Identification of major QTLs for drought tolerance in soybean, together with a novel candidate gene, GmUAA6.

Drought tolerance is a complex trait in soybean that is controlled by polygenetic quantitative trait loci (QTLs). In this study, wilting score, days-to-wilting, leaf relative water content, and leaf relative conductivity were used to identify QTLs associated with drought tolerance in recombinant inbred lines derived from a cross between a drought-sensitive variety, Lin, and a drought-tolerant variety, Meng. A total of 33 drought-tolerance QTLs were detected. Of these 17 were major QTLs. In addition, 15 were novel drought-tolerance QTLs. The most predominant QTL was on chromosome 11. This was detected in at least three environments. The overlapped mapping interval of the four measured traits was 0.2 cM in genetic distance (about 220 kb in physical length). Glyma.11g143500 (designated as GmUAA6), which encodes a UDP-N-acetylglucosamine transporter, was identified as the most likely candidate gene. The allele of GmUAA6 from Lin (GmUAA6Lin) was associated with improved soybean drought tolerance. Overexpression of GmUAA6Lin in Arabidopsis and soybean hairy roots enhanced drought tolerance. Furthermore, a 3-bp insertion/deletion (InDel) in the coding sequence of GmUAA6 explained up to 49.9% of the phenotypic variation in drought tolerance-related traits, suggesting that this InDel might be used in future marker-assisted selection of drought-tolerant lines in soybean breeding programs.

Identification of the domestication gene GmCYP82C4 underlying the major quantitative trait locus for the seed weight in soybean.

KEY MESSAGE: A major quantitative trait locus (QTL) for the hundred-seed weight (HSW) was identified and confirmed in the two distinct soybean populations, and the target gene GmCYP82C4 underlying this locus was identified that significantly associated with soybean seed weight, and it was selected during the soybean domestication and improvement process. Soybean is a major oil crop for human beings and the seed weight is a crucial goal of soybean breeding. However, only a limited number of target genes underlying the quantitative trait loci (QTLs) controlling seed weight in soybean are known so far. In the present study, six loci associated with hundred-seed weight (HSW) were detected in the first population of 573 soybean breeding lines by genome-wide association study (GWAS), and 64 gene models were predicted in these candidate QTL regions. The QTL qHSW_1 exhibits continuous association signals on chromosome four and was also validated by region association study (RAS) in the second soybean population (409 accessions) with wild, landrace, and cultivar soybean accessions. There were seven genes in qHSW_1 candidate region by linkage disequilibrium (LD) block analysis, and only Glyma.04G035500 (GmCYP82C4) showed specifically higher expression in flowers, pods, and seeds, indicating its crucial role in the soybean seed development. Significant differences in HSW trait were detected when the association panels are genotyped by single-nucleotide polymorphisms (SNPs) in putative GmCYP82C4 promoter region. Eight haplotypes were generated by six SNPs in GmCYP82C4 in the second soybean population, and two superior haplotypes (Hap2 and Hap4) of GmCYP82C4 were detected with average HSW of 18.27 g and 18.38 g, respectively. The genetic diversity of GmCYP82C4 was analyzed in the second soybean population, and GmCYP82C4 was most likely selected during the soybean domestication and improvement process, leading to the highest proportion of Hap2 of GmCYP82C4 both in landrace and cultivar subpopulations. The QTLs and GmCYP82C4 identified in this study provide novel genetic resources for soybean seed weight trait, and the GmCYP82C4 could be used for soybean molecular breeding to develop desirable seed weight in the future.

Exploring the role of FBXL fbxl gene family in Soybean: Implications for plant height and seed size regulation.

F-box proteins constitute a significant family in eukaryotes and, as a component of the Skp1p-cullin-F-box complex, are considered critical for cellular protein degradation and other biological processes in plants. Despite their importance, the functions of F-box proteins, particularly those with C-terminal leucine-rich repeat (LRR) domains, remain largely unknown in plants. Therefore, the present study conducted genome-wide identification and in silico characterization of F-BOX proteins with C-terminal LRR domains in soybean (Glycine max L.) (GmFBXLs). A total of 45 GmFBXLs were identified. The phylogenetic analysis showed that GmFBXLs could be subdivided into ten subgroups and exhibited a close relationship with those from Arabidopsis thaliana, Cicer aretineum, and Medicago trunculata. It was observed that most cis-regulatory elements in the promoter regions of GmFBXLs are involved in hormone signalling, stress responses, and developmental stages. In silico transcriptome data illustrated diverse expression patterns of the identified GmFBXLs across various tissues, such as shoot apical meristem, flower, green pods, leaves, nodules, and roots. Overexpressing (OE) GmFBXL12 in Tianlong No.1 cultivar resulted in a significant difference in seed size, number of pods, and number of seeds per plant, indicated a potential increase in yield compared to wild type. This study offers valuable perspectives into the role of FBXLs in soybean, serving as a foundation for future research. Additionally, the identified OE lines represent valuable genetic resources for enhancing seed-related traits in soybean.

Genome-wide characterization and functional analysis of class III peroxidase gene family in soybean reveal regulatory roles of GsPOD40 in drought tolerance.

Class III peroxidases (PODs) are plant-specific glycoproteins, that play essential roles in various plant physiological processes and defence responses. To date, scarce information is available about the POD gene family in soybean. Hence, the present study is the first comprehensive report about the genome-wide characterization of GmPOD gene family in soybean (Glycine max L.). Here, we identified a total of 124 GmPOD genes in soybean, that are unevenly distributed across the genome. Phylogenetic analysis classified them into six distinct sub-groups (A-F), with one soybean specific subgroup. Exon-intron and motif analysis suggested the existence of structural and functional diversity among the sub-groups. Duplication analysis identified 58 paralogous gene pairs; segmental duplication and positive/Darwinian selection were observed as the major factors involved in the evolution of GmPODs. Furthermore, RNA-seq analysis revealed that 23 out of a total 124 GmPODs showed differential expression between drought-tolerant and drought-sensitive genotypes under stress conditions; however, two of them (GmPOD40 and GmPOD42) revealed the maximum deregulation in all contrasting genotypes. Overexpression (OE) lines of GsPOD40 showed considerably higher drought tolerance compared to wild type (WT) plants under stress treatment. Moreover, the OE lines showed enhanced photosynthesis and enzymatic antioxidant activities under drought stress, resulting in alleviation of ROS induced oxidative damage. Hence, the GsPOD40 enhanced drought tolerance in soybean by regulating the key physiological and biochemical pathways involved in the defence response. Lastly, the results of our study will greatly assist in further functional characterization of GsPODs in plant growth and stress tolerance in soybean.

Unraveling the Diverse Roles of Neglected Genes Containing Domains of Unknown Function (DUFs): Progress and Perspective.

Domain of unknown function (DUF) is a general term for many uncharacterized domains with two distinct features: relatively conservative amino acid sequence and unknown function of the domain. In the Pfam 35.0 database, 4795 (24%) gene families belong to the DUF type, yet, their functions remain to be explored. This review summarizes the characteristics of the DUF protein families and their functions in regulating plant growth and development, generating responses to biotic and abiotic stress, and other regulatory roles in plant life. Though very limited information is available about these proteins yet, by taking advantage of emerging omics and bioinformatic tools, functional studies of DUF proteins could be utilized in future molecular studies.

Digs out and characterization of the resistance gene accountable to soybean mosaic virus in soybean (Glycine max (L.) Merrill).

KEY MESSAGE: A putative candidate gene conferring resistance to SMV strain SC1 was identified on chromosome 2, and the linked marker was validated in soybean cultivars Soybean mosaic, caused by the soybean mosaic virus, is the most common disease in soybean and a significant impediment to soybean production in the Huanghuai and Yangtze River regions of China. Kefeng No.1, a soybean cultivar, showed high resistance to soybean mosaic virus strain (SC1) collected from Huanghuai and Yangtze River regions. Genetic analysis based on the Mendelian genic population derived from the cross Kefeng No.1 × Nannong 1138-2 revealed that Kefeng No.1 possesses a single dominant gene. Furthermore, genetic fine-mapping using an F2 population containing 281 individuals delimited resistant gene to a genomic region of 186 kb flanked by SSR markers BS020610 and BS020620 on chromosome 2. Within this region, there were 14 genes based on the Williams 82 reference genome. According to sequence analysis, six of the 14 genes have amino acid differences, and one of these genes is the Rsv4 allele designated as Rsc1-DR. The functional analysis of candidate genes using the bean pod mottle virus (BPMV)-induced gene silencing (VIGS) system revealed that Rsc1-DR was accountable for Kefeng No.1's resistance to SMV-SC1. Based on the genome sequence of Rsc1-DR, an Insertion/Deletion (InDel) molecular marker, JT0212, was developed and genotyped using 100 soybean cultivars, and the coincidence rate was 89%. The study enriched our understanding of the SMV resistance mechanism. The marker developed in this study could be directly used by the soybean breeders to select the genotypes with favorable alleles for making crosses, and also it will facilitate marker-assisted selection of SMV resistance in soybean breeding.

Co-inoculation of Arbuscular Mycorrhizal Fungi and the Plant Growth-Promoting Rhizobacteria Improve Growth and Photosynthesis in Tobacco Under Drought Stress by Up-Regulating Antioxidant and Mineral Nutrition Metabolism.

Drought stress is a major environmental concern that limits crop growth on a large scale around the world. Significant efforts are required to overcome this issue in order to improve crop production. Therefore, the exciting role of beneficial microorganisms under stress conditions needs to be deeply explored. In this study, the role of two biotic entities, i.e., Arbuscular mycorrhizal fungi (AMF, Glomus versiforme) and plant growth-promoting rhizobacteria (PGPR, Bacillus methylotrophicus) inoculation in drought tolerance of tobacco (Nicotiana tabacum L.), was investigated. The present results showed that drought stress considerably reduced tobacco plant's growth and their physiological attributes. However, the plants co-inoculated with AMF and PGPR showed higher drought tolerance by bringing up significant improvement in the growth and biomass of tobacco plants. Moreover, the co-inoculation of AMF and PGPR considerably increased chlorophyll a, b, total chlorophylls, carotenoids, photosynthesis, and PSII efficiency by 96.99%, 76.90%, and 67.96% and 56.88%, 53.22%, and 33.43% under drought stress conditions, respectively. Furthermore, it was observed that drought stress enhanced lipid peroxidation and electrolyte leakage. However, the co-inoculation of AMF and PGPR reduced the electrolyte leakage and lipid peroxidation and significantly enhanced the accumulation of phenols and flavonoids by 57.85% and 71.74%. Similarly, the antioxidant enzymatic activity and the plant nutrition status were also considerably improved in co-inoculated plants under drought stress. Additionally, the AMF and PGPR inoculation also enhanced abscisic acid (ABA) and indole-3-acetic acid (IAA) concentrations by 67.71% and 54.41% in the shoots of tobacco plants. The current findings depicted that inoculation of AMF and PGPR (alone or in combination) enhanced the growth and mitigated the photosynthetic alteration with the consequent up-regulation of secondary metabolism, osmolyte accumulation, and antioxidant system.

Identification of major genomic regions for soybean seed weight by genome-wide association study.

The hundred-seed weight (HSW) is an important yield component and one of the principal breeding traits in soybean. More than 250 quantitative trait loci (QTL) for soybean HSW have been identified. However, most of them have a large genomic region or are environmentally sensitive, which provide limited information for improving the phenotype in marker-assisted selection (MAS) and identifying the candidate genes. Here, we utilized 281 soybean accessions with 58,112 single nucleotide polymorphisms (SNPs) to dissect the genetic basis of HSW in across years in the northern Shaanxi province of China through one single-locus (SL) and three multi-locus (ML) genome-wide association study (GWAS) models. As a result, one hundred and fifty-four SNPs were detected to be significantly associated with HSW in at least one environment via SL-GWAS model, and 27 of these 154 SNPs were detected in all (three) environments and located within 7 linkage disequilibrium (LD) block regions with the distance of each block ranged from 40 to 610 Kb. A total of 15 quantitative trait nucleotides (QTNs) were identified by three ML-GWAS models. Combined with the results of different GWAS models, the 7 LD block regions associated with HSW detected by SL-GWAS model could be verified directly or indirectly by the results of ML-GWAS models. Eleven candidate genes underlying the stable loci that may regulate seed weight in soybean were predicted. The significantly associated SNPs and the stable loci as well as predicted candidate genes may be of great importance for marker-assisted breeding, polymerization breeding, and gene discovery for HSW in soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01310-y.

NAC Transcription Factor GmNAC12 Improved Drought Stress Tolerance in Soybean.

NAC transcription factors (TFs) could regulate drought stresses in plants; however, the function of NAC TFs in soybeans remains unclear. To unravel NAC TF function, we established that GmNAC12, a NAC TF from soybean (Glycine max), was involved in the manipulation of stress tolerance. The expression of GmNAC12 was significantly upregulated more than 10-fold under drought stress and more than threefold under abscisic acid (ABA) and ethylene (ETH) treatment. In order to determine the function of GmNAC12 under drought stress conditions, we generated GmNAC12 overexpression and knockout lines. The present findings showed that under drought stress, the survival rate of GmNAC12 overexpression lines increased by more than 57% compared with wild-type plants, while the survival rate of GmNAC12 knockout lines decreased by at least 46%. Furthermore, a subcellular localisation analysis showed that the GmNAC12 protein is concentrated in the nucleus of the tobacco cell. In addition, we used a yeast two-hybrid assay to identify 185 proteins that interact with GmNAC12. Gene ontology (GO) and KEGG analysis showed that GmNAC12 interaction proteins are related to chitin, chlorophyll, ubiquitin-protein transferase, and peroxidase activity. Hence, we have inferred that GmNAC12, as a key gene, could positively regulate soybean tolerance to drought stress.

Identification of Novel Genomic Regions for Bacterial Leaf Pustule (BLP) Resistance in Soybean (Glycine max L.) via Integrating Linkage Mapping and Association Analysis.

Bacterial leaf pustule (BLP), caused by Xanthornonas axonopodis pv. glycines (Xag), is a worldwide disease of soybean, particularly in warm and humid regions. To date, little is known about the underlying molecular mechanisms of BLP resistance. The only single recessive resistance gene rxp has not been functionally identified yet, even though the genotypes carrying the gene have been widely used for BLP resistance breeding. Using a linkage mapping in a recombinant inbred line (RIL) population against the Xag strain Chinese C5, we identified that quantitative trait locus (QTL) qrxp-17-2 accounted for 74.33% of the total phenotypic variations. We also identified two minor QTLs, qrxp-05-1 and qrxp-17-1, that accounted for 7.26% and 22.26% of the total phenotypic variations, respectively, for the first time. Using a genome-wide association study (GWAS) in 476 cultivars of a soybean breeding germplasm population, we identified a total of 38 quantitative trait nucleotides (QTNs) on chromosomes (Chr) 5, 7, 8, 9,15, 17, 19, and 20 under artificial infection with C5, and 34 QTNs on Chr 4, 5, 6, 9, 13, 16, 17, 18, and 20 under natural morbidity condition. Taken together, three QTLs and 11 stable QTNs were detected in both linkage mapping and GWAS analysis, and located in three genomic regions with the major genomic region containing qrxp_17_2. Real-time RT-PCR analysis of the relative expression levels of five potential candidate genes in the resistant soybean cultivar W82 following Xag treatment showed that of Glyma.17G086300, which is located in qrxp-17-2, significantly increased in W82 at 24 and 72 h post-inoculation (hpi) when compared to that in the susceptible cultivar Jack. These results indicate that Glyma.17G086300 is a potential candidate gene for rxp and the QTLs and QTNs identified in this study will be useful for marker development for the breeding of Xag-resistant soybean cultivars.

Hydroperoxide lyase modulates defense response and confers lesion-mimic leaf phenotype in soybean (Glycine max (L.) Merr.).

Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are two important members of P450 enzymes metabolizing hydroperoxy fatty acid to produce jasmonates and aldehydes respectively, which function in response to diverse environmental and developmental stimuli. However, their exact roles in soybean have not been clarified. In present study, we identified a lesion-mimic mutant in soybean named NT302, which exhibits etiolated phenotype together with chlorotic and spontaneous lesions on leaves at R3 podding stage. The underlying gene was identified as GmHPL encoding hydroperoxide lyase by map-based cloning strategy. Sequence analysis demonstrated that a single nucleotide mutation created a premature termination codon (Gln20-Ter), which resulted in a truncated GmHPL protein in NT302. GmHPL RNA was significantly reduced in NT302 mutant, while genes in AOS branch of the 13-LOX pathway were up-regulated in NT302. The mutant exhibited higher susceptibility to bacterial leaf pustule (BLP) disease, but increased resistance against common cutworm (CCW) pest. GmHPL was significantly induced in response to MeJA, wounding, and CCW in wild type soybean. Virus induced gene silencing (VIGS) of GhHPL genes gave rise to similar lesion-mimic leaf phenotypes in upland cotton, coupled with upregulation of the expression of JA biosynthesis and JA-induced genes. Our study provides evidence that competition exist between HPL and AOS branches in 13-LOX pathway of the oxylipin metabolism in soybean, thereby plays essential roles in modulation of plant development and defense.

Genome-wide association study uncovers major genetic loci associated with seed flooding tolerance in soybean.

BACKGROUND: Seed flooding stress is one of the threatening environmental stressors that adversely limits soybean at the germination stage across the globe. The knowledge on the genetic basis underlying seed-flooding tolerance is limited. Therefore, we performed a genome-wide association study (GWAS) using 34,718 single nucleotide polymorphism (SNPs) in a panel of 243 worldwide soybean collections to identify genetic loci linked to soybean seed flooding tolerance at the germination stage. RESULTS: In the present study, GWAS was performed with two contrasting models, Mixed Linear Model (MLM) and Multi-Locus Random-SNP-Effect Mixed Linear Model (mrMLM) to identify significant SNPs associated with electrical conductivity (EC), germination rate (GR), shoot length (ShL), and root length (RL) traits at germination stage in soybean. With MLM, a total of 20, 40, 4, and 9 SNPs associated with EC, GR, ShL and RL, respectively, whereas in the same order mrMLM detected 27, 17, 13, and 18 SNPs. Among these SNPs, two major SNPs, Gm_08_11971416, and Gm_08_46239716 were found to be consistently connected with seed-flooding tolerance related traits, namely EC and GR across two environments. We also detected two SNPs, Gm_05_1000479 and Gm_01_53535790 linked to ShL and RL, respectively. Based on Gene Ontology enrichment analysis, gene functional annotations, and protein-protein interaction network analysis, we predicted eight candidate genes and three hub genes within the regions of the four SNPs with Cis-elements in promoter regions which may be involved in seed-flooding tolerance in soybeans and these warrant further screening and functional validation. CONCLUSIONS: Our findings demonstrate that GWAS based on high-density SNP markers is an efficient approach to dissect the genetic basis of complex traits and identify candidate genes in soybean. The trait associated SNPs could be used for genetic improvement in soybean breeding programs. The candidate genes could help researchers better understand the molecular mechanisms underlying seed-flooding stress tolerance in soybean.

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.

Comprehensive RNA-seq analysis revealed molecular pathways and genes associated with drought tolerance in wild soybean (Glycine soja Sieb. and Zucc.).

Drought stress at the germination stage is an important environmental stress limiting crop yield. Hence, our study investigated comparative root transcriptome profiles of four contrasting soybean genotypes viz., drought-tolerant (PI342618B/DTP and A214/DTL) and drought-sensitive (NN86-4/DSP and A195/DSL) under drought stress using RNA-Seq approach. A total of 4850 and 6272 differentially expressed genes (DEGs) were identified in tolerant (DTP and DTL) and sensitive (DSP and DSL) genotypes, respectively. Principle component analysis (PCA) and correlation analysis revealed higher correlation between DTP and DTL. Both gene ontology (GO) and MapMan analyses showed that the drought response was enriched in DEGs associated with water and auxin transport, cell wall/membrane, antioxidant activity, catalytic activity, secondary metabolism, signaling and transcription factor (TF) activities. Out of 981 DEGs screened from above terms, only 547 showed consistent opposite expression between contrasting genotypes. Twenty-eight DEGs of 547 were located on Chr.08 rich in QTLs and "Hotspot regions" associated with drought stress, and eight of them showed non-synonymous single nucleotide polymorphism. Hence, 10 genes (including above eight genes plus two hub genes) were predicated as possible candidates regulating drought tolerance, which needs further functional validation. Overall, the transcriptome profiling provided in-depth understanding about the genetic mechanism and candidate genes underlying drought tolerance in soybean.

Exploring genetic architecture for pod-related traits in soybean using image-based phenotyping.

Mature pod color (PC) and pod size (PS) served as important characteristics are used in the soybean breeding programs. However, manual phenotyping of such complex traits is time-consuming, laborious, and expensive for breeders. Here, we collected pod images from two different populations, namely, a soybean association panel (SAP) consisting of 187 accessions and an inter-specific recombinant inbred line (RIL) population containing 284 RILs. An image-based phenotyping method was developed and used to extract the pod color- and size-related parameters from images. Genome-wide association study (GWAS) and linkage mapping were performed to decipher the genetic control of pod color- and size-related traits across 2 successive years. Both populations exhibited wide phenotypic variations and continuous distribution in pod color- and size-related traits, indicating quantitative polygenic inheritance of these traits. GWAS and linkage mapping approaches identified the two major quantitative trait loci (QTL) underlying the pod color parameters, i.e., qPC3 and qPC19, located to chromosomes 3 and 19, respectively, and 12 stable QTLs for pod size-related traits across nine chromosomes. Several genes residing within the genomic region of stable QTL were identified as potential candidates underlying these pod-related traits based on the gene annotation and expression profiling data. Our results provide the useful information for fine-mapping/map-based cloning of QTL and marker-assisted selection of elite varieties with desirable pod traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01223-2.

Treatment of Complex Anal Fistula by Video-Assisted Anal Fistula Treatment Combined with Anal Fistula Plug: A Single-Center Study.

Objective. The surgical treatment of complex anal fistulae is very challenging because of the incidence of incontinence after traditional approaches. There are no studies on the role of video-assisted anal fistula treatment (VAAFT) combined with anal fistula plug (AFP) in the complex anal fistulae. The aim of this study was to demonstrate the efficacy of treating complex anal fistulae using VAAFT combined with AFP. Method. This was a retrospective, nonrandomized observational study. 57 consecutive patients with complex anal fistulae who had undergone the VAAFT with AFP in our hospital between April 2016 and December 2019 were included. The primary outcomes were the cure rate, recurrence rate, and Wexner incontinence scores; the secondary outcomes were surgery time, blood loss, wound healing time postoperatively, pain, and patient satisfaction. Results. All 57 patients completed the surgery and follow-up, with an average follow-up time of 28 months; 6 patients suffered with recurrence (recurrence rate: 10.5%). The average surgery time was 57.9 minutes, and the average wound healing time was 46 days. There were no severe postoperative complications, and anal sphincter function was protected in all patients. Conclusions. The treatment of complex anal fistula by VAAFT combined with AFP is safe and effective, has a high healing rate and few postoperative complications, and is a promising surgery that can effectively protect the patient's anal sphincter function.

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.

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.

Identification of quantitative trait nucleotides and candidate genes for soybean seed weight by multiple models of genome-wide association study.

BACKGROUND: Seed weight is a complex yield-related trait with a lot of quantitative trait loci (QTL) reported through linkage mapping studies. Integration of QTL from linkage mapping into breeding program is challenging due to numerous limitations, therefore, Genome-wide association study (GWAS) provides more precise location of QTL due to higher resolution and diverse genetic diversity in un-related individuals. RESULTS: The present study utilized 573 breeding lines population with 61,166 single nucleotide polymorphisms (SNPs) to identify quantitative trait nucleotides (QTNs) and candidate genes for seed weight in Chinese summer-sowing soybean. GWAS was conducted with two single-locus models (SLMs) and six multi-locus models (MLMs). Thirty-nine SNPs were detected by the two SLMs while 209 SNPs were detected by the six MLMs. In all, two hundred and thirty-one QTNs were found to be associated with seed weight in YHSBLP with various effects. Out of these, seventy SNPs were concurrently detected by both SLMs and MLMs on 8 chromosomes. Ninety-four QTNs co-localized with previously reported QTL/QTN by linkage/association mapping studies. A total of 36 candidate genes were predicted. Out of these candidate genes, four hub genes (Glyma06g44510, Glyma08g06420, Glyma12g33280 and Glyma19g28070) were identified by the integration of co-expression network. Among them, three were relatively expressed higher in the high HSW genotypes at R5 stage compared with low HSW genotypes except Glyma12g33280. Our results show that using more models especially MLMs are effective to find important QTNs, and the identified HSW QTNs/genes could be utilized in molecular breeding work for soybean seed weight and yield. CONCLUSION: Application of two single-locus plus six multi-locus models of GWAS identified 231 QTNs. Four hub genes (Glyma06g44510, Glyma08g06420, Glyma12g33280 & Glyma19g28070) detected via integration of co-expression network among the predicted candidate genes.

High-Resolution Mapping in Two RIL Populations Refines Major "QTL Hotspot" Regions for Seed Size and Shape in Soybean (Glycine max L.).

Seed size and shape are important traits determining yield and quality in soybean. However, the genetic mechanism and genes underlying these traits remain largely unexplored. In this regard, this study used two related recombinant inbred line (RIL) populations (ZY and K3N) evaluated in multiple environments to identify main and epistatic-effect quantitative trait loci (QTLs) for six seed size and shape traits in soybean. A total of 88 and 48 QTLs were detected through composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 15 QTLs were common among both methods; two of them were major (R2 > 10%) and novel QTLs (viz., qSW-1-1ZN and qSLT-20-1K3N). Additionally, 51 and 27 QTLs were identified for the first time through CIM and MCIM methods, respectively. Colocalization of QTLs occurred in four major QTL hotspots/clusters, viz., "QTL Hotspot A", "QTL Hotspot B", "QTL Hotspot C", and "QTL Hotspot D" located on Chr06, Chr10, Chr13, and Chr20, respectively. Based on gene annotation, gene ontology (GO) enrichment, and RNA-Seq analysis, 23 genes within four "QTL Hotspots" were predicted as possible candidates, regulating soybean seed size and shape. Network analyses demonstrated that 15 QTLs showed significant additive x environment (AE) effects, and 16 pairs of QTLs showing epistatic effects were also detected. However, except three epistatic QTLs, viz., qSL-13-3ZY, qSL-13-4ZY, and qSW-13-4ZY, all the remaining QTLs depicted no main effects. Hence, the present study is a detailed and comprehensive investigation uncovering the genetic basis of seed size and shape in soybeans. The use of a high-density map identified new genomic regions providing valuable information and could be the primary target for further fine mapping, candidate gene identification, and marker-assisted breeding (MAB).