Identification of Gene-Allele System Conferring Alkali-Tolerance at Seedling Stage in Northeast China Soybean Germplasm.

Salinization of cultivated soils may result in either high salt levels or alkaline conditions, both of which stress crops and reduce performance. We sampled genotypes included in the Northeast China soybean germplasm population (NECSGP) to identify possible genes that affect tolerance to alkaline soil conditions. In this study, 361 soybean accessions collected in Northeast China were tested under 220 mM NaHCO3:Na2CO3 = 9:1 (pH = 9.8) to evaluate the alkali-tolerance (ATI) at the seedling stage in Mudanjiang, Heilongjiang, China. The restricted two-stage multi-locus model genome-wide association study (RTM-GWAS) with gene-allele sequences as markers (6503 GASMs) based on simplified genome resequencing (RAD-sequencing) was accomplished. From this analysis, 132 main effect candidate genes with 359 alleles and 35 Gene × Environment genes with 103 alleles were identified, explaining 90.93% and 2.80% of the seedling alkali-tolerance phenotypic variation, respectively. Genetic variability of ATI in NECSGP was observed primarily within subpopulations, especially in ecoregion B, from which 80% of ATI-tolerant accessions were screened out. The biological functions of 132 candidate genes were classified into eight functional categories (defense response, substance transport, regulation, metabolism-related, substance synthesis, biological process, plant development, and unknown function). From the ATI gene-allele system, six key genes-alleles were identified as starting points for further study on understanding the ATI gene network.

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.

Identification of major quantitative trait loci and candidate genes for seed weight in soybean.

KEY MESSAGE: Four major quantitative trait loci for 100-seed weight were identified in a soybean RIL population under five environments, and the most likely candidate genes underlying these loci were identified. Seed weight is an important target of soybean breeding. However, the genes underlying the major quantitative trait loci (QTL) controlling seed weight remain largely unknown. In this study, a soybean population of 300 recombinant inbred lines (RILs) derived from a cross between PI595843 (PI) and WH was used to map the QTL and identify candidate genes for seed weight. The RIL population was genotyped through whole genome resequencing, and phenotyped for 100-seed weight under five environments. A total of 38 QTL were detected, and four major QTL, each explained at least 10% of the variation in 100-seed weight, were identified. Six candidate genes within these four major QTL regions were identified by analyses of their tissue expression patterns, gene annotations, and differential gene expression levels in soybean seeds during four developmental stages between two parental lines. Further sequence variation analyses revealed a C to T substitution in the first exon of the Glyma.19G143300, resulting in an amino acid change between PI and WH, and thus leading to a different predicted kinase domain, which might affect its protein function. Glyma.19G143300 is highly expressed in soybean seeds and encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Its predicted protein has typical domains of LRR-RLK family, and phylogenetic analyses reveled its similarity with the known LRR-RLK protein XIAO (LOC_Os04g48760), which is involved in controlling seed size. The major QTL and candidate genes identified in this study provide useful information for molecular breeding of new soybean cultivars with desirable seed weight.

Gene-allele system of shade tolerance in southern China soybean germplasm revealed by genome-wide association study using gene-allele sequence as markers.

KEY MESSAGE: Fifty-three shade tolerance genes with 281 alleles in the SCSGP were identified directly using gene-allele sequence as markers in RTM GWAS, from which optimized crosses, evolutionary motivators, and gene-allele networks were explored. Shade tolerance is a key for optimal cultivation of soybean inter/relay-cropped with corn. To explore the shade tolerance gene-allele system in the southern China soybean germplasm, we proposed using gene-allele sequence markers (GASMs) in a restricted two-stage multi-locus model genome-wide association study (GASM-RTM-GWAS). A representative sample with 394 accessions was tested for their shade tolerance index (STI), in Nanning, China. Through whole-genome re-sequencing, 47,586 GASMs were assembled. From GASM-RTM-GWAS, 53 main-effect STI genes with 281 alleles (2-13 alleles/gene) (totally 63 genes with 308 alleles, including 38 G × E genes with 191 alleles) were identified and then organized into a gene-allele matrix composed of eight submatrices corresponding to geo-seasonal subpopulations. The population featured mild STI changes (1.69 → 1.56-1.82) and mild gene-allele changes (92.5% alleles inherited, 0% alleles excluded, 7.5% alleles emerged) from the primitive (SAIII) to the derived seven subpopulations, but large transgressive recombination potentials and optimal crosses were predicted. The 63 STI genes were annotated into six biological categories (metabolic process, catalytic activity, response to stresses, transcription and translation, signal transduction and transport and unknown functions), interacted as gene networks. From the STI gene-allele system, 38 important alleles of 22 genes were nominated for further in-depth study. GASM-RTM-GWAS performed powerful and efficient in germplasm population genetic study comparing to other procedures through facilitating direct and thorough identification of its gene-allele system, from which genome-wide breeding by design could be achieved, and evolutionary motivators and gene-allele networks could be explored.

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

Soybean mosaic virus (SMV) strain SC11 was prevalent in middle China. Its resistance was controlled by a Mendelian single dominant gene RSC11K in soybean Kefeng-1. This study aimed at mapping RSC11K and identifying its candidate gene. RSC11K locus was mapped ~217 kb interval between two SNP-linkage-disequilibrium-blocks (Gm02_BLOCK_11273955_11464884 and Gm02_BLOCK_11486875_11491354) in W82.a1.v1 genome using recombinant inbred lines population derived from Kefeng-1 (Resistant) × NN1138-2 (Susceptible), but inserted with a ~245 kb segment in W82.a2.v1 genome. In the entire 462 kb RSC11K region, 429 SNPs, 142 InDels and 34 putative genes were identified with more SNPs/InDels distributed in non-functional regions. Thereinto, ten genes contained SNP/InDel variants with high and moderate functional impacts on proteins, among which Glyma.02G119700 encoded a typical innate immune receptor-like kinase involving in virus disease process and responded to SMV inoculation, therefore was recognized as RSC11K's candidate gene. The novel RSC11K locus and candidate genes may help developing SMV resistance germplasm.

The Identification of a Quantative Trait Loci-Allele System of Antixenosis against the Common Cutworm (Spodoptera litura Fabricius) at the Seedling Stage in the Chinese Soybean Landrace Population.

Common cutworm (CCW) is an omnivorous insect causing severe yield losses in soybean crops. The seedling-stage mini-tray identification system with the damaged leaf percentage (DLP) as an indicator was used to evaluate antixenosis against CCW in the Chinese soybean landrace population (CSLRP) under three environments. Using the innovative restricted two-stage multi-locus genome-wide association study procedure (RTM-GWAS), 86 DLP QTLs with 243 alleles (2-11/QTL) were identified, including 66 main-effect loci with 203 alleles and 57 QTL-environment interaction loci with 172 alleles. Among the main-effect loci, 12 large-contribution loci (R2 ≥ 1%) explained 25.45% of the phenotypic variation (PV), and 54 small-contribution loci (R2 < 1%) explained 16.55% of the PV. This indicates that the CSLRP can be characterized with a DLP QTL-allele system complex that has not been found before, except for a few individual QTLs without alleles involved. From the DLP QTL-allele matrix, the recombination potentials expressed in the 25th percentile of the DLP of all possible crosses were predicted to be reduced by 41.5% as the maximum improvement and 14.2% as the maximum transgression, indicating great breeding potential in the antixenosis of the CSLRP. From the QTLs, 62 candidate genes were annotated, which were involved in eight biological function categories as a gene network of the DLP. Changing from susceptible to moderate plus resistant varieties in the CSLRP, 26 QTLs had 32 alleles involved, in which 19 genes were annotated from 25 QTL-alleles, including eight increased negative alleles on seven loci and 11 decreased positive alleles on 11 loci, showing the major genetic constitution changes for the antixenosis enhancement at the seedling stage in the CSLRP.

An Improved Genome-Wide Association Procedure Explores Gene-Allele Constitutions and Evolutionary Drives of Growth Period Traits in the Global Soybean Germplasm Population.

In soybeans (Glycine max (L.) Merr.), their growth periods, DSF (days of sowing-to-flowering), and DFM (days of flowering-to-maturity) are determined by their required accumulative day-length (ADL) and active temperature (AAT). A sample of 354 soybean varieties from five world eco-regions was tested in four seasons in Nanjing, China. The ADL and AAT of DSF and DFM were calculated from daily day-lengths and temperatures provided by the Nanjing Meteorological Bureau. The improved restricted two-stage multi-locus genome-wide association study using gene-allele sequences as markers (coded GASM-RTM-GWAS) was performed. (i) For DSF and its related ADLDSF and AATDSF, 130-141 genes with 384-406 alleles were explored, and for DFM and its related ADLDFM and AATDFM, 124-135 genes with 362-384 alleles were explored, in a total of six gene-allele systems. DSF shared more ADL and AAT contributions than DFM. (ii) Comparisons between the eco-region gene-allele submatrices indicated that the genetic adaptation from the origin to the geographic sub-regions was characterized by allele emergence (mutation), while genetic expansion from primary maturity group (MG)-sets to early/late MG-sets featured allele exclusion (selection) without allele emergence in addition to inheritance (migration). (iii) Optimal crosses with transgressive segregations in both directions were predicted and recommended for breeding purposes, indicating that allele recombination in soybean is an important evolutionary drive. (iv) Genes of the six traits were mostly trait-specific involved in four categories of 10 groups of biological functions. GASM-RTM-GWAS showed potential in detecting directly causal genes with their alleles, identifying differential trait evolutionary drives, predicting recombination breeding potentials, and revealing population gene networks.

Differential SW16.1 allelic effects and genetic backgrounds contributed to increased seed weight after soybean domestication.

Although seed weight has increased following domestication from wild soybean (Glycine soja) to cultivated soybean (Glycine max), the genetic basis underlying this change is unclear. Using mapping populations derived from chromosome segment substitution lines of wild soybean, we identified SW16.1 as the causative gene underlying a major quantitative trait locus controlling seed weight. SW16.1 encodes a nucleus-localized LIM domain-containing protein. Importantly, the GsSW16.1 allele from wild soybean accession N24852 had a negative effect on seed weight, whereas the GmSW16.1 allele from cultivar NN1138-2 had a positive effect. Gene expression network analysis, reverse-transcription quantitative polymerase chain reaction, and promoter-luciferase reporter transient expression assays suggested that SW16.1 regulates the transcription of MT4, a positive regulator of seed weight. The natural variations in SW16.1 and other known seed weight genes were analyzed in soybean germplasm. The SW16.1 polymorphism was associated with seed weight in 247 soybean accessions, showing much higher frequency of positive-effect alleles in cultivated soybean than in wild soybean. Interestingly, gene allele matrix analysis of the known seed weight genes revealed that G. max has lost 38.5% of the G. soja alleles and that most of the lost alleles had negative effects on seed weight. Our results suggest that eliminating negative alleles from G. soja led to a higher frequency of positive alleles and changed genetic backgrounds in G. max, which contributed to larger seeds in cultivated soybean after domestication from wild soybean. Our findings provide new insights regarding soybean domestication and should assist current soybean breeding programs.

Growth period QTL-allele constitution of global soybeans and its differential evolution changes in geographic adaptation versus maturity group extension.

Soybean (Glycine max (L.) Merr.) has been disseminated globally as a photoperiod/temperature-sensitive crop with extremely diverse days to flowering (DTF) and days to maturity (DTM) values. A population with 371 global varieties covering 13 geographic regions and 13 maturity groups (MGs) was analyzed for its DTF and DTM QTL-allele constitution using restricted two-stage multi-locus genome-wide association study (RTM-GWAS). Genotypes with 20 701 genome-wide SNPLDBs (single-nucleotide polymorphism linkage disequilibrium blocks) containing 55 404 haplotypes were observed, and 52 DTF QTLs and 59 DTM QTLs (including 29 and 21 new ones) with 241 and 246 alleles (two to 13 per locus) were detected, explaining 84.8% and 74.4% of the phenotypic variance, respectively. The QTL-allele matrix characterized with all QTL-allele information of each variety in the global population was established and subsequently separated into geographic and MG set submatrices. Direct comparisons among them revealed that the genetic adaptation from the origin to geographic subpopulations was characterized by new allele/new locus emergence (mutation) but little allele exclusion (selection), while that from the primary MG set to emerged early and late MG sets was characterized by allele exclusion without allele emergence. The evolutionary changes involved mainly 72 DTF and 71 DTM alleles on 28 respective loci, 10-12 loci each with three to six alleles being most active. Further recombination potential for faster maturation (12-21 days) or slower maturation (14-56 days) supported allele convergence (recombination) as a constant genetic factor in addition to migration (inheritance). From the QTLs, 44 DTF and 36 DTM candidate genes were annotated and grouped respectively into nine biological processes, indicating multi-functional DTF/DTM genes are involved in a complex gene network. In summary, we identified QTL-alleles relatively thoroughly using RTM-GWAS for direct matrix comparisons and subsequent analysis.

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.

Identification of QTLs and joint QTL segments of leaflet traits at different canopy layers in an interspecific RIL population of soybean.

KEY MESSAGE: A leaflet trait on different canopy layers may have different QTLs; leaflet trait QTLs may cluster to form joint QTL segments; all canopy layer QTLs form a complete QTL system for a leaflet trait. As the main part of the plant canopy structure, leaf/leaflet size and shape affect the plant architecture and yield. To explore the leaflet trait QTL system, a population composed of 199 recombinant inbred lines derived from Changling (annual wild, narrow leaflet) and Yiqianli (landrace, broad leaflet) with their parents was tested for leaflet length (LL), width (LW) and length to width (LLW). The population was genotyped with specific-locus amplified fragment sequencing (SLAF-seq) and applied for linkage mapping of the leaflet traits. The results showed that the leaflet traits varied greatly even within a plant, which supported a stratified leaflet sampling strategy to evaluate these traits at top, middle and bottom canopy layers. Altogether, 13 LL, 10 LW and 9 LLW in a total of 32 plus 3 duplicated QTLs were identified, in which, 17 QTLs were new ones, and 48.6%, 28.6% and 22.8% of QTLs were from the top, middle and bottom layers, respectively, indicating the genetic importance of the top layer leaves. Since a leaflet trait may have layer-specific QTLs, all layer QTLs form a complete QTL system. Five QTL clusters each with their QTL supporting intervals overlapped were designated as joint QTL segments (JQSs). In JQS-16, with its linkage map further validated using PCR markers, two QTLs, qLW-16-1 and qLLW-16-1 of the top layer leaflet, were identified six QTL·times. Six candidate genes were predicted, with Glyma.16G127900 as the most potential one for LW and LLW. Three PCR markers, Gm16PAV0653, BARCSOYSSR_16_0796 and YC-16-3, were suggested for marker-assisted selection for LW and LLW in JQS-16.

CRISPR-Cas9 based stress tolerance: New hope for abiotic stress tolerance in chickpea (Cicer arietinum).

Plants are subjected to biotic and abiotic stresses regularly, which irreparably harm agricultural production. Eco-friendly and sustainable technology to deal with this challenge is to breed abiotic stress tolerant cultivars. To generate crop plants conferring resistance against stresses, conventional breeding was used in the past, but because of the complex heredity of abiotic stress tolerance traits, such techniques remain insufficient in making greater enhancement. Genome-engineering based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein9) has shown enormous potential in developing climate-resilient cultivars. Likewise, the development of chickpea transgenic lines by knockout of 4CL and REV7 genes exhibits drought tolerance which establishes a foundation for future studies in chickpea. In addition, the CRISPR-Cas9 system can boost yield potential under abiotic stress situations by producing non-transgenic plants having the required characteristics. This review article discusses the validation of gene function based on the CRISPR-Cas9 for the development of abiotic stress-tolerant crop plants, emphasizing the chickpea to open the new ventures of generating abiotic stress-tolerant chickpea varieties.

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.

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.

Evolutionary QTL-allele changes in main stem node number among geographic and seasonal subpopulations of Chinese cultivated soybeans.

The main stem node number (MSN) is a trait related to geographic adaptation, plant architecture and yield potential of soybean. The QTL-allele constitution of the Chinese Cultivated Soybean Population (CCSP) was identified using the RTM-GWAS (restricted two-stage multi-locus genome-wide association study) procedure, from which a QTL-allele matrix was established and then separated into submatrices to explore the genetic structure, evolutionary differentiation, breeding potential and candidate genes of MSN in CCSP. The MSN of 821 accessions varied from 8.8 to 31.1, with an average of 16.3 in Nanjing, China (32.07° N, 118.62° E), where the MSNs of all the materials could be evaluated in a standardized manner. Among the six geo-seasonal subpopulations, the MSN varied from 21.7 in a southern summer-autumn-sowing subpopulation (SA-IV) down to 13.5 in a northeastern spring-sowing subpopulation (SP-I). The materials were genotyped with restriction site-associated DNA-sequencing. Totally 142 main-effect QTLs (73.24% new) with 560 alleles contributing 72.98% to the phenotypic variance were identified. The evolutionary QTL-allele changes in MSN from SA-IV through SP-I showed that inheritance (78.93% of alleles) was the primary factor influencing the evolution of this trait, followed by allele emergence (19.64% alleles), allele exclusion (1.43% alleles), and recombination among retained alleles. In the evolutionary changes, 70 QTLs, including 12 newly emerged QTLs, with 118 alleles were involved. An increase potential of 2-8 nodes was predicted and 112 candidate genes were annotated and preliminarily verified with χ2-tests in the CCSP. The RTM-GWAS showed powerful in detecting QTL-allele system, assessing evolution factors, predicting optimal crosses and identifying candidate genes in a germplasm population.

Natural variation in the promoter of GsERD15B affects salt tolerance in soybean.

Salt stress has detrimental effects on crop growth and yield, and the area of salt-affected land is increasing. Soybean is a major source of vegetable protein, oil and feed, but considered as a salt-sensitive crop. Cultivated soybean (Glycine max) is domesticated from wild soybean (G. soja) but lost considerable amount of genetic diversity during the artificial selection. Therefore, it is important to exploit the gene pool of wild soybean. In this study, we identified 34 salt-tolerant accessions from wild soybean germplasm and found that a 7-bp insertion/deletion (InDel) in the promoter of GsERD15B (early responsive to dehydration 15B) significantly affects the salt tolerance of soybean. GsERD15B encodes a protein with transcriptional activation function and contains a PAM2 domain to mediate its interaction with poly(A)-binding (PAB) proteins. The 7-bp deletion in GsERD15B promoter enhanced the salt tolerance of soybean, with increased up-regulation of GsERD15B, two GmPAB genes, the known stress-related genes including GmABI1, GmABI2, GmbZIP1, GmP5CS, GmCAT4, GmPIP1:6, GmMYB84 and GmSOS1 in response to salt stress. We propose that natural variation in GsERD15B promoter affects soybean salt tolerance, and overexpression of GsERD15B enhanced salt tolerance probably by increasing the expression levels of genes related to ABA-signalling, proline content, catalase peroxidase, dehydration response and cation transport.

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.

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.

Identifying Wild Versus Cultivated Gene-Alleles Conferring Seed Coat Color and Days to Flowering in Soybean.

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

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.