New statistical selection method for pleiotropic variants associated with both quantitative and qualitative traits.

BACKGROUND: Identification of pleiotropic variants associated with multiple phenotypic traits has received increasing attention in genetic association studies. Overlapping genetic associations from multiple traits help to detect weak genetic associations missed by single-trait analyses. Many statistical methods were developed to identify pleiotropic variants with most of them being limited to quantitative traits when pleiotropic effects on both quantitative and qualitative traits have been observed. This is a statistically challenging problem because there does not exist an appropriate multivariate distribution to model both quantitative and qualitative data together. Alternatively, meta-analysis methods can be applied, which basically integrate summary statistics of individual variants associated with either a quantitative or a qualitative trait without accounting for correlations among genetic variants. RESULTS: We propose a new statistical selection method based on a unified selection score quantifying how a genetic variant, i.e., a pleiotropic variant associates with both quantitative and qualitative traits. In our extensive simulation studies where various types of pleiotropic effects on both quantitative and qualitative traits were considered, we demonstrated that the proposed method outperforms the existing meta-analysis methods in terms of true positive selection. We also applied the proposed method to a peanut dataset with 6 quantitative and 2 qualitative traits, and a cowpea dataset with 2 quantitative and 6 qualitative traits. We were able to detect some potentially pleiotropic variants missed by the existing methods in both analyses. CONCLUSIONS: The proposed method is able to locate pleiotropic variants associated with both quantitative and qualitative traits. It has been implemented into an R package 'UNISS', which can be downloaded from http://github.com/statpng/uniss.

QTL mapping reveals key factors related to the isoflavone contents and agronomic traits of soybean (Glycine max).

BACKGROUND: Soybean is a valuable source of edible protein and oil, as well as secondary metabolites that can be used in food products, cosmetics, and medicines. However, because soybean isoflavone content is a quantitative trait influenced by polygenes and environmental interactions, its genetic basis remains unclear. RESULTS: This study was conducted to identify causal quantitative trait loci (QTLs) associated with soybean isoflavone contents. A mutant-based F2 population (190 individuals) was created by crossing the Korean cultivar Hwanggeum with low isoflavone contents (1,558 µg g-1) and the soybean mutant DB-088 with high isoflavone contents (6,393 µg g-1). A linkage map (3,049 cM) with an average chromosome length of 152 cM was constructed using the 180K AXIOM® SoyaSNP array. Thirteen QTLs related to agronomic traits were mapped to chromosomes 2, 3, 11, 13, 19, and 20, whereas 29 QTLs associated with isoflavone contents were mapped to chromosomes 1, 3, 8, 11, 14, 15, and 17. Notably, the qMGLI11, qMGNI11, qADZI11, and qTI11, which located Gm11_9877690 to Gm11_9955924 interval on chromosome 11, contributed to the high isoflavone contents and explained 11.9% to 20.1% of the phenotypic variation. This QTL region included four candidate genes, encoding ß-glucosidases 13, 14, 17-1, and 17-2. We observed significant differences in the expression levels of these genes at various seed developmental stages. Candidate genes within the causal QTLs were functionally characterized based on enriched GO terms and KEGG pathways, as well as the results of a co-expression network analysis. A correlation analysis indicated that certain agronomic traits (e.g., days to flowering, days to maturity, and plant height) are positively correlated with isoflavone content. CONCLUSIONS: Herein, we reported that the major QTL associated with isoflavone contents was located in the interval from Gm11_9877690 to Gm11_9955924 (78 kb) on chromosome 11. Four ß-glucosidase genes were identified that may be involved in high isoflavone contents of soybean DB-088. Thus, the mutant alleles from soybean DB-088 may be useful for marker-assisted selection in developing soybean lines with high isoflavone contents and superior agronomic traits.

Morpho-physiological and genetic characteristics of a salt-tolerant mutant line in soybean (Glycine max L.).

KEY MESSAGE: One major quantitative trait loci and candidate gene for salt tolerance were identified on chromosome 3 from a new soybean mutant derived from gamma-ray irradiation, which will provide a new genetic resource for improving soybean salt tolerance. Soil salinity is a worldwide problem that reduces crop yields, but the development of salt-tolerant crops can help overcome this challenge. This study was conducted with the purpose of evaluating the morpho-physiological and genetic characteristics of a new salt-tolerant mutant KA-1285 developed using gamma-ray irradiation in soybean (Glycine max L.). The morphological and physiological responses of KA-1285 were compared with salt-sensitive and salt-tolerant genotypes after treatment with 150 mM NaCl for two weeks. In addition, a major salt tolerance quantitative trait locus (QTL) was identified on chromosome 3 in this study using the Daepung X KA-1285 169 F2:3 population, and a specific deletion was identified in Glyma03g171600 (Wm82.a2.v1) near the QTL region based on re-sequencing analysis. A kompetitive allele-specific PCR (KASP) marker was developed based on the deletion of Glyma03g171600 which distinguished the wild-type and mutant alleles. Through the analysis of gene expression patterns, it was confirmed that Glyma03g171700 (Wm82.a2.v1) is a major gene that controls salt tolerance functions in Glyma03g32900 (Wm82.a1.v1). These results suggest that the gamma-ray-induced mutant KA-1285 has the potential to be employed for the development of a salt-tolerant cultivar and provide useful information for genetic research related to salt tolerance in soybeans.

Genome-Wide Association Studies of Seven Root Traits in Soybean (Glycine max L.) Landraces.

Soybean [Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global climate, developing crops that are resistant to climatic extremes and produce viable yields under predicted climatic conditions will be essential in the coming decades. To develop such crops, it will be necessary to gain a thorough understanding of the genetic basis of agronomic and plant root traits. As plant roots generally lie beneath the soil surface, detailed observations and phenotyping throughout plant development present several challenges, and thus the associated traits have tended to be ignored in genomics studies. In this study, we phenotyped 357 soybean landraces at the early vegetative (V2) growth stages and used a 180 K single-nucleotide polymorphism (SNP) soybean array in a genome-wide association study (GWAS) conducted to determine the phenotypic relationships among root traits, elucidate the genetic bases, and identify significant SNPs associated with root trait-controlling genomic regions/loci. A total of 112 significant SNP loci/regions were detected for seven root traits, and we identified 55 putative candidate genes considered to be the most promising. Our findings in this study indicate that a combined approach based on SNP array and GWAS analyses can be applied to unravel the genetic basis of complex root traits in soybean, and may provide an alternative high-resolution marker strategy to traditional bi-parental mapping. In addition, the identified SNPs, candidate genes, and diverse variations in the root traits of soybean landraces will serve as a valuable basis for further application in genetic studies and the breeding of climate-resilient soybeans characterized by improved root traits.

Quantitative Trait Loci (QTL) Analysis of Seed Protein and Oil Content in Wild Soybean (Glycine soja).

Soybean seeds consist of approximately 40% protein and 20% oil, making them one of the world's most important cultivated legumes. However, the levels of these compounds are negatively correlated with each other and regulated by quantitative trait loci (QTL) that are controlled by several genes. In this study, a total of 190 F2 and 90 BC1F2 plants derived from a cross of Daepung (Glycine max) with GWS-1887 (G. soja, a source of high protein), were used for the QTL analysis of protein and oil content. In the F2:3 populations, the average protein and oil content was 45.52% and 11.59%, respectively. A QTL associated with protein levels was detected at Gm20_29512680 on chr. 20 with a likelihood of odds (LOD) of 9.57 and an R2 of 17.2%. A QTL associated with oil levels was also detected at Gm15_3621773 on chr. 15 (LOD: 5.80; R2: 12.2%). In the BC1F2:3 populations, the average protein and oil content was 44.25% and 12.14%, respectively. A QTL associated with both protein and oil content was detected at Gm20_27578013 on chr. 20 (LOD: 3.77 and 3.06; R2 15.8% and 10.7%, respectively). The crossover to the protein content of BC1F3:4 population was identified by SNP marker Gm20_32603292. Based on these results, two genes, Glyma.20g088000 (S-adenosyl-l-methionine-dependent methyltransferases) and Glyma.20g088400 (oxidoreductase, 2-oxoglutarate-Fe(II) oxygenase family protein), in which the amino acid sequence had changed and a stop codon was generated due to an InDel in the exon region, were identified.

Transcriptome Analysis of Differentially Expressed Genes Associated with Salt Stress in Cowpea (Vigna unguiculata L.) during the Early Vegetative Stage.

Cowpea (Vigna unguiculata (L.), 2n = 22) is a tropical crop grown in arid and semiarid regions that is tolerant to abiotic stresses such as heat and drought. However, in these regions, salt in the soil is generally not eluted by rainwater, leading to salt stress for a variety of plant species. This study was conducted to identify genes related to salt stress using the comparative transcriptome analysis of cowpea germplasms with contrasting salt tolerance. Using the Illumina Novaseq 6000 platform, 1.1 billion high-quality short reads, with a total length of over 98.6 billion bp, were obtained from four cowpea germplasms. Of the differentially expressed genes identified for each salt tolerance type following RNA sequencing, 27 were shown to exhibit significant expression levels. These candidate genes were subsequently narrowed down using reference-sequencing analysis, and two salt stress-related genes (Vigun_02G076100 and Vigun_08G125100) with single-nucleotide polymorphism (SNP) variation were selected. Of the five SNPs identified in Vigun_02G076100, one that caused significant amino acid variation was identified, while all nucleotide variations in Vigun_08G125100 was classified as missing in the salt-resistant germplasms. The candidate genes and their variation, identified in this study provide, useful information for the development of molecular markers for cowpea breeding programs.

Fine-mapping and candidate gene analysis for the foxglove aphid resistance gene Raso2 from wild soybean PI 366121.

KEY MESSAGE: The foxglove aphid resistance gene Raso2 from PI 366121 was fine-mapped to 77 Kb region, and one candidate gene was identified. The foxglove aphid (FA: Aulacorthum solani Kaltenbach) is an important insect pest that causes serious yield losses in soybean. The FA resistance gene Raso2 from wild soybean PI 366121 was previously mapped to a 13 cM interval on soybean chromosome 7. However, fine-mapping of Raso2 was needed to improve the effectiveness of marker-assisted selection (MAS) and to eventually clone it. The objectives of this study were to fine-map Raso2 from PI 366121 using Axiom® 180 K SoyaSNP array, to confirm the resistance and inheritance of Raso2 in a different background, and to identify candidate gene(s). The 105 F4:8 recombinant inbred lines were used to fine-map the gene and to test antibiosis and antixenosis of Raso2 to FA. These efforts resulted in the mapping of Raso2 on 1 cM interval which corresponds to 77 Kb containing eight annotated genes based on the Williams 82 reference genome assembly (Wm82.a2.v1). Interestingly, all nonsynonymous substitutions were in Glyma.07g077700 which encodes the disease resistance protein containing LRR domain and expression of the gene in PI 366121 was significantly higher than that in Williams 82. In addition, distinct SNPs within Glyma.07g077700 that can distinguish PI 366121 and diverse FA-susceptible soybeans were identified. We also confirmed that Raso2 presented the resistance to FA and the Mendelian inheritance for single dominant gene in a different background. The results of this study would provide fundamental information on MAS for development of FA-resistant cultivars as well as functional study and cloning of the candidate gene in soybean.

Analysis of genetic diversity and relationships of Perilla frutescens using novel EST-SSR markers derived from transcriptome between wild-type and mutant Perilla.

BACKGROUND: Perilla frutescens (Lamiaceae) is distributed in East Asia and is classified into var. frutescens and crispa. P. frutescens is multipurpose crop for human health because of a variety of secondary metabolites such as phenolic compound and essential oil. However, a lack of genetic information has hindered the development and utilization of Perilla genotypes. METHODS AND RESULTS: This study was performed to develop expressed sequence tag-simple sequence repeat (EST-SSR) markers from P. frutescens var. crispa (wild type) and Antisperill (a mutant cultivar) and used them to assess the genetic diversity of, and relationships among, 94 P. frutescens genotypes. We obtained 65 Gb of sequence data comprising 632,970 transcripts by de novo RNA-sequencing. Of the 14,780 common SSRs, 102 polymorphic EST-SSRs were selected using in silico polymerase chain reaction (PCR). Overall, successful amplification from 58 EST-SSRs markers revealed remarkable genetic diversity and relationships among 94 P. frutescens genotypes. In total, 268 alleles were identified, with an average of 4.62 alleles per locus (range 2-11 alleles/locus). The average polymorphism information content (PIC) value was 0.50 (range 0.04-0.86). In phylogenetic and population structure analyses, the genotypes formed two major groups: Group I (var. crispa) and Group II (var. frutescens). CONCLUSION: This results suggest that 58 novel EST-SSR markers derived from wild-type cultivar (var. crispa) and its mutant cultivar (Antisperill) have potential uses for population genetics and recombinant inbred line mapping analyses, which will provide comprehensive insights into the genetic diversity and relationship of P. frutescens.

The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures.

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3'H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-ß-cyclodextrin (MeßCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 µM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeßCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeßCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

Selection of mutants with high linolenic acid contents and characterization of fatty acid desaturase 2 and 3 genes during seed development in soybean (Glycine max).

BACKGROUND: Soybean seeds contain 18-24% lipids, which are made up of 85% polyunsaturated fatty acids. Two of these (linoleic and linolenic acids) comprise essential fatty acids that are not synthesized in humans and animals. Linolenic acid plays a vital role in the maintenance of brain function and is a source of docosahexaenoic acid for retinal and nerve tissue, with its physiological functions being a focus of attention. RESULTS: We developed mutant soybean populations via gamma irradiation of Korean cultivars Danbaek and Daepung and evaluated the linolenic acid content of 78 and 154 M9 mutant progenies. We selected the four mutant lines with the highest linolenic acid contents based on 2 years of investigation of fatty acids. The selected mutant lines had linolenic acid contents that were 33.9% to 67.7% higher than those of the original cultivars and exhibited increased fatty acid desaturase (FAD) gene expression levels during seed development. We also identified nucleotide polymorphisms of FAD genes in the four mutant lines. CONCLUSION: The present study found that linolenic acid content is related to significantly increased expression levels of the FAD3C and FAD3D genes in the endoplasmic reticulum, which was uncovered by radiation mutation breeding of soybean. © 2019 Society of Chemical Industry.

Molecular characterization of proton beam-induced mutations in soybean using genotyping-by-sequencing.

Proton beam irradiation is a next-generation technique to develop mutant crop varieties. The mutagenic effects and molecular mechanisms of radiation are important multi-disciplinary research subjects. This study was conducted to investigate the types of mutations induced in the soybean genome by proton beam irradiation. In total, 22 plants, including 10 M2 plants treated with proton beam irradiation at 118 and 239 Gy, each, and two wild-type plants (Daepung) were sequenced by genotyping-by-sequencing (GBS). In total, 7453 single nucleotide polymorphisms (SNPs) were detected in the 20 M2 plants, compared with the two wild-type controls. The SNP frequency was 1/36,976 bp with proton beam irradiation at 118 Gy, and 1/32,945 bp at 239 Gy. Of these, 3569 SNPs were detected in genic regions. We observed that proton beam irradiation induced more substitutions than small insertion-deletions (INDELs). Based on the mutagenic effect of proton beam irradiation, the frequency of transition mutations was shown to be higher than that of transversions. The proton beam-induced SNPs were distributed uniformly in most of the chromosomes. Gene ontology (GO) analysis showed that there were many genes involved in protein metabolic process under biological process, intracellular membrane-bounded organelle under cellular component, and nucleic acid binding under molecular function. This study could provide valuable information for investigating the potential mechanisms of mutation, and guidance for developing soybeans cultivars using mutation breeding.

Development, validation and genetic analysis of a large soybean SNP genotyping array.

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under-use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high-density genetic mapping and genome-wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome-wide association analyses. More than four million high-quality SNPs identified from high-depth genome re-sequencing of 16 soybean accessions and low-depth genome re-sequencing of 31 soybean accessions were used to select 180,961 SNPs for creation of the Axiom(®) SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170,223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene-rich chromosomal regions suggest that this array may be suitable for genome-wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.

Identification of candidate genes for an early-maturing soybean mutant by genome resequencing analysis.

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 (GI), Glya02g33040 (AGL18), Glyma17g11040 (TOC1), and Glyma14g10530 (ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding.

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. 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 F5:6, F5:7, and F5:8 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.

Selection and molecular characterization of a lipoxygenase-free soybean mutant line induced by gamma irradiation.

KEY MESSAGE: A lipoxygenase-free soybean mutant line (H70) induced by gamma ray was selected and its detailed information about the lipoxygenase was analyzed by comparison of DNA sequence. Soybean seeds contain three lipoxygenase enzymes, which induce a beany or grassy flavor. The elimination of lipoxygenases can reduce the poor stability and off-flavors of soybean oil and protein products. In this study, we selected a soybean mutant (H70) in which the three lipoxygenases had been mutated using gamma rays. To obtain detailed information about the lipoxygenase, we investigated the sequences of the Lox1, Lox2 and Lox3 genes in H70 compared to the original cultivar, Hwanggum. Comparisons of the sequences of the Lox1 and Lox2 genes in H70 with those in a line with normal lipoxygenase (HG) showed that the mutations in these genes affected a highly conserved group of six histidine residues necessary for enzymatic activity. Lox1 in H70 contained a 74 bp deletion in exon 8, creating a stop codon that prematurely terminates translation. A single point mutation (T-A) in exon 8 of Lox2 changed histidine (H532, one of the iron-binding ligands essential for Lox2 activity) to glutamine. The mutation in the Lox3 gene in H70 was a single-point mutation in exon 6 (A-G), which changed the amino acid from histidine to arginine. This amino acid alteration in Lox3 was located in the N-terminal barrel, which might play a role in molecular recognition during catalysis and/or proteolysis. These results suggest that gene analysis based on DNA sequencing could be useful for elucidating the lipoxygenase content in soybean mutant lines. Additionally, the soybean mutant line selected in this study could be used to develop soybean cultivars with improved flavor.

Transcriptome profiling in response to different types of ionizing radiation and identification of multiple radio marker genes in rice.

Ionizing radiation (IR) affects gene expression from plant genomes. To monitor the genome-wide transcriptional changes induced by three types of IR, we used the rice Affymetrix GeneChip microarray to identify genes that are up- or down-regulated by gamma rays (GAs), cosmic rays (CRs) and ion beams (IBs). The overall expression patterns in rice seedlings generated from seeds exposed to GAs and IBs were similar but differed for CRs exposure. Expression profiles of genes involved in metabolic pathways and cellular response were identified using MapMan analysis. This result revealed that IRs induced gene expression related to sucrose-starch metabolisms; sugar and starch accumulation was significantly increased in response to three types of IR in rice. In addition, we compared the genes commonly up- or down-regulated by exposure to three types of IR and identified 53 candidate radio marker genes (RMGs) that were differentially regulated by radiation exposure but not by other stresses. Among these genes, we selected six RMGs commonly applicable to different types of IR by specific coexpression networks using the algorithm for the reconstruction of accurate cellular networks (aracne) and confirmed the expression of these genes by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Our results provided insight into the mechanisms of the responses to different types of IR and identified multiple marker genes to predict sensitivity to three types of IR.

Differentially expressed genes in response to gamma-irradiation during the vegetative stage in Arabidopsis thaliana.

Biochemical and physiological processes in plants are affected by gamma-irradiation, which causes significant changes in gene transcripts and expression. To identify the differentially expressed Arabidopsis genes in response to gamma-irradiation, we performed a microarray analysis with rosette leaves during the vegetative stage. Arabidopsis plants were exposed to a wide spectrum doses of gamma ray (100, 200, 300, 400, 800, 1200, 1600 or 2000 Gy) for 24 h. At the dose range from 100 to 400 Gy, irradiated plants were found to be shorter than controls after 8 days of irradiation, while doses over 800 Gy caused severe growth retardation. Therefore, 100 and 800 Gy were selected as adequate doses for microarray analysis to identify differentially expressed genes. Among the 20,993 genes used as microarray probes, a total number of 496 and 1,042 genes were up-regulated and down-regulated by gamma-irradiation, respectively (P < 0.05). We identified the characteristics of the genes that were up-and down-regulated fourfold higher genes by gamma irradiation according to The arabidopsis information resource gene ontology. To confirm the microarray results, we performed a northern blot and quantitative real-time PCR with several selected genes that had a large difference in expression after irradiation. In particular, genes associated with lipid transfer proteins, histones and transposons were down-regulated by 100 and/or 800 Gy of gamma irradiation. The expression patterns of selected genes were generally in agreement with the microarray results, although there were quantitative differences in the expression levels.

Physiological changes and anti-oxidative responses of Arabidopsis plants after acute and chronic γ-irradiation.

To identify the effects of acute and chronic γ-irradiation in Arabidopsis plants, physiological responses and antioxidant-related gene expression were investigated. Seedlings were exposed to 200 Gy of γ-irradiation in acute manner for 1 or 24 h (A1 and A24) or in chronic manner for 1, 2, or 3 weeks (C1 W, C2 W, and C3 W). Plant height, silique number, and silique length in A1 and A24 irradiated plants were significantly reduced when compared to non-irradiated plants. Silique number decreased in response to both acute and chronic irradiation, except with the C3 W treatment, and the number of trichomes dramatically increased in A1 and C1 W. Electron spin resonance signal intensities increased in A1 and in all chronically irradiated plants, but decreased in the A24-treated plant. To investigate the effects of acute and chronic γ-irradiation on antioxidant enzymes, we examined activity of four antioxidant enzymes: catalase (CAT), peroxidase (POD), ascorbate peroxidase, and superoxide dismutase. In general, POD and CAT activities decreased in response to acute and chronic γ-irradiation. Oligonucleotide microarrays were used to investigate transcriptional changes after irradiation. Several genes related to reactive oxygen species signaling were up-regulated after acute and chronic exposure, including genes encoding heat shock factors, zinc finger proteins, NADPH oxidase, WRKY DNA-binding proteins, and calcium binding proteins. Taken together, our data indicate that the responses and activation of antioxidant systems prompted by irradiation exposure are dependent upon the γ-ray dose rate.

Differentially Expressed Genes Related to Isoflavone Biosynthesis in a Soybean Mutant Revealed by a Comparative Transcriptomic Analysis.

Soybean [Glycine max (L.) Merr.] isoflavones, which are secondary metabolites with various functions, are included in food, cosmetics, and medicine. However, the molecular mechanisms regulating the glycosylation and malonylation of isoflavone glycoconjugates remain unclear. In this study, we conducted an RNA-seq analysis to compare soybean genotypes with different isoflavone contents, including Danbaek and Hwanggeum (low-isoflavone cultivars) as well as DB-088 (high-isoflavone mutant). The transcriptome analysis yielded over 278 million clean reads, representing 39,156 transcripts. The analysis of differentially expressed genes (DEGs) detected 2654 up-regulated and 1805 down-regulated genes between the low- and high-isoflavone genotypes. The putative functions of these 4459 DEGs were annotated on the basis of GO and KEGG pathway enrichment analyses. These DEGs were further analyzed to compare the expression patterns of the genes involved in the biosynthesis of secondary metabolites and the genes encoding transcription factors. The examination of the relative expression levels of 70 isoflavone biosynthetic genes revealed the HID, IFS, UGT, and MAT expression levels were significantly up/down-regulated depending on the genotype and seed developmental stage. These expression patterns were confirmed by quantitative real-time PCR. Moreover, a gene co-expression analysis detected potential protein-protein interactions, suggestive of common functions. The study findings provide valuable insights into the structural genes responsible for isoflavone biosynthesis and accumulation in soybean seeds.

Transcriptome Profiling of a Soybean Mutant with Salt Tolerance Induced by Gamma-ray Irradiation.

Salt stress is a significant abiotic stress that reduces crop yield and quality globally. In this study, we utilized RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs) in response to salt stress induced by gamma-ray irradiation in a salt-tolerant soybean mutant. The total RNA library samples were obtained from the salt-sensitive soybean cultivar Kwangan and the salt-tolerant mutant KA-1285. Samples were taken at three time points (0, 24, and 72 h) from two tissues (leaves and roots) under 200 mM NaCl. A total of 967,719,358 clean reads were generated using the Illumina NovaSeq 6000 platform, and 94.48% of these reads were mapped to 56,044 gene models of the soybean reference genome (Glycine_max_Wm82.a2.v1). The DEGs with expression values were compared at each time point within each tissue between the two soybeans. As a result, 296 DEGs were identified in the leaves, while 170 DEGs were identified in the roots. In the case of the leaves, eight DEGs were related to the phenylpropanoid biosynthesis pathway; however, in the roots, Glyma.03G171700 within GmSalt3, a major QTL associated with salt tolerance in soybean plants, was differentially expressed. Overall, these differences may explain the mechanisms through which mutants exhibit enhanced tolerance to salt stress, and they may provide a basic understanding of salt tolerance in soybean plants.