Transcriptome Analysis Reveals the Genes Related to Pollen Abortion in a Cytoplasmic Male-Sterile Soybean (Glycine max (L.) Merr.).

Cytoplasmic male sterility (CMS) lays a foundation for the utilization of heterosis in soybean. The soybean CMS line SXCMS5A is an excellent CMS line exhibiting 100% male sterility. Cytological analysis revealed that in SXCMS5A compared to its maintainer SXCMS5B, its tapetum was vacuolated and abnormally developed. To identify the genes and metabolic pathways involving in pollen abortion of SXCMS5A, a comparative transcriptome analysis was conducted between SXCMS5A and SXCMS5B using flower buds. A total of 372,973,796 high quality clean reads were obtained from 6 samples (3 replicates for each material), and 840 differentially expressed genes (DEGs) were identified, including 658 downregulated and 182 upregulated ones in SXCMS5A compared to SXCMS5B. Among them, 13 DEGs, i.e., 12 open reading frames (ORFs) and 1 COX2, were mitochondrial genome genes in which ORF178 and ORF103c were upregulated in CMS lines and had transmembrane domain(s), therefore, identified as CMS candidate mitochondrial genes of SXCMS5A. Furthermore, numerous DEGs were associated with pollen wall development, carbohydrate metabolism, sugar transport, reactive oxygen species (ROS) metabolism and transcription factor. Some of them were further confirmed by quantitative real time PCR analysis between CMS lines with the same cytoplasmic source as SXCMS5A and their respective maintainer lines. The amount of soluble sugar and adenosine triphosphate and the activity of catalase and ascorbic acid oxidase showed that energy supply and ROS scavenging decreased in SXCMS5A compared to SXCMS5B. These findings provide valuable information for further understanding the molecular mechanism regulating the pollen abortion of soybean CMS.

Discovery and characterization of differentially expressed soybean miRNAs and their targets during soybean mosaic virus infection unveils novel insight into Soybean-SMV interaction.

BACKGROUND: Soybean mosaic virus (SMV) is one of the most devastating pathogens of soybean. MicroRNAs (miRNAs) are a class of non-coding RNAs (21-24 nucleotides) which are endogenously produced by the plant host as part of a general gene expression regulatory mechanisms, but also play roles in regulating plant defense against pathogens. However, miRNA-mediated plant response to SMV in soybean is not as well documented. RESULT: In this study, we analyzed 18 miRNA libraries, including three biological replicates from two soybean lines (Resistant and susceptible lines to SMV strain SC3 selected from the near-isogenic lines of Qihuang No. 1 × Nannong1138-2) after virus infection at three different time intervals (0 dpi, 7 dpi and 14 dpi). A total of 1,092 miRNAs, including 608 known miRNAs and 484 novel miRNAs were detected. Differential expression analyses identified the miRNAs profile changes during soybean-SMV interaction. Then, miRNAs potential target genes were predicted via data mining, and functional annotation was done by Gene Ontology (GO) analysis. The expression patterns of several miRNAs were validated by quantitative real-time PCR. We also validated the miRNA-target gene interaction by agrobacterium-mediated transient expression in Nicotiana benthamiana. CONCLUSION: We have identified a large number of miRNAs and their target genes and also functional annotations. We found that multiple miRNAs were differentially expressed in the two lines and targeted a series of NBS-LRR resistance genes. It is worth mentioning that many of these genes exist in the previous fine-mapping interval of the resistance gene locus. Our study provides additional information on soybean miRNAs and an insight into the role of miRNAs during SMV-infection in soybean.

Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean.

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Comparative Transcriptome Analysis of Two Contrasting Soybean Varieties in Response to Aluminum Toxicity.

: Aluminum (Al) toxicity is a major factor limiting crop productivity on acid soils. Soybean (Glycine max) is an important oil crop and there is great variation in Al tolerance in soybean germplasms. However, only a few Al-tolerance genes have been reported in soybean. Therefore, the purpose of this study was to identify candidate Al tolerance genes by comparative transcriptome analysis of two contrasting soybean varieties in response to Al stress. Two soybean varieties, M90-24 (M) and Pella (P), which showed significant difference in Al tolerance, were used for RNA-seq analysis. We identified a total of 354 Al-tolerance related genes, which showed up-regulated expression by Al in the Al-tolerant soybean variety M and higher transcript levels in M than P under Al stress. These genes were enriched in the Gene Ontology (GO) terms of cellular glucan metabolic process and regulation of transcription. Five out of 11 genes in the enriched GO term of cellular glucan metabolic process encode cellulose synthases, and one cellulose synthase gene (Glyma.02G205800) was identified as the key hub gene by co-expression network analysis. Furthermore, treatment of soybean roots with a cellulose biosynthesis inhibitor decreased the Al tolerance, indicating an important role of cellulose production in soybean tolerance to Al toxicity. This study provides a list of candidate genes for further investigation on Al tolerance mechanisms in soybean.

Double mutation of two homologous genes YL1 and YL2 results in a leaf yellowing phenotype in soybean [Glycine max (L.) Merr].

KEY MESSAGE: Two homologous, chloroplast located CAAX proteases were identified to be functional redundancy in determining soybean leaf color, and they probably play essential roles in regulating light harvesting and absorption during photosynthesis process. Leaf color mutants are ideal materials for studying photosynthesis and chlorophyll metabolism. The soybean [Glycine max (L.) Merr.] yellowing leaf (yl) variation is a recombinant mutant characterized by yellow foliage, which derived from the specific cross between green seed-coated and yellow seed-coated soybean varieties. Molecular cloning and subsequent gene silencing revealed that the yellow leaf trait of yl was controlled by two recessive nuclear genes, glyma11g04660 and glyma01g40650, named as YL1 and YL2 respectively, and the latter was confirmed to be same as the earlier reported green seed-coat gene G. Both YL1 and YL2 belonged to chloroplast-located proteases possessing Abi domain, and these genes were expressed in various tissues, especially in young leaves. In yl, the expression of YL1 and YL2 were suppressed in most tissues, and the young leaf of yl presented an increased maximal photochemical efficiency (Fv/Fm) as well as enhanced net photosynthesis activity (Pn), indicating that YL1 and YL2 are involved in light absorption regulation during photosynthesis process. Collectively, the identification and description of YL1 and YL2 in our study provides insights for the regulatory mechanism of photosynthesis process, and these findings will further assist to clarify the close relationship between photosynthesis and chlorophyll metabolism.

Natural variation and selection in GmSWEET39 affect soybean seed oil content.

Soybean (Glycine max) is a major contributor to the world oilseed production. Its seed oil content has been increased through soybean domestication and improvement. However, the genes underlying the selection are largely unknown. The present contribution analyzed the expression patterns of genes in the seed oil quantitative trait loci with strong selective sweep signals, then used association, functional study and population genetics to reveal a sucrose efflux transporter gene, GmSWEET39, controlling soybean seed oil content and under selection. GmSWEET39 is highly expressed in soybean seeds and encodes a plasma membrane-localized protein. Its expression level is positively correlated with soybean seed oil content. The variation in its promoter and coding sequence leads to different natural alleles of this gene. The GmSWEET39 allelic effects on total oil content were confirmed in the seeds of soybean recombinant inbred lines, transgenic Arabidopsis, and transgenic soybean hairy roots. The frequencies of its superior alleles increased from wild soybean to cultivated soybean, and are much higher in released soybean cultivars. The findings herein suggest that the sequence variation in GmSWEET39 affects its relative expression and oil content in soybean seeds, and GmSWEET39 has been selected to increase seed oil content during soybean domestication and improvement.

Overexpression of Peroxidase Gene GsPRX9 Confers Salt Tolerance in Soybean.

Peroxidases play prominent roles in antioxidant responses and stress tolerance in plants; however, their functions in soybean tolerance to salt stress remain unclear. Here, we investigated the role of a peroxidase gene from the wild soybean (Glycine soja), GsPRX9, in soybean tolerance to salt stress. GsPRX9 gene expression was induced by salt treatment in the roots of both salt-tolerant and -sensitive soybean varieties, and its relative expression level in the roots of salt-tolerant soybean varieties showed a significantly higher increase than in salt-sensitive varieties after NaCl treatment, suggesting its possible role in soybean response to salt stress. GsPRX9-overexpressing yeast (strains of INVSc1 and G19) grew better than the control under salt and H2O2 stress, and GsPRX9-overexpressing soybean composite plants showed higher shoot fresh weight and leaf relative water content than control plants after NaCl treatment. Moreover, the GsPRX9-overexpressing soybean hairy roots had higher root fresh weight, primary root length, activities of peroxidase and superoxide dismutase, and glutathione level, but lower H2O2 content than those in control roots under salt stress. These findings suggest that the overexpression of the GsPRX9 gene enhanced the salt tolerance and antioxidant response in soybean. This study would provide new insights into the role of peroxidase in plant tolerance to salt stress.

Identifying a wild allele conferring small seed size, high protein content and low oil content using chromosome segment substitution lines in soybean.

KEY MESSAGE: A wild soybean allele conferring 100-seed weight, protein content and oil content simultaneously was fine-mapped to a 329-kb region on Chromosome 15, in which Glyma.15g049200 was predicted a candidate gene. Annual wild soybean characterized with small 100-seed weight (100SW), high protein content (PRC), low oil content (OIC) may contain favourable alleles for broadening the genetic base of cultivated soybeans. To evaluate these alleles, a population composed of 195 chromosome segment substitution lines (SojaCSSLP4), with wild N24852 as donor and cultivated NN1138-2 as recurrent parent, was tested. In SojaCSSLP4, 10, 9 and 8 wild segments/QTL were detected for 100SW, PRC and OIC, respectively. Using a backcross-derived secondary population, one segment for the three traits (q100SW15, qPro15 and qOil15) and one for 100SW (q100SW18.2) were fine-mapped into a 329-kb region on chromosome 15 and a 286-kb region on chromosome 18, respectively. Integrated with the transcription data in SoyBase, 42 genes were predicted in the 329-kb region where Glyma.15g049200 showed significant expression differences at all seed development stages. Furthermore, the Glyma.15g049200 segments of the two parents were sequenced and compared, which showed two base insertions in CDS (coding sequence) in the wild N24852 comparing to the NN1138-2. Since only Glyma.15g049200 performed differential CDS between the two parents but related to the three traits, Glyma.15g049200 was predicted a pleiotropic candidate gene for 100SW, PRC and OIC. The functional annotation of Glyma.15g049200 indicated a bidirectional sucrose transporter belonging to MtN3/saliva family which might be the reason that this gene provides a same biochemical basis for 100SW, PRC and OIC, therefore, is responsible for the three traits. This result may facilitate isolation of the specific gene and provide prerequisite for understanding the other two pleiotropic QTL.

Dynamic Transcriptome Changes Related to Oil Accumulation in Developing Soybean Seeds.

Soybean is one of the most important oil crops in the world. Revealing the molecular basis and exploring key candidate genes for seed oil synthesis has great significance for soybean improvement. In this study, we found that oil accumulation rates and gene expression levels changed dynamically during soybean seed development. The expression levels of genes in metabolic pathways such as carbon fixation, photosynthesis, glycolysis, and fatty acid biosynthesis were significantly up-regulated during the rapid accumulation of oil in developing soybean seeds. Through weighted correlation network analysis, we identified six co-expression modules associated with soybean seed oil content and the pink module was the most positively correlated (r = 0.83, p = 7 × 10-4) network. Through the integration of differential expression and co-expression analysis, we predicted 124 candidate genes potentially affecting soybean seed oil content, including seven genes in lipid metabolism pathway, two genes involved in glycolysis, one gene in sucrose metabolism, and 12 genes belonged to transcription factors as well as other categories. Among these, three genes (GmABI3b, GmNFYA and GmFAD2-1B) have been shown to control oil and fatty acid content in soybean seeds, and other newly identified candidate genes would broaden our knowledge to understand the molecular basis for oil accumulation in soybean seeds.

Genome-Wide Detection of Major and Epistatic Effect QTLs for Seed Protein and Oil Content in Soybean Under Multiple Environments Using High-Density Bin Map.

Seed protein and oil content are the two important traits determining the quality and value of soybean. Development of improved cultivars requires detailed understanding of the genetic basis underlying the trait of interest. However, it is prerequisite to have a high-density linkage map for precisely mapping genomic regions, and therefore the present study used high-density genetic map containing 2267 recombination bin markers distributed on 20 chromosomes and spanned 2453.79 cM with an average distance of 1.08 cM between markers using restriction-site-associated DNA sequencing (RAD-seq) approach. A recombinant inbred line (RIL) population of 104 lines derived from a cross between Linhefenqingdou and Meng 8206 cultivars was evaluated in six different environments to identify main- and epistatic-effect quantitative trait loci (QTLs)as well as their interaction with environments. A total of 44 main-effect QTLs for protein and oil content were found to be distributed on 17 chromosomes, and 15 novel QTL were identified for the first time. Out of these QTLs, four were major and stable QTLs, viz., qPro-7-1, qOil-8-3, qOil-10-2 and qOil-10-4, detected in at least two environments plus combined environment with R² values >10%. Within the physical intervals of these four QTLs, 111 candidate genes were screened for their direct or indirect involvement in seed protein and oil biosynthesis/metabolism processes based on gene ontology and annotation information. Based on RNA sequencing (RNA-seq) data analysis, 15 of the 111 genes were highly expressed during seed development stage and root nodules that might be considered as the potential candidate genes. Seven QTLs associated with protein and oil content exhibited significant additive and additive × environment interaction effects, and environment-independent QTLs revealed higher additive effects. Moreover, three digenic epistatic QTLs pairs were identified, and no main-effect QTLs showed epistasis. In conclusion, the use of a high-density map identified closely linked flanking markers, provided better understanding of genetic architecture and candidate gene information, and revealed the scope available for improvement of soybean quality through marker assisted selection (MAS).

Detecting the QTL-Allele System of Seed Oil Traits Using Multi-Locus Genome-Wide Association Analysis for Population Characterization and Optimal Cross Prediction in Soybean.

Soybean is one of the world's major vegetative oil sources, while oleic acid and linolenic acid content are the major quality traits of soybean oil. The restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS), characterized with error and false-positive control, has provided a potential approach for a relatively thorough detection of whole-genome QTL-alleles. The Chinese soybean landrace population (CSLRP) composed of 366 accessions was tested under four environments to identify the QTL-allele constitution of seed oil, oleic acid and linolenic acid content (SOC, OAC, and LAC). Using RTM-GWAS with 29,119 SNPLDBs (SNP linkage disequilibrium blocks) as genomic markers, 50, 98, and 50 QTLs with 136, 283, and 154 alleles (2-9 per locus) were detected, with their contribution 82.52, 90.31, and 83.86% to phenotypic variance, corresponding to their heritability 91.29, 90.97, and 90.24% for SOC, OAC, and LAC, respectively. The RTM-GWAS was shown to be more powerful and efficient than previous single-locus model GWAS procedures. For each trait, the detected QTL-alleles were organized into a QTL-allele matrix as the population genetic constitution. From which the genetic differentiation among 6 eco-populations was characterized as significant allele frequency differentiation on 28, 56, and 30 loci for the three traits, respectively. The QTL-allele matrices were also used for genomic selection for optimal crosses, which predicted transgressive potential up to 24.76, 40.30, and 2.37% for the respective traits, respectively. From the detected major QTLs, 38, 27, and 25 candidate genes were annotated for the respective traits, and two common QTL covering eight genes were identified for further study.

Comparative analysis of circular RNAs between soybean cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B by high-throughput sequencing.

BACKGROUND: Cytoplasmic male sterility (CMS) is a natural phenomenon of pollen abortion caused by the interaction between cytoplasmic genes and nuclear genes. CMS is a simple and effective pollination control system, and plays an important role in crop heterosis utilization. Circular RNAs (circRNAs) are a vital type of non-coding RNAs, which play crucial roles in microRNAs (miRNAs) function and post-transcription control. To explore the expression profile and possible functions of circRNAs in the soybean CMS line NJCMS1A and its maintainer NJCMS1B, high-throughput deep sequencing coupled with RNase R enrichment strategy was conducted. RESULTS: CircRNA libraries were constructed from flower buds of NJCMS1A and its maintainer NJCMS1B with three biological replicates. A total of 2867 circRNAs were identified, with 1009 circRNAs differentially expressed between NJCMS1A and NJCMS1B based on analysis of high-throughput sequencing. Of the 12 randomly selected circRNAs with different expression levels, 10 showed consistent expression patterns based on high-throughput sequencing and quantitative real-time PCR analyses. Tissue specific expression patterns were also verified with two circRNAs by quantitative real-time PCR. Most parental genes of differentially expressed circRNAs were mainly involved in biological processes such as metabolic process, biological regulation, and reproductive process. Moreover, 83 miRNAs were predicted from the differentially expressed circRNAs, some of which were strongly related to pollen development and male fertility; The functions of miRNA targets were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and the target mRNAs were significantly enriched in signal transduction and programmed cell death. Furthermore, a total of 165 soybean circRNAs were predicted to contain at least one internal ribosome entry site (IRES) element and an open reading frame, indicating their potential to encode polypeptides or proteins. CONCLUSIONS: Our study indicated that the circRNAs might participate in the regulation of flower and pollen development, which could provide a new insight into the molecular mechanisms of CMS in soybean.

Chicken Toes-Like Leaf and Petalody Flower (CTP) is a novel regulator that controls leaf and flower development in soybean.

A soybean mutant displaying chicken toes-like leaves and petalody flowers was identified as being caused by a single recessive gene, termed ctp. Using heterozygous-inbred recombinants, this gene was fine-mapped to a 37-kb region harbouring three predicted genes on chromosome 5. The gene Glyma05g022400.1 was detected to have a 33-bp deletion in its first exon that was responsible for ctp. Validation for this gene was provided by the fact that the 33-bp deletion-derived marker I2 completely co-segregated with the phenotypes of 96 F10-derived residual heterozygous lines and 2200 fine-mapping individuals, and by the fact that the orthologue NbCTP in Nicotiana benthamiana also influenced leaf and flower development under virus-induced gene silencing. In terms of characterization, the CTPs shared highly conserved domains specifically in higher plants, GmCTP was alternatively spliced, and it was expressed in multiple organs, especially in lateral meristems. GmCTP was localized to the nucleus and other regions and performed transcriptional activity. In ctp, the expression levels and splicing patterns were dramatically disrupted, and many key regulators in leaf and/or floral developmental pathways were interrupted. Thus, CTP is a novel and critical pleiotropic regulator of leaf and flower development that participates in multiple regulation pathways, and may play key roles in lateral organ differentiation as a putative novel transcription regulator.

Identification of Major Quantitative Trait Loci for Seed Oil Content in Soybeans by Combining Linkage and Genome-Wide Association Mapping.

Soybean oil is the most widely produced vegetable oil in the world and its content in soybean seed is an important quality trait in breeding programs. More than 100 quantitative trait loci (QTLs) for soybean oil content have been identified. However, most of them are genotype specific and/or environment sensitive. Here, we used both a linkage and association mapping methodology to dissect the genetic basis of seed oil content of Chinese soybean cultivars in various environments in the Jiang-Huai River Valley. One recombinant inbred line (RIL) population (NJMN-RIL), with 104 lines developed from a cross between M8108 and NN1138-2, was planted in five environments to investigate phenotypic data, and a new genetic map with 2,062 specific-locus amplified fragment markers was constructed to map oil content QTLs. A derived F2 population between MN-5 (a line of NJMN-RIL) and NN1138-2 was also developed to confirm one major QTL. A soybean breeding germplasm population (279 lines) was established to perform a genome-wide association study (GWAS) using 59,845 high-quality single nucleotide polymorphism markers. In the NJMN-RIL population, 8 QTLs were found that explained a range of phenotypic variance from 6.3 to 26.3% in certain planting environments. Among them, qOil-5-1, qOil-10-1, and qOil-14-1 were detected in different environments, and qOil-5-1 was further confirmed using the secondary F2 population. Three loci located on chromosomes 5 and 20 were detected in a 2-year long GWAS, and one locus that overlapped with qOil-5-1 was found repeatedly and treated as the same locus. qOil-5-1 was further localized to a linkage disequilibrium block region of approximately 440 kb. These results will not only increase our understanding of the genetic control of seed oil content in soybean, but will also be helpful in marker-assisted selection for breeding high seed oil content soybean and gene cloning to elucidate the mechanisms of seed oil content.

Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

Characterization of a soybean mosaic virus variant causing different diseases in Glycine max and Nicotiana benthamiana.

We discovered a soybean mosaic virus (SMV) variant (4278-1) that caused systemic infections in Nicotiana benthamiana plants, resulting in stem stunting and leaf shriveling. The virus had a particle morphology and incubation period similar to those of other SMV isolates but differed from them in the leaf symptoms it caused when infecting soybean and N. benthamiana. The genome of this variant consisted of a 9994-nt single-stranded RNA, which was different from most of the other known SMV isolates (approximately 9600 nt). Interestingly, we found evidence that two recombination events (nt 1-476 and nt 1145-1349) had occurred between 4278-1 and a watermelon mosaic virus analogue (WMV analogue), in the 5' untranslated region and the P1 cistron.

Different responses of soybean cyst nematode resistance between two RIL populations derived from Peking x 7605 under two ecological sites.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a plant-parasitic nematode that feeds on the roots of soybean and most economically devastating pathogen of soybean (Glycine max (L.) Merr.) worldwide. Host plant resistance is the most effective control method. To understand SCN resistance in different environments, two recombinant-inbred lines (RILs) populations NJ(RN)P7 (217 F2:8:11 lines) and JN(RN)P7 (248 F2:7:9 lines) were developed from the cross of the cultivars Peking x 7605 in Nanjing and Jinan, respectively, and examined in this study. Peking is resistant to SCN race 1 (HG types 2.5.7), while 7605 is highly susceptible. Chi-square test of frequency distribution of families' female index (FI) showed that resistance to SCN was significantly different between NJ(RN)P7 and JN(RN)P7 populations. Three recessive genes conditioned the inheritance of resistance to SCN race 1 in both populations, but significant difference was detected for the mean of FI on two populations (DM= -16.68, P< 0.01). This indicated that natural selection may affect resistance to SCN. By analysing the variation of phenotype, the genetic structure of the two populations was determined to be different. The inheritance and variation of resistance were confirmed by simple sequence repeat (SSR) markers. For the two populations, 10 SSR markers showed polymorphism of resistant and susceptible DNA bulks. Some markers associated with the resistance of SCN races 1, 2, 3 and 5, and two markers, Satt163 and Satt309, reportedly related to rgh1 were detected both in NJ(RN)P7 and JN(RN)P7 populations. The results support the view that a disease acts as a selective force on plant resistance characteristics, which may alter the relative fitness of resistance alleles.

Genome-wide analysis of MATE transporters and expression patterns of a subgroup of MATE genes in response to aluminum toxicity in soybean.

BACKGROUND: Multidrug and toxic compound extrusion (MATE) family is an important group of the multidrug efflux transporters that extrude organic compounds, transporting a broad range of substrates such as organic acids, plant hormones and secondary metabolites. However, genome-wide analysis of MATE family in plant species is limited and no such studies have been reported in soybean. RESULTS: A total of 117 genes encoding MATE transporters were identified from the whole genome sequence of soybean (Glycine max), which were denominated as GmMATE1 - GmMATE117. These 117 GmMATE genes were unevenly localized on soybean chromosomes 1 to 20, with both tandem and segmental duplication events detected, and most genes showed tissue-specific expression patterns. Soybean MATE family could be classified into four subfamilies comprising ten smaller subgroups, with diverse potential functions such as transport and accumulation of flavonoids or alkaloids, extrusion of plant-derived or xenobiotic compounds, regulation of disease resistance, and response to abiotic stresses. Eight soybean MATE transporters clustered together with the previously reported MATE proteins related to aluminum (Al) detoxification and iron translocation were further analyzed. Seven stress-responsive cis-elements such as ABRE, ARE, HSE, LTR, MBS, as well as a cis-element of ART1 (Al resistance transcription factor 1), GGNVS, were identified in the upstream region of these eight GmMATE genes. Differential gene expression analysis of these eight GmMATE genes in response to Al stress helps us identify GmMATE75 as the candidate gene for Al tolerance in soybean, whose relative transcript abundance increased at 6, 12 and 24 h after Al treatment, with more fold changes in Al-tolerant than Al-sensitive cultivar, which is consistent with previously reported Al-tolerance related MATE genes. CONCLUSIONS: A total of 117 MATE transporters were identified in soybean and their potential functions were proposed by phylogenetic analysis with known plant MATE transporters. The cis-elements and expression patterns of eight soybean MATE genes related to Al detoxification/iron translocation were analyzed, and GmMATE75 was identified as a candidate gene for Al tolerance in soybean. This study provides a first insight on soybean MATE family and their potential roles in soybean response to abiotic stresses especially Al toxicity.

Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2-4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.