Harnessing the power of microbes: Enhancing soybean growth in an acidic soil through AMF inoculation rather than P-fertilization.

The low phosphorus (P) availability of acidic soils severely limits leguminous plant growth and productivity. Improving the soil P nutritional status can be achieved by increasing the P-content through P-fertilization or stimulating the mineralization of organic P via arbuscular mycorrhizal fungi (AMF) application; however, their corresponding impacts on plant and soil microbiome still remain to be explored. Here, we examined the effects of AMF-inoculation and P-fertilization on the growth of soybean with different P-efficiencies, as well as the composition of rhizo-microbiome in an acidic soil. The growth of recipient soybean NY-1001, which has a lower P-efficiency, was not significantly enhanced by AMF-inoculation or P-fertilization. However, the plant biomass of higher P-efficiency transgenic soybean PT6 was significantly increased by 46.74%-65.22% through AMF-inoculation. Although there was no discernible difference in plant biomass between PT6 and NY-1001 in the absence of AMF-inoculation and P-fertilization, PT6 had approximately 1.9-2.5 times the plant biomass of NY-1001 after AMF-inoculation. Therefore, the growth advantage of higher P-efficiency soybean was achieved through the assistance of AMF rather than P-fertilization in available P-deficient acidic soil. Most nitrogen (N)-fixing bacteria and some functional genes related to N-fixation were abundant in endospheric layer, as were the P-solubilizing Pseudomonas plecoglossicida, and annotated P-metabolism genes. These N-fixing and P-solubilizing bacteria were positive correlated with each other. Lastly, the two most abundant phytopathogenic fungi species accumulated in endospheric layer, they exhibited positive correlations with N-fixing bacteria, but displayed negative interactions with the majority of the other dominant non-pathogenic genera with potential antagonistic activity.

A novel in-situ-process technique constructs whole circular cpDNA library.

BACKGROUND: The chloroplast genome (cp genome) is directly related to the study and analysis of molecular phylogeny and evolution of plants in the phylogenomics era. The cp genome, whereas, is highly plastic and exists as a heterogeneous mixture of sizes and physical conformations. It is advantageous to purify/enrich the circular chloroplast DNA (cpDNA) to reduce sequence complexity in cp genome research. Large-insert, ordered DNA libraries are more practical for genomics research than conventional, unordered ones. From this, a technique of constructing the ordered BAC library with the goal-insert cpDNA fragment is developed in this paper. RESULTS: This novel in-situ-process technique will efficiently extract circular cpDNA from crops and construct a high-quality cpDNA library. The protocol combines the in-situ chloroplast lysis for the high-purity circular cpDNA with the in-situ substitute/ligation for the high-quality cpDNA library. Individually, a series of original buffers/solutions and optimized procedures for chloroplast lysis in-situ is different than bacterial lysis in-situ; the in-situ substitute/ligation that reacts on the MCE membrane is suitable for constructing the goal-insert, ordered cpDNA library while preventing the large-insert cpDNA fragment breakage. The goal-insert, ordered cpDNA library is arrayed on the microtiter plate by three colonies with the definite cpDNA fragment that are homologous-corresponds to the whole circular cpDNA of the chloroplast. CONCLUSION: The novel in-situ-process technique amply furtherance of research in genome-wide functional analysis and characterization of chloroplasts, such as genome sequencing, bioinformatics analysis, cloning, physical mapping, molecular phylogeny and evolution.

Multi-enzymatic systems synergize new RCA technique amplified super-long dsDNA from DNA circle.

BACKGROUND: In the field of DNA amplification, there are great challenges in the effectively amplify of long-chain amplification, especially amplification up to several hundred kb level. RESULTS: A novel technique for the unbiased whole genome amplification from a thimbleful of DNA circles, such as low as 10 ng/ 10 µL of the circular cpDNA or low as 5 ng/ 10 µL of the plasmid, is developed, which can amplify an abundance of the whole genome sequences. Specifically, the new technique that combines rolling-amplification and triple-enzyme system presents a tightly controlled process of a series of buffers/reactions and optimized procedures, that applies from the primer-template duplexes to the Elution step. The result of this technique provides a new approach for extending RCA capacity, where it can reach 200 kb from the circular cpDNA amplification and 150 kb from the plasmid DNA amplification, that demonstrates superior breadth and evenness of genome coverage, high reproducibility, small amplification bias with the amplification efficiency. SIGNIFICANCE AND NOVELTY: This new technique will develop into one of the powerful tools for isothermal DNA amplification in vitro, genome sequencing/analysis, phylogenetic analysis, physical mapping, and other molecular biology applications.

A small heat shock protein GmHSP18.5a improves the male fertility restorability of cytoplasmic male sterility-based restorer line under high temperature stress in soybean.

Small heat shock protein (sHSP) is involved in high temperature (HT) stress response. However, the function of sHSPs in regulating male fertility of soybean under HT stress remains largely unknown. Here, we identified a sHSP gene, GmHSP18.5a, which was responded to HT stress during flowering in cytoplasmic male sterility (CMS)-based restorer line of soybean. Moreover, GmHSFA6b turned out to directly activated the expression of GmHSP18.5a by binding to the heat shock cis-element in its promoter. Overexpression of GmHSP18.5a increased male fertility in transgenic Arabidopsis, soybean CMS-based restorer line and its hybrid F1 with CMS line under HT stress. Reactive oxygen species (ROS) content detection revealed that GmHSP18.5a promoted the ROS scavenging ability of Arabidopsis inflorescence and soybean flower bud under HT stress. Enzyme activity assay and gene expression analysis indicated that GmHS18.5a mainly increased the activity of antioxidant enzymes and the expression level of ROS metabolism-related genes under HT stress. Our results indicated that GmHSP18.5a improved the male fertility restorability of CMS-based restorer line in soybean by regulating ROS metabolic pathway and reducing ROS accumulation. Our findings not only revealed the molecular mechanism of sHSP regulating the male fertility of soybean under HT stress, but also provided a theoretical basis for creating strong restorer line with thermotolerance.

The miR156b-GmSPL2b module mediates male fertility regulation of cytoplasmic male sterility-based restorer line under high-temperature stress in soybean.

High-temperature (HT) stress at flowering stage causes significant damage to soybean, including pollen abortion and fertilization failure, but few genes involved in male fertility regulation under HT stress in soybean have been characterized. Here, we demonstrated that miR156b-GmSPL2b module involved in male fertility regulation of soybean cytoplasmic male sterility (CMS)-based restorer line under HT stress. Overexpression of miR156b decreased male fertility in soybean CMS-based restorer line and its hybrid F1 with CMS line under HT stress. RNA-seq analysis found that miR156b mediated male fertility regulation in soybean under HT stress by regulating the expression of pollen development and HT response related genes. Metabolomic analysis of miR156bOE revealed reduction in flavonoid content under HT stress. Integrated transcriptomic and metabolomic analysis showed that the overexpression of miR156b caused flavonoid metabolism disorder in soybean flower bud under HT stress. Knockout of GmSPL2b also decreased the thermotolerance of soybean CMS-based restorer line during flowering. Moreover, GmSPL2b turned out to be directly bounded to the promoter of GmHSFA6b. Further verification indicated that GmHSFA6b overexpression enhanced HT tolerance in Arabidopsis during flowering. Substance content and gene expression analysis revealed that miR156b-GmSPL2b may mediate reactive oxygen species clearance by regulating flavonoid metabolism, thus participating in the regulation of male fertility in soybean under HT stress. This study not only provided important progress for understanding the molecular mechanism of miR156b-GmSPL2b regulating the male fertility of soybean CMS-based restorer line under HT stress, but also provided genetic resources and theoretical basis for creating HT-tolerant strong restorer lines.

Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L.

Brassica napus L. (canola, oil seed rape) is one of the world's most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants' vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study's findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

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.

Genotype imputation for soybean nested association mapping population to improve precision of QTL detection.

KEY MESSAGE: Software for high imputation accuracy in soybean was identified. Imputed dataset could significantly reduce the interval of genomic regions controlling traits, thus greatly improve the efficiency of candidate gene identification. Genotype imputation is a strategy to increase marker density of existing datasets without additional genotyping. We compared imputation performance of software BEAGLE 5.0, IMPUTE 5 and AlphaPlantImpute and tested software parameters that may help to improve imputation accuracy in soybean populations. Several factors including marker density, extent of linkage disequilibrium (LD), minor allele frequency (MAF), etc., were examined for their effects on imputation accuracy across different software. Our results showed that AlphaPlantImpute had a higher imputation accuracy than BEAGLE 5.0 or IMPUTE 5 tested in each soybean family, especially if the study progeny were genotyped with an extremely low number of markers. LD extent, MAF and reference panel size were positively correlated with imputation accuracy, a minimum number of 50 markers per chromosome and MAF of SNPs > 0.2 in soybean line were required to avoid a significant loss of imputation accuracy. Using the software, we imputed 5176 soybean lines in the soybean nested mapping population (NAM) with high-density markers of the 40 parents. The dataset containing 423,419 markers for 5176 lines and 40 parents was deposited at the Soybase. The imputed NAM dataset was further examined for the improvement of mapping quantitative trait loci (QTL) controlling soybean seed protein content. Most of the QTL identified were at identical or at similar position based on initial and imputed datasets; however, QTL intervals were greatly narrowed. The resulting genotypic dataset of NAM population will facilitate QTL mapping of traits and downstream applications. The information will also help to improve genotyping imputation accuracy in self-pollinated crops.

Genome-wide identification of PME genes, evolution and expression analyses in soybean (Glycine max L.).

BACKGROUND: Pectin methylesterase (PME) is one of pectin-modifying enzyme that affects the pectin homeostasis in cell wall and regulates plant growth and diverse biological processes. The PME genes have been well explored and characterized in different plants. Nevertheless, systematic research on the soybean (Glycine max L.) PME genes remain lacking. RESULTS: We identified 127 Glycine max PME genes (GmPME) from the soybean Wm82.a2.v1 genome, which unevenly distributed on 20 soybean chromosomes. Phylogenetic analysis classified the GmPME genes into four clades (Group I, Group II, Group III and Group IV). GmPME gene members in the same clades displayed similar gene structures and motif patterns. The gene family expansion analysis demonstrated that segmental duplication was the major driving force to acquire novel GmPME genes compared to the tandem duplication events. Further synteny and evolution analyses showed that the GmPME gene family experienced strong purifying selective pressures during evolution. The cis-element analyses together with the expression patterns of the GmPME genes in various tissues suggested that the GmPME genes broadly participate in distinct biological processes and regulate soybean developments. Importantly, based on the transcriptome data and quantitative RT-PCR validations, we examined the potential roles of the GmPME genes in regulating soybean flower bud development and seed germination. CONCLUSION: In conclusion, we provided a comprehensive characterization of the PME genes in soybean, and our work laid a foundation for the functional study of GmPME genes in the future.

Transcription Factor GmWRKY46 Enhanced Phosphate Starvation Tolerance and Root Development in Transgenic Plants.

Phosphorus (P) is one of the essential macronutrients, whose deficiency limits the growth and development of plants. In this study, we investigated the possible role of GmWRKY46 in the phosphate (Pi) starvation stress tolerance of soybean. GmWRKY46 belonged to the group III subfamily of the WRKY transcription factor family, which was localized in the nucleus and had transcriptional activator activity. GmWRKY46 could be strongly induced by Pi starvation, especially in soybean roots. Overexpression of GmWRKY46 significantly enhanced tolerance to Pi starvation and lateral root development in transgenic Arabidopsis. RNA-seq analysis showed that overexpression of GmWRKY46 led to change in many genes related to energy metabolisms, stress responses, and plant hormone signal transduction in transgenic Arabidopsis. Among these differential expression genes, we found that overexpression of AtAED1 alone could enhance the tolerance of transgenic Arabidopsis to Pi starvation. Y1H and ChIP-qPCR analyses showed that GmWRKY46 could directly bind to the W-box motif of the AtAED1 promoter in vitro and in vivo. Furthermore, results from intact soybean composite plants with GmWRKY46 overexpression showed that GmWRKY46 was involved in hairy roots development and subsequently affected plant growth and Pi uptake. These results provide a basis for the molecular genetic breeding of soybean tolerant to Pi starvation.

Gm6PGDH1, a Cytosolic 6-Phosphogluconate Dehydrogenase, Enhanced Tolerance to Phosphate Starvation by Improving Root System Development and Modifying the Antioxidant System in Soybean.

Phosphorus plays an important role in plant growth and development, and is an important limiting factor for crop yield. Although previous studies have shown that 6-phosphogluconate dehydrogenase (6PGDH) plays an important role in plant resistance to adversity, its response to low phosphorus (P) stress remains unknown. In this study, we reported the cloning and characterization of a cytosolic 6PGDH gene, Gm6PGDH1, which enhanced the tolerance to phosphate (Pi) starvation by improving root system development and modifying the antioxidant system in transgenic plants. Gm6PGDH1 was highly expressed in the root at full bloom stage, and strongly induced by Pi starvation. The results from intact soybean composite plant and soybean plant, both containing a Gm6PGDH1-overexpressing construct, showed that Gm6PGDH1 was involved in root system development, and subsequently affected P uptake under Pi-deficient conditions. Meanwhile, the accumulation of reactive oxygen species (ROS) in the root tip of transgenic soybean was reduced, and the activity of ROS-scavenging enzymes was enhanced compared with those of the wild type under Pi-deficient conditions. Interestingly, we found that the overexpression of Gm6PGDH1 weakened the response of several other important Pi-answer genes to Pi starvation, such as some purple acid phosphatases (PAPs) and redox-related genes. In addition, the results from a virus-induced gene silencing (VIGS) indicated that Gm6PGDH1 might have functional redundancy in soybean, and the results from a heterogeneous transformation system showed that overexpressing Gm6PGDH1 also enhanced tolerance to Pi starvation in transgenic Arabidopsis. Together, these results suggested the great potential of Gm6PGDH1 in crop breeding for low Pi tolerance.

Transcriptomic analysis of salt tolerance-associated genes and diversity analysis using indel markers in yardlong bean (Vigna unguiculata ssp. sesquipedialis).

BACKGROUND: High salinity is a devastating abiotic stresses for crops. To understand the molecular basis of salinity stress in yardlong bean (Vigna unguiculata ssp. sesquipedalis), and to develop robust markers for improving this trait in germplasm, whole transcriptome RNA sequencing (RNA-seq) was conducted to compare the salt-tolerant variety Suzi 41 and salt-sensitive variety Sujiang 1419 under normal and salt stress conditions. RESULTS: Compared with controls, 417 differentially expressed genes (DEGs) were identified under exposure to high salinity, including 42 up- and 11 down-regulated DEGs in salt-tolerant Suzi 41 and 186 up- and 197 down-regulated genes in salt-sensitive Sujiang 1419, validated by qRT-PCR. DEGs were enriched in "Glycolysis/Gluconeogenesis" (ko00010), "Cutin, suberine and wax biosynthesis" (ko00073), and "phenylpropanoid biosynthesis" (ko00940) in Sujiang 1419, although "cysteine/methionine metabolism" (ko00270) was the only pathway significantly enriched in salt-tolerant Suzi 41. Notably, AP2/ERF, LR48, WRKY, and bHLH family transcription factors (TFs) were up-regulated under high salt conditions. Genetic diversity analysis of 84 yardlong bean accessions using 26 InDel markers developed here could distinguish salt-tolerant and salt-sensitive varieties. CONCLUSIONS: These findings show a limited set of DEGs, primarily TFs, respond to salinity stress in V. unguiculata, and that these InDels associated with salt-inducible loci are reliable for diversity analysis.

A small RNA of miR2119b from soybean CMS line acts as a negative regulator of male fertility in transgenic Arabidopsis.

The miR2119 is involved in the growth, development and abiotic stress response of some legumes, including Medicago truncatula, Phaseolus vulgaris and soybean (Glycine max (L.) Merr.). Our previous small RNA sequencing analysis showed that miR2119b was up-regulated in the flower buds of soybean cytoplasmic male sterile (CMS) line compared with its maintainer line, but the role and mechanism of miR2119b in the regulation of soybean male fertility are still unclear. In this study, the gma-miR2119b and its target gene alcohol dehydrogenase 1.3b (ADH1.3b) were characterized and found to be highly expressed in the flowers of soybean CMS line and its maintainer. Transgenic Arabidopsis plants overexpressing gma-miR2119b exhibit male fertility abnormalities, including pollen fertility and germination rate decreased. Enzyme activity detection found the ADH and catalase (CAT) enzyme activities in inflorescence of gma-miR2119b overexpressed plants were lower than those of wild-type. Bioinformatics and gene expression analysis showed that gma-miR2119b/GmADH1.3b module was responsive to high temperature (HT) stress during flowering. After HT stress, the gma-miR2119b overexpressed plants showed male sterility, including shorter filament, sterile pollen, indehiscent anther and non seed. Moreover, some key genes involved in HT response and reactive oxygen species (ROS) signal regulation pathway, including heat shock protein70, galactinol synthase 1 and CAT, showed down-regulated expression in transgenic plants under HT stress, suggesting that gma-miR2119b regulates male fertility via HT-ROS signaling pathway under HT stress. It was speculated that the gma-miR2119b acted as a negative regulator of male fertility in plants by regulating ADH1, HT-induced and ROS scavenging genes expression.

Confirmation of GmPPR576 as a fertility restorer gene of cytoplasmic male sterility in soybean.

In soybean, heterosis achieved through the three-line system has been gradually applied in breeding to increase yield, but the underlying molecular mechanism remains unknown. We conducted a genetic analysis using the pollen fertility of offspring of the cross NJCMS1A×NJCMS1C. All the pollen of F1 plants was semi-sterile; in F2, the ratio of pollen-fertile plants to pollen-semi-sterile plants was 208:189. This result indicates that NJCMS1A is gametophyte sterile, and the fertility restoration of NJCMS1C to NJCMS1A is a quality trait controlled by a single gene locus. Using bulked segregant analysis, the fertility restorer gene Rf in NJCMS1C was located on chromosome 16 between the markers BARCSOYSSR_16_1067 and BARCSOYSSR_16_1078. Sequence analysis of genes in that region showed that GmPPR576 was non-functional in rf cultivars. GmPPR576 has one functional allele in Rf cultivars but three non-functional alleles in rf cultivars. Phylogenetic analysis showed that the GmPPR576 locus evolved rapidly with the presence of male-sterile cytoplasm. GmPPR576 belongs to the RFL fertility restorer gene family and is targeted to the mitochondria. GmPPR576 was knocked out in soybean N8855 using CRISPR/Cas9. The T1 plants showed sterile pollen, and T2 plants produced few pods at maturity. The results indicate that GmPPR576 is the fertility restorer gene of NJCMS1A.

Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.).

The three-amino-acid-loop-extension (TALE) superfamily genes broadly existed in plants, which played important roles in plant growth, development and abiotic stress responses. In this study, we identified 68 Glycine max TALE (GmTALE) superfamily members. Phylogenetic analysis divided the GmTALE superfamily into the BEL1-like (BLH/BELL homeodomain) and the KNOX (KNOTTED-like homeodomain) subfamilies. Moreover, the KNOX subfamily could be further categorized into three clades (KNOX Class I, KNOX Class II and KNOX Class III). The GmTALE genes showed similarities in the gene structures in the same subfamily or clade, whose coding proteins exhibited analogous motif and conserved domain compositions. Besides, synteny analyses and evolutionary constraint evaluations of the TALE members among soybean and different species provided more clues for GmTALE superfamily evolution. The cis-element analyses in gene promoter regions and relevant gene expression profiling revealed different regulating roles of GmTALE genes during soybean plant development, saline and dehydration stresses. Genome-wide characterization, evolution, and expression profile analyses of GmTALE genes can pave the way for future gene functional research and facilitate their roles for applications in genetic improvement on soybean in saline and dehydration stresses.

Heat-Responsive miRNAs Participate in the Regulation of Male Fertility Stability in Soybean CMS-Based F1 under High Temperature Stress.

MicroRNAs (miRNAs), a class of noncoding small RNAs (sRNAs), are widely involved in the response to high temperature (HT) stress at both the seedling and flowering stages. To dissect the roles of miRNAs in regulating male fertility in soybean cytoplasmic male sterility (CMS)-based F1 under HT, sRNA sequencing was performed using flower buds from HT-tolerant and HT-sensitive CMS-based F1 combinations (NF1 and YF1, respectively). A total of 554 known miRNAs, 59 new members of known miRNAs, 712 novel miRNAs, and 1145 target genes of 580 differentially expressed miRNAs (DEMs) were identified under normal temperature and HT conditions. Further integrated analysis of sRNA and transcriptome sequencing found that 21 DEMs and 15 differentially expressed target genes, such as gma-miR397a/Laccase 2, gma-miR399a/Inorganic phosphate transporter 1-4, and gma-miR4413a/PPR proteins, mitochondrial-like, were negatively regulated under HT stress. Furthermore, all members of the gma-miR156 family were suppressed by HT stress in both NF1 and YF1, but were highly expressed in YF1 under HT condition. The negative correlation between gma-miR156b and its target gene squamosa promoter-binding protein-like 2b was confirmed by expression analysis, and overexpression of gma-miR156b in Arabidopsis led to male sterility under HT stress. With these results, we proposed that miRNAs play an important role in the regulation of male fertility stability in soybean CMS-based F1 under HT stress.

Comparative analysis of mitochondrial genomes of soybean cytoplasmic male-sterile lines and their maintainer lines.

In soybean, only one mitochondrial genome of cultispecies has been completely obtained. To explore the effect of mitochondrial genome on soybean cytoplasmic male sterility (CMS), two CMS lines and three maintainer lines were used for sequencing. Comparative analysis showed that mitochondrial genome of the CMS line was more compact than that of its maintainer line, but genes were highly conserved. Conserved and unique sequence coexisted in the genomes. Mitochondrial genomes contained different sequence lengths and copy numbers of repeats between CMS line and maintainer line. Large and short repeats mediated intramolecular and intermolecular recombination in mitochondria. Unique sequences and genes were also involved in recombination process and constituted a complex network. orf178 and orf261 were identified as CMS-associated candidate genes. They had sequence characteristics of reported CMS genes in other crops and could be transcribed in CMS lines but not in maintainer lines. This report reveals mitochondrial genome of soybean CMS lines and compares complete mitochondrial sequence between CMS lines and their maintainer lines. The information will be helpful in further understanding the characteristics of soybean mitochondrial genome and the mechanism underlying CMS.

Genome-wide identification and characterization of GRAS genes in soybean (Glycine max).

BACKGROUND: GRAS proteins are crucial transcription factors, which are plant-specific and participate in various plant biological processes. Thanks to the rapid progress of the whole genome sequencing technologies, the GRAS gene families in different plants have been broadly explored and studied. However, comprehensive research on the soybean (Glycine max) GRAS gene family is relatively lagging. RESULTS: In this study, 117 Glycine max GRAS genes (GmGRAS) were identified. Further phylogenetic analyses showed that the GmGRAS genes could be categorized into nine gene subfamilies: DELLA, HAM, LAS, LISCL, PAT1, SCL3, SCL4/7, SCR and SHR. Gene structure analyses turned out that the GmGRAS genes lacked introns and were relatively conserved. Conserved domains and motif patterns of the GmGRAS members in the same subfamily or clade exhibited similarities. Notably, the expansion of the GmGRAS gene family was driven both by gene tandem and segmental duplication events. Whereas, segmental duplications took the major role in generating new GmGRAS genes. Moreover, the synteny and evolutionary constraints analyses of the GRAS proteins among soybean and distinct species (two monocots and four dicots) provided more detailed evidence for GmGRAS gene evolution. Cis-element analyses indicated that the GmGRAS genes may be responsive to diverse environmental stresses and regulate distinct biological processes. Besides, the expression patterns of the GmGRAS genes were varied in various tissues, during saline and dehydration stresses and during seed germination processes. CONCLUSIONS: We conducted a systematic investigation of the GRAS genes in soybean, which may be valuable in paving the way for future GmGRAS gene studies and soybean breeding.

Correction to: Molecular mapping of a novel male-sterile gene msNJ in soybean [Glycine max (L.) Merr.].

The Acknowledgement section of the original publication gave a wrong grant number. The correct Acknowledgement should read.

Comparative Transcriptomics Analysis and Functional Study Reveal Important Role of High-Temperature Stress Response Gene GmHSFA2 During Flower Bud Development of CMS-Based F1 in Soybean.

High-temperature (HT) is one of the most important environmental factors that negatively impact the yield of some soybean cytoplasmic male sterility (CMS)-based hybrid (F1) combinations. The response of soybean to HT, especially at the male organ development stage, is poorly understood. To investigate the molecular mechanisms of the response from soybean CMS-based F1 male organ to HT, a detailed transcriptomics analysis was performed during flower bud development of soybean HT-tolerant and HT-sensitive CMS-based F1 combinations (NF1 and YF1) under normal-temperature and HT conditions. Obvious HT damage was observed by subjecting YF1 with HT, such as indehiscent anthers and decreased pollen fertility, whereas the male fertility of NF1 was normal. In total, 8,784 differentially expressed genes (DEGs) were found to respond to HT stress, which were mainly associated with anther/pollen wall development, carbohydrate metabolism and sugar transport, and auxin signaling. The quantitative real-time PCR (qRT-PCR) analysis and substance content detection also revealed that HT caused male fertility defects in YF1 by altering pectin metabolism, auxin, and sugar signaling pathways. Most importantly, the sugar signaling-PIF-auxin signaling pathway may underlie the instability of male fertility in YF1 under HT. Furthermore, HT induced the expression of heat shock factor (HSF) and heat shock protein (HSP) gene families. Overexpression of GmHSFA2 in Arabidopsis can promote the expression of HT protective genes (such as HSP20) by binding to the HSE motifs in their promoters, so as to improve the HT tolerance during flowering. Our results indicated that GmHSFA2 acted as a positive regulator, conferring HT tolerance improvement in soybean CMS-based F1. GmHSFA2 may be directly involved in the activation of male fertility protection mechanism in the soybean CMS-based F1 under HT stress.