Soybean Mutant Research Articles

Genetic Analysis and Gene Mapping for a Short-Petiole Mutant in Soybean (Glycine max (L.) Merr.)

Short petiole is a valuable trait for the improvement of plant canopy of ideotypes with high yield. Here, we identified a soybean mutant line derived short petiole (dsp) with extremely short petiole in the field, which is obviously different from most short-petiole lines identified previously. Genetic analysis on 941 F2 individuals and subsequent segregation analysis of 184 F2:3 and 172 F3:4 families revealed that the dsp mutant was controlled by two recessive genes, named as dsp1 and dsp2. Map-based cloning showed that these two recessive genes were located on two nonhomologous regions of chromosome 07 and chromosome 11, of which the dsp1 locus was mapped at a physical interval of 550.5-Kb on chromosome 07 near to centromere with flanking markers as BARCSOYSSR_07_0787 and BARCSOYSSR_07_0808; whereas, the dsp2 locus was mapped to a 263.3-Kb region on chromosome 11 with BARCSOYSSR_11_0037 and BARCSOYSSR_11_0043 as flanking markers. A total of 36 and 33 gene models were located within the physical genomic interval of dsp1 and dsp2 loci, respectively. In conclusion, the present study identified markers linked with genomic regions responsible for short-petiole phenotype of soybean, which can be effectively used to develop ideal soybean cultivars through marker-assisted breeding.

Characterization of a new sg-5 variant with reduced biosynthesis of group A saponins in soybean (Glycine max (L.) Merr.)

Triterpenoid saponins are major secondary metabolites in soybean; they are classified into group A and DDMP saponins. Group A saponins cause bitterness and an astringent aftertaste, whereas DDMP saponins are less bitter and more beneficial to human health. Therefore, a need exists for genetic improvement to develop bitter-free soybeans without losing other beneficial saponins. In this study, an ethyl methanesulfonate (EMS)–induced mutant, PE1327, was isolated and characterized as having a reduced level of group A saponins by chromatography. The PE1327 phenotype, which exhibits no accumulation of group A saponins, resembles that of a previously reported sg-5 wild soybean mutant whose biosynthesis is controlled by the Sg-5 (Glyma.15G243300) gene. The Sg-5 gene sequence in PE1327 revealed a single-nucleotide polymorphism (G1036A) that causes an amino acid change from Glu346 to Lys346 in the sg-5 protein. Multiple alignment analysis of cytochrome P450 enzymes from numerous organisms revealed that Glu346 is highly conserved in eukaryotes, including plants and animals, but not in prokaryotes. The predicted 3D model of the Sg-5 protein showed that the Glu346 residue is located in the J helix and is likely involved in the linkage between the J and K helices and the stabilization of the J-K loop, suggesting that the amino acid substitution from Glu346 to Lys346 in the PE1327 mutant results in hypofunction of Sg-5. Co-segregation analysis revealed that the Sg-5 locus is tightly linked to reduced group A saponin biosynthesis in PE1327 and that the novel sg-5 variant is recessive to Sg-5.

Molecular characterization of high stearic acid soybean mutants and post-transcriptional control of GmSACPD genes in the mutant with a single nucleotide deletion

Soybean (Glycine max) oil normally contains 2%–4% stearic acid. Increased saturated fatty acid levels elevate the melting point of soybean oil and improve oxidative stability. Using X-ray irradiation, we isolated six high stearic acid mutants and investigated mutations in the gene encoding Δ9-stearoyl-ACP-desaturase (SACPD). Our analyses of the KK2 mutant revealed an approximately 155-kbp deletion in the GmSACPD-B region. We also identified a frameshift mutation in the KK24 mutant, corresponding with a base-pair deletion in the GmSACPD-C coding region. The other four mutants M2, M18, MM106, and MS827 also had 2.2–21.8-kbp deletions in the GmSACPD-C region. Thus, we designed co-dominant marker sets for KK2 and KK24 mutant alleles using multiplex polymerase chain reactions and the CEL I mismatch detection method, respectively. In addition, we quantified the degradation of GmSACPD-C mRNA with a single base-pair deletion and related it to nonsense-mediated mRNA decay and simultaneous reductions in GmSACPD-A and -B mRNA levels. This molecular mechanism may be exploited to introduce silent mutations in duplicate genes of soybean plants and other organisms.

Genomic changes and biochemical alterations of seed protein and oil content in a subset of fast neutron induced soybean mutants

BackgroundSoybean is subjected to genetic manipulation by breeding, mutation, and transgenic approaches to produce value-added quality traits. Among those genetic approaches, mutagenesis through fast neutrons radiation is intriguing because it yields a variety of mutations, including single/multiple gene deletions and/or duplications. Characterizing the seed composition of the fast neutron mutants and its relationship with gene mutation is useful towards understanding oil and protein traits in soybean.ResultsFrom a large population of fast neutron mutagenized plants, we selected ten mutants based on a screening of total oil and protein content using near infra-red spectroscopy. These ten mutants were regrown, and the seeds were analyzed for oil by GC-MS, protein profiling by SDS-PAGE and gene mapping by comparative genomic hybridization. The mutant 2R29C14Cladecr233cMN15 (nicknamed in this study as L10) showed higher protein and lower oil content compared to the wild type, followed by three other lines (nicknamed in this study as L03, L05, and L06). We characterized the fatty acid methyl esters profile of the trans-esterified oil and found the presence of five major fatty acids (palmitic, stearic, oleic, linoleic, and linolenic acids) at varying proportions among the mutants. Protein profile using SDS-PAGE of the ten mutants did exhibit discernable variation between storage (glycinin and β-conglycinin) and anti-nutritional factor (trypsin inhibitor) proteins. In addition, we physically mapped the position of the gene deletions or duplications in each mutant using comparative genomic hybridization.ConclusionCharacterization of oil and protein profile in soybean fast neutron mutants will assist scientist and breeders to develop new value-added soybeans with improved protein and oil quality traits.

Gamma radiation to produce soybean mutants for better plant performance and chemical composition of seeds

The supply the constant demand for new soybean cultivar, it is necessary to select superior plants in populations of high genetic variability. To generate new plant variants which would perform satisfactorily in the field and produce seeds of better composition, gamma rays can be used. Therefore, soybean seeds from line VX04-6828 were irradiated in a 60Co source (gamma rays) in doses: 0 (control); 50; 150 and 250 Gy, and were seeded to produce the M1 generation. Potential plants of each row were identified and evaluated for their agronomic performance. The seeds of the selected plants were sown, producing the plants of M2 generation and again selected and evaluated, including the chemical composition of their seeds. It was concluded that the use of gamma radiation increases the variability in soybean, with consequent increase in the probabilities of identification of new mutants, useful to breeding programs that aim at better agronomic performance and gains in the composition of the seeds. Better results are observed when 50 to 150 Gy is applied on soybean dry seeds.

Raman Spectroscopic and Chemometric Investigation of Lipid-Protein Ratio Contents of Soybean Mutants.

Seeds belonging to fourth generation mutants (M4) of Ataem-7 cultivar (A7) variety and S04-05 (S) breeding line salt-tolerant soybeans were investigated by Raman spectroscopy, complemented by chemometrics methods, in order to evaluate changes induced by mutations in the relative lipid-protein contents, and to find fast, efficient strategies for discrimination of the mutants and the control groups based on their Raman spectra. It was concluded that gamma irradiation caused an increase in the lipid to protein ratio of the studied Ataem-7 variety mutants, while it led to a decrease of this ratio in the investigated S04-05 breeding line mutants. These results were found to be in agreement with data obtained by reflectance spectrum analysis of the seeds in the full ultraviolet to near-infrared spectral region and suggest the possibility of developing strategies where gamma irradiation can be used as a tool to improve mutant soybean plants targeted to different applications, either enriched in proteins or in lipids. Ward's clustering and principal component analysis showed a clear discrimination between mutants and controls and, in the case of the studied S-type species, discrimination between the different mutants. The grouping scheme is also found to be in agreement with the compositional information extracted from the analysis of the lipid-protein contents of the different samples.

Evaluasi Ukuran dan Bentuk Biji Genotip-genotip Mutan Kedelai Generasi M4

Soybean was one of the most important crop plants in Indonesia, but the need has not been fulfilled yet because its productivity was low. This study aims to obtain genotypes of M4 soybean mutant generation based on phenotypic characters of seed size and seed shape. The experiment was carried out in the experimental methods without spatial design, used 19 soybean mutant genotypes on M 4 generation and Arjasari variety as a control. Eight characters of seed size (seed length, seed thickness, seed width, seed dimension, and 100 seed weight) and seed shape (seed length–thickness ratio, seed length-width ratio, and seed thickness–width ratio) have observed. Data analyzed with F-test, z-test, cluster analysis, and correlation analysis. The results showed that the seed size and seed shape characters had narrow intrapopulation variations. Eight genotypes of soybean mutants, namely MRAR 14-11-17-5, MRAR 15-17-31-11, MRAR 26-6-21-8, MRAR 29-12-26-11, MRAR 29-15-33 -7, MRAR 29-15-36-4, MRAR 29-15-36-8, and MRAR 29-15-30-10, have more significant seed size characters compared to Arjasari varieties. The MRAR genotypes 15-17-31-11 and MRAR 29-12-27-8 have a more rounded seed shape than the Arjasari variety. The seed size and seed shape characters correlated very strongly with each other. Seed size characters do not associate with seed shape characters.

Screening populations for copy number variation using genotyping-by-sequencing: a proof of concept using soybean fast neutron mutants

BackgroundThe effective use of mutant populations for reverse genetic screens relies on the population-wide characterization of the induced mutations. Genome- and population-wide characterization of the mutations found in fast neutron populations has been hindered, however, by the wide range of mutations generated and the lack of affordable technologies to detect DNA sequence changes. In this study, we therefore aimed to test whether genotyping-by-sequencing (GBS) technology could be used to characterize copy number variation (CNV) induced by fast neutrons in a soybean mutant population.ResultsWe called CNVs from GBS data in 79 soybean mutants and assessed the sensitivity and precision of this approach by validating our results against array comparative genomic hybridization (aCGH) data for 19 of these mutants as well as targeted PCR and ddPCR assays for a representative subset of the smallest events detected by GBS. Our GBS pipeline detected 55 of the 96 events found by aCGH, with approximate detection thresholds of 60 kb, 500 kb and 1 Mb for homozygous deletions, hemizygous deletions and duplications, respectively. Among the whole set of 79 mutants, the GBS data revealed 105 homozygous deletions, 32 hemizygous deletions and 19 duplications. This included several extremely large events, exhibiting maximum sizes of ~ 11.2 Mb for a homozygous deletion, ~ 11.6 Mb for a hemizygous deletion, and ~ 50 Mb for a duplication.ConclusionsThis study provides a proof of concept that GBS can be used as an affordable high-throughput method for assessing CNVs in fast neutron mutants. The modularity of this GBS approach allows combining as many different libraries or sequencing runs as is necessary for reaching the goals of a particular study. This method should enable the low-cost genome-wide characterization of hundreds to thousands of individuals in fast neutron mutant populations or any population with large genomic deletions and duplications.

Effective identification of soybean candidate genes involved in resistance to soybean cyst nematode via direct whole genome re-sequencing of two segregating mutants.

Three soybean candidate genes involved in resistance to soybean cyst nematode race 4 were identified via direct whole genome re-sequencing of two segregating mutants. The genes conferring resistance to soybean cyst nematode (SCN) race 4 (Hg type 1.2.3.5.7) in soybean (Glycine max L. Merr.) remains unknown. Next generation sequencing-based methods identify a wide range of targets, it is difficult to identify genes underlying traits. Use of the MutMap and QTL-seq methods to identify trait candidate genes needs backcrossing and is very time-consuming. Here we report a simple method to effectively identify candidate genes involved in resistance to SCN race 4. Two ethane methylsulfonate mutagenized mutants of soybean 'PI 437654', whose SCN race 4-infection phenotype altered, were selected. Six relevant whole genomes were re-sequenced, and then calling of genomic variants (SNPs and InDels) was conducted and compared to 'Williams 82'. The comparison eliminated many genomic variants from the mutant lines that overlapped two non-phenotypic but mutant progeny plants, wild-type PI 437654 and 'Zhonghuang 13'. Finally, only 27 mutations were found among 10 genes. Of these 10 genes, 3 genes, Glyma.09g054000, Glyma.16g065700 and Glyma.18g192200 were overlapped between two phenotypic mutant progeny plants. Therefore, the three genes may be the candidate genes involved in resistance of PI 437654 to soybean cyst nematode race 4. This method simplifies the effective identification of candidate genes.

Characterization and Rapid Gene-Mapping of Leaf Lesion Mimic Phenotype of spl-1 Mutant in Soybean (Glycine max (L.) Merr.).

In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (spl-1) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (lm1 locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5′ or 3′ UTR. Hence, the data of the present study are in favor of Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.

Hypernodulating soybean mutant line nod4 lacking 'Autoregulation of Nodulation' (AON) has limited root-to-shoot water transport capacity.

Although hypernodulating phenotype mutants of legumes, such as soybean, possess a high leaf N content, the large number of root nodules decreases carbohydrate availability for plant growth and seed yield. In addition, under conditions of high air vapour pressure deficit (VPD), hypernodulating plants show a limited capacity to replace water losses through transpiration, resulting in stomatal closure, and therefore decreased net photosynthetic rates. Here, we used hypernodulating (nod4) (282.33 ± 28.56 nodules per plant) and non-nodulating (nod139) (0 nodules per plant) soybean mutant lines to determine explicitly whether a large number of nodules reduces root hydraulic capacity, resulting in decreased stomatal conductance and net photosynthetic rates under high air VPD conditions. Plants were either inoculated or not inoculated with Bradyrhizobium diazoefficiens (strain BR 85, SEMIA 5080) to induce nitrogen-fixing root nodules (where possible). Absolute root conductance and root conductivity, plant growth, leaf water potential, gas exchange, chlorophyll a fluorescence, leaf 'greenness' [Soil Plant Analysis Development (SPAD) reading] and nitrogen content were measured 37 days after sowing. Besides the reduced growth of hypernodulating soybean mutant nod4, such plants showed decreased root capacity to supply leaf water demand as a consequence of their reduced root dry mass and root volume, which resulted in limited absolute root conductance and root conductivity normalized by leaf area. Thereby, reduced leaf water potential at 1300 h was observed, which contributed to depression of photosynthesis at midday associated with both stomatal and non-stomatal limitations. Hypernodulated plants were more vulnerable to VPD increases due to their limited root-to-shoot water transport capacity. However, greater CO2 uptake caused by the high N content can be partly compensated by the stomatal limitation imposed by increased VPD conditions.

Evaluation of yield stability of black soybean mutant lines in ten environments

To obtain national high yielding varieties, it is necessary to evaluate black soybean yield stability in various environments. This study aims to evaluate yield stability and to study its genotype and environment interaction. Genetic material used in this study was five mutant lines of black soybean derived from the irradiation of the Cikuray variety with a dose of 200 Gy. Evaluation of yield stability was carried out jointly with 10 black soybean breeding lines from Balitkabi and Padjadjaran University. Two control varieties of Cikuray and Detam 1 were included in the evaluation. The test was carried out in ten locations across Java Island. Estimation of stability was conducted by the method of Finlay-Wilkinson and Eberhart-Russell. The results showed that genotype and environment significantly contributed to yield variation respectively. Genotype by environment interactions were existed which could affect genotype performance in the testing environment. Four out of five mutant lines showed significant higher yield than other genotypes and control varieties, which ranged from 2.34-2.42 ton/ha. Stability analysis indicated genotypes of G8, G14 and control variety of Detam 1 were stable genotypes and high yield above the general average with average yields of 2.36, 2.09 and 2.21 tons / ha, respectively.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins.

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean.

Food contamination by cadmium (Cd) is a serious threat to human health. Thus, it is imperative to prevent Cd accumulation in staple crops like soybean. The development of low Cd accumulating cultivars is an effective solution. To this end, it is essential to identify the gene(s) controlling seed Cd accumulation. Although Glyma.09G055600 (GmHMA3) seems to be associated with Cd accumulation in soybean, it has not been established if it is responsible for seed Cd accumulation. In the present study, the effect of GmHMA3 on seed Cd accumulation in soybean was validated using three independent GmHMA3 mutants isolated from an ethyl methanesulfonate-induced soybean mutant library. Each of mutant had an amino acid substitution in GmHMA3 and segregating progenies were developed by crossing the original cultivar with each of the three mutants. The relationship between these three mutations and seed Cd accumulation was investigated. While two of them significantly increased seed Cd accumulation corresponding to previous reports of a natural missense mutation in GmHMA3, the other slightly decreased seed Cd accumulation. Overall, these results indicate that GmHMA3 is responsible for seed Cd accumulation in soybean.

Impact of Cross-Breeding of Low Phytic Acid MIPS1 and IPK1 Soybean ( Glycine max L. Merr.) Mutants on Their Contents of Inositol Phosphate Isomers.

The knowledge on consequences of cross-breeding of induced low phytic acid ( lpa) soybean ( Glycine max L. Merr.) mutants on the contents of phytic acid (InsP6) and lower inositol phosphate isomers (InsP2-InsP5) in the resulting progenies is limited. Therefore, MIPS1 and IPK1 lpa soybean mutants were crossed with wild-type (WT) cultivars or among themselves to generate homozygous lpa and WT progenies and double lpa mutants. The lpa trait of the MIPS1 mutant was not altered by cross-breeding with a WT cultivar; lpa progenies had InsP6 reductions of about 44% compared to WT progenies. IPK1 progenies showed pronounced accumulations of specific InsP3-InsP5 isomers (up to 12.4 mg/g) compared to the progenitor lpa mutant (4.7 mg/g); the extent of InsP6 reduction (43-71%) was depending on the WT crossing parent. Double mutants exhibited the most pronounced InsP6 reductions (up to 87%), accompanied by moderate accumulations of InsP3-InsP5 (2.5 mg/g). Cross-breeding offers the potential to modulate the amounts of both InsP6 and InsP3-InsP5 contents in lpa soybean mutants and thus to improve their nutritional quality.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean (Glycine max (L.) Merr.) to soybean cyst nematode (SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four- and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCN-infection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wild-type soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Impact of Mutations on the Binding Pocket of Soybean Lipoxygenase: Implications for Proton-Coupled Electron Transfer.

Soybean lipoxygenase catalyzes a proton-coupled electron transfer (PCET) reaction and serves as a prototype for hydrogen tunneling in enzymes due to the unusually high kinetic isotope effect and significant modulation of the rate constant and kinetic isotope effect by mutation. Herein these experimental observations are interpreted in the context of changes to the substrate binding pocket in microsecond molecular dynamics simulations of wild-type and mutant soybean lipoxygenase. The binding pocket exhibits an hourglass shape with residues L546 and L754 bracketing the bottleneck, positioning the linoleic acid substrate for PCET. Mutation of I553 to less bulky residues slightly increases the width of the bottleneck and the volume of the binding pocket. Mutating L546 or L754 to a smaller residue also enlarges this width and volume, and mutating both has an even more dramatic effect. This analysis illustrates how mutation of the substrate binding pocket can be used as a strategy to tune the kinetics.

Characteristics of mutant soybean lines at Satt100 and Satt319 loci linked with E7 gene

Aim. Аnalysis of genetic polymorphism of microsatellite loci Satt100 and Satt319 in 10 lines obtained by chemical mutagenesis, and 4 parental varieties – Oksana, Femida, Zolotysta, Podil’s’ka 416. Satt100 and Satt319 are flanking E7gene, which determines sensitivity of soybean to the length of the day in ripening phase. Methods. DNA were isolated from soybean seeds using the kit NeoPrep100 DNA. PCR were performed with microsatellites Satt100 and Satt319. The PCR products were fractionated in 7% polyacrylamide gels. Results. In the investigated lines were detected 5 alleles at the Satt100 locus and 3 alleles at the Satt319 locus. 42.9 % of the varieties and lines in this study were carriers of the «E» allele for Satt100. According to analysis of locus Satt319 64.2 % of varieties and lines were the carriers of «B» allele. Conclusions. Variety Oksana, mutant lines Oksana M №12, Femida M №29 – are carriers of the dominant allele E7. Variety Zolotysta and five mutant lines have recessive allele e7. In 75 % cases we have detected changes of alleles at microsatellite loci in mutant lines in comparison with parental forms. We assume that mutagenic reagents could affect at generative organs of soybean and lead totheir open flowering and crosspollinations.
 Keywords: Glycine max (L.), soybean, gene E7, microsatellite markers, photoperiodic sensitivity.

Characterization of an EMS-induced soybean mutant with an increased content of Af saponin and a new component Ab-δ in the seed hypocotyl

Saponins are one of the components present in the soybean seeds that have various functional properties. The chemical structures and concentration of soyasaponins affect the taste of the processed soyfood, thereby limiting its industrial applications. Therefore, it is important to understand saponin biosynthesis to explore natural and artificial variation in the saponin components, which can be modified to suit its application. The objective of the present study was to identify and characterize an EMS-induced soybean mutant with an altered saponin composition from a pool of 892 M4 lines. The mutant PE1905 showed an increased content of saponin Af (336.0%). The content of saponin Ab, DDMP-αg, and DDMP-βg was decreased in the mutant PE1905 by 89.3, 24.8, and 63.1%, respectively compared to the wild-type Pungsannamul. Additionally, four new components were detected in the mutant PE1905 that were absent in the wild type. Of these, the compound 4 (designated as Ab-δ) had the highest concentration, and therefore it was further characterized by HPLC and LC-PDA/MS/MS analysis to know the chemical structure, and molecular weight and formula. Considering these details, along with the alterations in the saponin Af and Ab concentrations, it was presumed that the Ab-δ acts as a precursor for the synthesis of saponin Af and Ab. Thus, we predicted a biosynthetic pathway from the Ab-δ to Ab saponin. The inheritance analysis showed that the concentration of saponin Ab-δ is controlled by a single recessive gene in the mutant PE1905. The results from the present study would be helpful in understanding the mechanisms behind altered seed saponin composition in soybeans.

Reaction Mechanism of Prephenate Dehydrogenase from the Alternative Tyrosine Biosynthesis Pathway in Plants.

Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine-insensitive biosynthesis of 4-hydroxyphenylpyruvate, a precursor to tyrosine. The three-dimensional structure of soybean PDH1 was recently solved in complex with the NADP+ cofactor. This structure allowed for the identification of both the cofactor- and ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active-site mutants of soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.