Correlation Analysis of New Soybean [<i>Glycine max</i> (L.) Merr] Gene Gm15G117700 with Oleic Acid

The fatty acid dehydrogenase gene plays an important role in regulating the oleic acid content in soybean. Genome-wide association study screened out soybean oleic acid related gene Gm15G117700. A fragment size of 693bp was obtained by PCR amplification of the gene and, it was connected by seamless cloning technology to the pMD18T cloning vector. Based on the gene sequence cloned, bioinformatic analysis of gene protein was performed. The overexpression vector of Gm15G117700 and the CRISPR/Cas9 gene editing vector were constructed. The positive plants were obtained by Agrobacterium-mediated transformation of soybean cotyledon nodes and T₂ plants were identified by conventional PCR, QT-PCR and Southern blot hybridization. 10 copies of high and low oleic acid seeds were selected for QT-PCR to identify the expression content of Gm15G117700 gene in different soybeans, and finally near-infrared spectroscopy analyzer was used to identify the oleic acid quality of soybeans. T₂ RT-PCR identification showed that overexpression was reduced by 3.94%, and gene editing was increased by 3.49%. It is determined that the Gm15G117700 gene may belong to a regulatory gene, a minor gene that can promote the conversion to linoleic acid content in soybean oleic acid synthesis. The gene cloning and its functional verification was not reported yet. This is the first report by PCR amplification of soybean Gm15G117700 genes and gene expression vector. Improving the content of oleic acid in soybean lay a foundation for researchers. Therefore;this study clearly identified the function of soybean Gm15G117700 gene and its role played in oleic acid synthesis and metabolism.     

Bifurcation and Enhancement of Autonomous-Nonautonomous Retrotransposon Partnership through LTR Swapping in Soybean

Long terminal repeat (LTR) retrotransposons, the most abundant genomic components in flowering plants, are classifiable into autonomous and nonautonomous elements based on their structural completeness and transposition capacity. It has been proposed that selection is the major force for maintaining sequence (e.g., LTR) conservation between nonautonomous elements and their autonomous counterparts. Here, we report the structural, evolutionary, and expression characterization of a giant retrovirus-like soybean (Glycine max) LTR retrotransposon family, SNARE. This family contains two autonomous subfamilies, SARE^A and SARE^B, that appear to have evolved independently since the soybean genome tetraploidization event ∼13 million years ago, and a nonautonomous subfamily, SNRE, that originated from SARE^A. Unexpectedly, a subset of the SNRE elements, which amplified from a single founding SNRE element within the last ∼3 million years, have been dramatically homogenized with either SARE^A or SARE^B primarily in the LTR regions and bifurcated into distinct subgroups corresponding to the two autonomous subfamilies. We uncovered evidence of region-specific swapping of nonautonomous elements with autonomous elements that primarily generated various nonautonomous recombinants with LTR sequences from autonomous elements of different evolutionary lineages, thus revealing a molecular mechanism for the enhancement of preexisting partnership and the establishment of new partnership between autonomous and nonautonomous elements.     

Quantitative trait loci controlling aluminum tolerance in soybean: candidate gene and single nucleotide polymorphism marker discovery

Aluminum (Al) toxicity is an important abiotic stress that affects soybean production in acidic soils throughout the world. Development of Al-tolerant cultivars is an efficient and environmentally friendly solution to the problem. A previous report identified quantitative trait loci (QTL) for Al tolerance inherited from PI 416937, using restriction fragment length polymorphism markers, in a population of Young × PI 416937. The population was genotyped with 162 simple sequence repeats to enhance the power of QTL detection and enable the selection of candidate genes for functional marker development. Two QTL that explained 54 % of the phenotypic variation in root extension under Al stress conditions (HIAL) were refined on chromosomes (chr) Gm08 and Gm16. Three QTL located on chr Gm08, Gm16 and Gm19 explained 59 % of the phenotypic variation in root extension as a percent of control (PC). Two major QTL, designated qAL_HIAL_08 and qAL_PC_08, controlling HIAL and PC, respectively, were mapped to the same genomic region on chr Gm08 and inherited their favorable allele from PI 416937. These QTL explained 45 and 41 % of phenotypic variation in HIAL and PC, respectively. Six homologues for citrate synthase (CS) genes were found in the soybean genome sequence at chr Gm02, Gm08, Gm14, Gm15, and Gm18. Sixteen single nucleotide polymorphisms (SNPs) were identified in the CS homologue on chr Gm08. A SimpleProbe assay of Glyma08g42400-SNP was developed for the major QTL on chr Gm08. The SNPs identified from this region could be used for marker-assisted selection of Al tolerance.     

Evolutionary Divergence of TNL Disease-Resistant Proteins in Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and Common Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>)

Disease-resistant genes (R genes) encode proteins that are involved in protecting plants from their pathogens and pests. Availability of complete genome sequences from soybean and common bean allowed us to perform a genome-wide identification and analysis of the Toll interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat (TNL) proteins. Hidden Markov model (HMM) profiling of all protein sequences resulted in the identification of 117 and 77 regular TNL genes in soybean and common bean, respectively. We also identified TNL gene homologs with unique domains, and signal peptides as well as nuclear localization signals. The TNL genes in soybean formed 28 clusters located on 10 of the 20 chromosomes, with the majority found on chromosome 3, 6 and 16. Similarly, the TNL genes in common bean formed 14 clusters located on five of the 11 chromosomes, with the majority found on chromosome 10. Phylogenetic analyses of the TNL genes from Arabidopsis, soybean and common bean revealed less divergence within legumes relative to the divergence between legumes and Arabidopsis. Syntenic blocks were found between chromosomes Pv10 and Gm03, Pv07 and Gm10, as well as Pv01 and Gm14. The gene expression data revealed basal level expression and tissue specificity, while analysis of available microRNA data showed 37 predicted microRNA families involved in targeting the identified TNL genes in soybean and common bean.     

Soybean Matrix Metalloproteinase <Emphasis Type="Italic">Gm2-MMP</Emphasis> Relates to Growth and Development and Confers Enhanced Tolerance to High Temperature and Humidity Stress in Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. It has been speculated that plant MMPs are involved in plant growth, development, and stress response. However, the biological function of MMPs in higher plants still remains elusive. In the present study, a MMP gene Gm2-MMP was isolated and characterized. It encoded a 357 amino acids protein and contained a common domain structure of MMPs. Subcellular localization of Gm2-MMP-GFP fusion protein indicated that Gm2-MMP was located in plasma membrane. Gm2-MMP was found to show higher expression levels in mature seeds, mature leaves, or old leaves than in other organs and was continuously expressed from seed development to maturation stage. Additionally, Gm2-MMP participated in response to HTH stress in leaves and developing seeds (R7 period) of soybean. The overexpression of Gm2-MMP in Arabidopsis affected the growth and development of leaves, enhanced the tolerance to HTH stress in leaves and developing seeds, and improved the vitality of seed. Twenty-eight candidate proteins interacted with Gm2-MMP were identified from the cDNA library of soybean developing seed under HTH stress by yeast two-hybrid (Y2H) screen. Our results suggested that Gm2-MMP is related to growth and development and confers enhanced HTH stress tolerance in plants. This will be helpful for us in further understanding of the biological functions of MMP family in plant.     

Sequence Level Analysis of Recently Duplicated Regions in Soybean [Glycine max (L.) Merr.] Genome

A single recessive gene, rxp, on linkage group (LG) D2 controlsbacterial leaf-pustule resistance in soybean. We identifiedtwo homoeologous contigs (GmA and GmA') composed of five bacterialartificial chromosomes (BACs) during the selection of BAC clonesaround Rxp region. With the recombinant inbred line populationfrom the cross of Pureunkong and Jinpumkong 2, single-nucleotidepolymorphism and simple sequence repeat marker genotyping wereable to locate GmA' on LG A1. On the basis of information inthe Soybean Breeders Toolbox and our results, parts of LG A1and LG D2 share duplicated regions. Alignment and annotationrevealed that many homoeologous regions contained kinases andproteins related to signal transduction pathway. Interestingly,inserted sequences from GmA and GmA' had homology with transposaseand integrase. Estimation of evolutionary events revealed thatspeciation of soybean from Medicago and the recent divergenceof two soybean homoeologous regions occurred at 60 and 12 millionyears ago, respectively. Distribution of synonymous substitutionpatterns, K_s, yielded a first secondary peak (mode K_s = 0.10–0.15)followed by two smaller bulges were displayed between soybeanhomologous regions. Thus, diploidized paleopolyploidy of soybeangenome was again supported by our study.     

Mapping Locus RSC11K and predicting candidate gene resistant to Soybean mosaic virus strain SC11 through linkage analysis combined with genome resequencing of the parents in soybean

Soybean mosaic virus (SMV) strain SC11 was prevalent in middle China. Its resistance was controlled by a Mendelian single dominant gene R_SC11K in soybean Kefeng-1. This study aimed at mapping R_SC11K and identifying its candidate gene. R_SC11K locus was mapped ~217 kb interval between two SNP-linkage-disequilibrium-blocks (Gm02_BLOCK_11273955_11464884 and Gm02_BLOCK_11486875_11491354) in W82.a1.v1 genome using recombinant inbred lines population derived from Kefeng-1 (Resistant) × NN1138-2 (Susceptible), but inserted with a ~245 kb segment in W82.a2.v1 genome. In the entire 462 kb R_SC11K region, 429 SNPs, 142 InDels and 34 putative genes were identified with more SNPs/InDels distributed in non-functional regions. Thereinto, ten genes contained SNP/InDel variants with high and moderate functional impacts on proteins, among which Glyma.02G119700 encoded a typical innate immune receptor-like kinase involving in virus disease process and responded to SMV inoculation, therefore was recognized as R_SC11K's candidate gene. The novel R_SC11K locus and candidate genes may help developing SMV resistance germplasm.     

Localization of Structural and Regulatory Genes for Phosphodiesterase in Wheat (TRITICUM AESTIVUM)

Genes (Pde-A3; Pde-B3; Pde-D3) for phosphodiesterase (PDE; E.C. 3.1.4.1.) isoenzymes in hexaploid wheat were located on the three homoeologous chromosomes of group 3 by testing the electrophoretic banding pattern of monosomic, nullisomic and nullisomic/tetrasomic compensation lines of "Chinese Spring" variety. In plants nullisomic for chromosome 5B, the 3D structural gene is not expressed and this lack of expression can be overcome by four doses of either homoeologous chromosome 5A or 5D. Our data conclusively indicate that there are genes on group 5 chromosomes which positively control the expression of the 3D structural gene. In addition, the expression of the "regulatory genes" is dosage dependent. Thus, our study reveals a complex interaction of the three genomes of wheat for regulation of PDE gene expression.     

SNP assay to detect the ‘Hyuuga’ red-brown lesion resistance gene for Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus.     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

Characterization of the Jembrana Disease Virus tat Gene and the cis- and trans-Regulatory Elements in Its Long Terminal Repeats

Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDV tat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides −68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide −196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.     

Meta-analysis to refine map position and reduce confidence intervals for delayed-canopy-wilting QTLs in soybean

Slow canopy wilting in soybean has been identified as a potentially beneficial trait for ameliorating drought effects on yield. Previous research identified QTLs for slow wilting from two different biparental populations, and this information was combined with data from three other populations to identify nine QTL clusters for slow wilting on Gm02, Gm05, Gm11, Gm 14, Gm17, and Gm19. The QTL cluster on Gm14 was eliminated because these QTLs appeared to be false positives. In the present research, QTLs from these remaining eight clusters were compiled onto the soybean consensus map for meta-QTL analysis. Five model selection criteria were used to determine the most appropriate number of meta-QTLs at these eight chromosomal regions. For a QTL cluster on Gm02, two meta-QTLs were identified, whereas for the remaining seven QTL clusters the single meta-QTL model was most appropriate. Thus, the analysis identified nine meta-QTLs associated with slow wilting. Meta-analysis decreased the confidence intervals from an average of 21.4 cM for the eight QTL clusters to 10.8 cM for the meta-QTLs. Averaged R² values of the nine meta-QTLs in eight QTL clusters were 0.13 and ranged from 0.09 to 0.22. Meta-QTLs on Gm11 and Gm19 had the highest R² values (0.22 and 0.20, respectively).     

Genome-Wide Association Studies Identifies Seven Major Regions Responsible for Iron Deficiency Chlorosis in Soybean (Glycine max)

Iron deficiency chlorosis (IDC) is a yield limiting problem in soybean (Glycine max (L.) Merr) production regions with calcareous soils. Genome-wide association study (GWAS) was performed using a high density SNP map to discover significant markers, QTL and candidate genes associated with IDC trait variation. A stepwise regression model included eight markers after considering LD between markers, and identified seven major effect QTL on seven chromosomes. Twelve candidate genes known to be associated with iron metabolism mapped near these QTL supporting the polygenic nature of IDC. A non-synonymous substitution with the highest significance in a major QTL region suggests soybean orthologs of FRE1 on Gm03 is a major gene responsible for trait variation. NAS3, a gene that encodes the enzyme nicotianamine synthase which synthesizes the iron chelator nicotianamine also maps to the same QTL region. Disease resistant genes also map to the major QTL, supporting the hypothesis that pathogens compete with the plant for Fe and increase iron deficiency. The markers and the allelic combinations identified here can be further used for marker assisted selection.     

Identification of quantitative trait loci controlling linolenic acid concentration in PI483463 (Glycine soja)

Key message

The QTLs controlling alpha-linolenic acid concentration from wild soybean were mapped on nine soybean chromosomes with various phenotypic variations. New QTLs for alpha-linolenic acid were detected in wild soybean.

Abstract

Alpha-linolenic acid (ALA) is a polyunsaturated fatty acid desired in human and animal diets. Some wild soybean (Glycine soja) genotypes are high in ALA. The objective of this study was to identify quantitative trait loci (QTLs) controlling ALA concentration in a wild soybean accession, PI483463. In total, 188 recombinant inbred lines of F_5:6, F_5:7, and F_5:8 generations derived from a cross of wild soybean PI483463 (~15 % ALA) and cultivar Hutcheson (~9 % ALA) were planted in four environments. Harvested seeds were used to measure fatty acid concentration. Single nucleotide polymorphism markers of the universal soybean linkage panel (USLP 1.0) and simple sequence repeat markers were used for molecular genotyping. Nine putative QTLs were identified that controlled ALA concentration by model-based composite interval mapping and mapped to different soybean chromosomes. The QTLs detected in four environments explained 2.4–7.9 % of the total phenotypic variation (PV). Five QTLs, qALA5_3, qALA6_1, qALA14_1, qALA15_1, and qALA17_1, located on chromosomes 5, 6, 14, 15, and 17 were identified by model-based composite interval mapping and composite interval mapping in two individual environments. Among them, qALA6_1 showed the highest contribution to the PV with 10.0–10.2 % in two environments. The total detected QTLs for additive and epistatic effects explained 52.4 % of the PV for ALA concentration. These findings will provide useful information for understanding genetic structure and marker-assisted breeding programs to increase ALA concentration in seeds derived from wild soybean PI483463.     

Identification of germplasm with stacked QTL underlying seed traits in an inbred soybean population from cultivars Essex and Forrest

Soybean (Glycine max (L.) Merr.) seed provides valuable oil (~200 g/kg) and protein (~400 g/kg) co-products. Seed composition variations result from several quantitative trait loci (QTL) that act through development. The objectives here were to identify loci underlying seed traits in the Essex × Forrest (EF94)-derived recombinant inbred line (RIL) population which has low frequencies of marker polymorphisms. Seed weight, protein, and oil were measured over 3 years: 2001, 2003, and 2005. Essex’s seeds were larger (141 mg/seed), higher in protein (406 g/kg), and lower in oil (190 g/kg) than Forrest’s (115 mg/seed, 395 g protein/kg, and 203 g oil/kg). Marker analysis included 413 markers for trait associations used for ANOVA, interval mapping, and composite interval mapping. Eleven QTL in nine genomic regions were associated (LOD >2; P < 0.0077) with seed traits. Two QTL, for mean protein and seed size, were clustered on linkage group (LG) E (chromosome Gm16). QTL for protein content alone were found on LG C2 (Gm6), LG D1b (Gm2), LG H (Gm12), and LG I (Gm20). The alleles from Essex, the high-protein parent, underlay higher protein (4–10 g/kg) at four of five loci. A QTL for mean oil was found on LG A2 (Gm18) and on LG I (Gm 20). The alleles from Forrest underlay higher oil (3–4 g/kg). Five separate QTL for mean seed weight were found on LG A1 (Gm05), LG N (Gm15), two on LG B1 (Gm11) and one on LG N (Gm3). The alleles from Essex underlay greater seed weight (0.4–0.66 g/100 seeds). The QTL positions were consistent with reported loci. Germplasm that contained all five beneficial alleles at the QTL underlying protein was significantly higher in protein and yield than Essex (409.7–412.3 g/kg) and included RILs 49 and 62. The germplasm identified can be useful for further breeding of the many traits and QTL measured in each line.