavone content in soybean seeds. The genome-wide association studies (GWAS) using 3VmrMLM model combined the isoflavone phenotype with 23,131 single nucleotide polymorphisms (SNPs), we identified 97 genetic loci related to isoflavone traits in single environment and 20 genetic loci related to isoflavone traits in multiple environments, after that screened 6 potential genes related to total isoflavone content, daidzein, glycitein and genistein. Glyma.09G037300 and Glyma.18G111200 related to multiple traits of soybean isoflavones are highly expressed in soybean seeds. Haplotype analysis shows that Glyma.09G037300 and Glyma.18G111200 are higher expression in the high isoflavone populations than low isoflavone populations. Transient overexpression of Glyma.18G111200 increased the content of total isoflavones in hairy roots by 30–51%. These results revealed the genetic basis of soybean isoflavones, provided a series of predictable potential genes affecting the synthesis and accumulation of soybean isoflavones, and updated the research on soybean isoflavone related genes and genetic related loci. This provide a valuable basis for the functional study of soybean isoflavones.

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.

Genome-wide identification and expression analysis of diacylglycerol acyltransferase genes in soybean (Glycine max).

Soybean (Glycine max) is a major protein and vegetable oil source. In plants, diacylglycerol acyltransferase (DGAT) can exert strong flux control, which is rate-limiting for triacylglycerol biosynthesis in seed oil formation. Here, we identified soybean DGAT genes via a bioinformatics method, thereby laying a solid foundation for further research on their function. Based on our bioinformatics analyses, including gene structure, protein domain characteristics, and phylogenetic analysis, 26 DGAT putative gene family members unevenly distributed on 12 of the 20 soybean chromosomes were identified and divided into the following four groups: DGAT1, DGAT2, WS/DGAT, and cytoplasmic DGAT. The Ka/Ks ratio of most of these genes indicated a significant positive selection pressure. DGAT genes exhibited characteristic expression patterns in soybean tissues. The differences in the structure and expression of soybean DGAT genes revealed the diversity of their functions and the complexity of soybean fatty acid metabolism. Our findings provide important information for research on the fatty acid metabolism pathway in soybean. Furthermore, our results will help identify candidate genes for potential fatty acid-profile modifications to improve soybean seed oil content. This is the first time that in silico studies have been used to report the genomic and proteomic characteristics of DGAT in soybean and the effect of its specific expression on organs, age, and stages.

Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family

Δ12-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.

Two soybean homologues of TERMINAL FLOWER 1 control flowering time under long day conditions

Flowering time is a key agronomic trait that directly affect the adaptation and yield of soybean. After whole genome duplications, about 75% of genes being represented by multiple copies in soybean. There are four TERMINAL FLOWER 1 (TFL1) genes in soybean, and the TFL1b (Dt1) has been characterized as the determinant of stem growth habit. The function of other TFL1 homologs in soybean is still unclear. Here, we generated knockout mutants by CRISPR/Cas9 genome editing technology and found that the tfl1c/tfl1d double mutants flowered significantly earlier than wild-type plants. We investigated that TFL1c and TFL1d could physically interact with the bZIP transcription factor FDc1 and bind to the promoter of APETALA1a (AP1a). RNA-seq and qRT-PCR analyses indicated that TFL1c and TFL1d repressed the expressions of the four AP1 homologs and delayed the flowering time in soybean. The two genes play important roles in the regulation of flowering time in soybean and mainly act as the flowering inhibitors under long-day conditions. Our results identify novel components in the flowering-time regulation network of soybean and will be invaluable for molecular breeding of improved soybean yield.

Registration of 64 soybean germplasm lines with all combinations of five soybean aphid resistance genes in two genetic backgrounds

AbstractSoybean aphid is a serious pest in North America, and infestations can cause economically significant losses. Aphid resistance genes have been identified and mapped with genetic markers, and the deployment of cultivars with pyramids of these genes could offer durable resistance. There is a need, however, for soybean [Glycine max (L.) Merr.] lines with different gene pyramids to be developed and tested to improve our understanding of what combinations provide the most resistance across aphid biotypes. We backcrossed the resistance genes Rag1, Rag2, Rag3, Rag4, and Rag6 into the background of two soybean cultivars, and lines with all 32 combinations of these five genes were developed in each background. These lines, LD19‐APH01–LD19‐APH64 (Reg. no. GP‐459–GP‐522, PI 701410–PI 701473), were evaluated for agronomic traits in the field and lines from one background were evaluated for resistance to four soybean aphid biotypes. There were significant differences among the performance of the backcross lines for agronomic traits; however, yield performance for most of the backcross lines did not significantly differ from the recurrent parent. Those lines with the Rag6 resistance gene and many gene pyramids showed a high level of resistance to all four aphid biotypes. Because these lines have the resistance genes in a mostly uniform background, they will be a useful source of germplasm for researchers studying soybean aphid isolates and the interaction between resistance genes and aphid isolates, as well as for breeders who are developing germplasm with resistance gene pyramids.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

Evaluate the guide RNA effectiveness via Agrobacterium-mediated transient assays in Nicotiana benthamiana.

CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the Agrobacterium-mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in Nicotiana benthamiana and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting Nicotiana benthamiana and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting NbEDS1, NbWRKY70, GmKTI1, and GmKTI3 had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting NbNDR1 was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.

GmERF54, an ERF Transcription Factor, Negatively Regulates the Resistance of Soybean to the Common Cutworm (Spodoptera litura Fabricius)

Soybean is attacked by various herbivorous insect pest species during the whole course of its life cycle in the field. It is important for soybean production to improve insect resistance by identifying and utilizing soybean endogenous insect-resistant genes. The ethylene-responsive transcription factor (ERF) plays a significant role in plant biotic and abiotic stresses; however, few studies focus on its role in insect resistance in soybean. Here, based on our previous common cutworm (CCW)-induced soybean transcriptome data, a soybean ERF gene GmERF54 was cloned, which responded to CCW feeding. Transcriptional analysis revealed that GmERF54 was ubiquitous in all soybean tissues and was expressed differently in insect-resistant and insect-susceptible soybean cultivars. RNA interference of GmERF54 increased the resistance to CCW, while the overexpression of GmERF54 decreased the resistance to CCW in transgenic soybean hairy roots compared with their controls. GmERF54 was localized to the nucleus, had transcriptional activation activity, and interacted with AP2/ERF GmPLT2. Several putative hormone response elements were predicted in the promoter sequence of GmERF54. Four putative elements were only found in the GmERF54 promoter sequence of insect-resistant cultivar Wanxianbaidongdou (WX), but not in the insect-susceptible cultivar Nannong 99-10 (99-10). GmERF54 promoter sequences of WX and 99-10 were cloned into the pCAMBIA1381z vector containing the β-glucuronidase (GUS) gene to generate GmERF54WX:GUS and GmERF5499-10:GUS recombinant vectors, respectively. GUS staining of soybean hairy roots containing GmERF54WX:GUS and GmERF5499-10:GUS showed that GmERF54 was induced by CCW attack and both MeJA (methyl jasmonate) and IAA (indole-3-acetic acid) treatments. Alleles in insect-resistant and insect-sensitive cultivars responded to these inductions differently. Overall, our results reveal that GmERF54 may be involved in the regulation of soybean resistance to CCW.

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana.

Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

Genome-Wide Identification and Expression Analysis of the Ammonium Transporter Family Genes in Soybean.

Ammonium transporters (AMTs) are responsible for ammonium absorption and utilization in plants. As a high-nitrogen-demand crop and a legume, soybean can also obtain ammonium from symbiotic root nodules in which nitrogen-fixing rhizobia convert atmospheric nitrogen (N2) into ammonium. Although increasing evidence implicates vital roles of ammonium transport in soybean, no systematic analyses of AMTs in soybean (named GmAMTs) or functional analyses of GmAMTs are available. In this study, we aimed to identify all GmAMT family genes and gain a better understanding of the characteristics of GmAMT genes in soybean. Here, due to the improved genome assembly and annotation of soybean, we tried to generate a phylogenetic tree of 16 GmAMTs based on new information. Consistent with reported data, GmAMT family members can be divided into two subfamilies of GmAMT1 (6 genes) and GmAMT2 (10 genes). Interestingly, unlike Arabidopsis, which has only one AMT2, soybean has substantially increased the number of GmAMT2s, suggesting enhanced demand for ammonium transport. These genes were distributed on nine chromosomes, of which GmAMT1.3, GmAMT1.4, and GmAMT1.5 were three tandem repeat genes. The gene structures and conserved protein motifs of the GmAMT1 and GmAMT2 subfamilies were different. All the GmAMTs were membrane proteins with varying numbers of transmembrane domains ranging from 4 to 11. Promoter analysis found that these GmAMT genes have phytohormone-, circadian control-, and organ expression-related cis-elements in their promoters, and notably, there were nodulation-specific and nitrogen-responsive elements in the promoters of the GmAMT1 and GmAMT2 genes. Further expression data showed that these GmAMT family genes exhibited different spatiotemporal expression patterns across tissues and organs. In addition, GmAMT1.1, GmAMT1.2, GmAMT2.2, and GmAMT2.3 were responsive to nitrogen treatment, while GmAMT1.2, GmAMT1.3, GmAMT1.4, GmAMT1.5, GmAMT1.6, GmAMT2.1, GmAMT2.2, GmAMT2.3, GmAMT3.1, and GmAMT4.6 showed circadian rhythms in transcription. RT-qPCR validated the expression patterns of GmAMTs in response to different forms of nitrogen and exogenous ABA treatments. Gene expression analysis also confirmed that GmAMTs are regulated by key nodulation gene GmNINa, indicating a role of GmAMTs in symbiosis. Together, these data indicate that GmAMTs may differentially and/or redundantly regulate ammonium transport during plant development and in response to environmental factors. These findings provide a basis for future research on the functions of GmAMTs and the mechanisms through which GmAMTs regulate ammonium metabolism and nodulation in soybean.

Detection of Soybean-Derived Components in Dairy Products Using Proofreading Enzyme-Mediated Probe Cleavage Coupled with Ladder-Shape Melting Temperature Isothermal Amplification (Proofman–LMTIA)

Food adulteration is a serious problem all over the world. Establishing an accurate, sensitive and fast detection method is an important part of identifying food adulteration. Herein, a sequence-specific ladder-shape melting temperature isothermal amplification (LMTIA) assay was reported to detect soybean-derived components using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. The results showed that, under the optimal temperature of 57 °C, the established Proofman-LMTIA method for the detection of soybean-derived components in dairy products was sensitive to 1 pg/μL, with strong specificity, and could distinguish soybean genes from those of beef, mutton, sunflower, corn, walnut, etc. The established Proofman-LMTIA detection method was applied to the detection of actual samples of cow milk and goat milk. The results showed that the method was accurate, stable and reliable, and the detection results were not affected by a complex matrix without false positives or false negatives. It was proved that the method could be used for the detection and identification of soybean-derived components in actual dairy products samples.

Integrative pathway and network analysis provide insights on flooding-tolerance genes in soybean

Soybean is highly sensitive to flooding and extreme rainfall. The phenotypic variation of flooding tolerance is a complex quantitative trait controlled by many genes and their interaction with environmental factors. We previously constructed a gene-pool relevant to soybean flooding-tolerant responses from integrated multiple omics and non-omics databases, and selected 144 prioritized flooding tolerance genes (FTgenes). In this study, we proposed a comprehensive framework at the systems level, using competitive (hypergeometric test) and self-contained (sum-statistic, sum-square-statistic) pathway-based approaches to identify biologically enriched pathways through evaluating the joint effects of the FTgenes within annotated pathways. These FTgenes were significantly enriched in 36 pathways in the Gene Ontology database. These pathways were related to plant hormones, defense-related, primary metabolic process, and system development pathways, which plays key roles in soybean flooding-induced responses. We further identified nine key FTgenes from important subnetworks extracted from several gene networks of enriched pathways. The nine key FTgenes were significantly expressed in soybean root under flooding stress in a qRT-PCR analysis. We demonstrated that this systems biology framework is promising to uncover important key genes underlying the molecular mechanisms of flooding-tolerant responses in soybean. This result supplied a good foundation for gene function analysis in further work.

A novel soybean malectin-like receptor kinase-encoding gene, GmMLRK1, provides resistance to soybean mosaic virus.

Soybean mosaic virus (SMV) severely damages soybean [Glycine max (L.) Merr.] yield and seed quality. Moreover, the underlying genetic determinants of resistance to SMV remain largely unknown. Here, we performed a genome-wide association study (GWAS) of SMV resistance in a panel of 219 diverse soybean accessions across four environments and identified a new resistance-related gene, GmMLRK1, at the major resistance locus Rsv4 on chromosome 2. GmMLRK1 encodes a malectin-like receptor kinase (RK) that was induced earlier and to a greater degree in leaves of the SMV-resistant cultivar Kefeng No. 1 than in those of the susceptible cultivar Nannong 1138-2 after inoculation. We demonstrated that soybean plants overexpressing GmMLRK1 show broad-spectrum resistance to both strains SC7 and SC3 on the basis of reduced viral accumulation, increased reactive oxygen species production, and local cell death associated with the hypersensitive response. In contrast, GmMLRK1 knockout mutants were more susceptible to both pathotypes. Haplotype analysis revealed the presence of five haplotypes (H1-H5) within the soybean population, and only H1 provided SMV resistance, which was independent of its tightly linked SMV resistance gene RNase-H at the same locus. These results report a novel gene that adds new understanding of SMV resistance and can be used for breeding resistant soybean accessions.

MADS-Box Subfamily Gene GmAP3 from Glycine max Regulates Early Flowering and Flower Development.

AP3 has been studied and is reported to affect structural changes in floral organs in various plants. However, the function of the soybean AP3 genes in flower development is unknown. Here, the full-length cDNA sequence of GmAP3 was obtained by RACE and it was verified that it belongs to the MADS-box subfamily by a bioinformatics analysis. The expression of GmAP3 is closely related to the expression of essential enzyme genes related to flower development. Yeast two-hybrid assays demonstrated that GmAP3 interacts with AP1 to determine the identity of flower organ development. A follow-up analysis showed that overexpression of the GmAP3 gene advanced flowering time and resulted in changes in floral organ morphology. The average flowering time of overexpressed soybean and tobacco plants was 6-8 days earlier than that of wild-type plants, and the average flowering time of gene-edited soybean and tobacco plants was 6-11 days later than that of wild-type plants. In conclusion, GmAP3 may directly or indirectly affect the flower development of soybean. The results of this study lay the foundation for further research on the biological functions of MADS transcriptional factors in soybeans.