An pair of an atypical NLR encoding genes confer Asian soybean rust resistance in soybean.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

PH13 improves soybean shade traits and enhances yield for high-density planting at high latitudes.

Shading in combination with extended photoperiods can cause exaggerated stem elongation (ESE) in soybean, leading to lodging and reduced yields when planted at high-density in high-latitude regions. However, the genetic basis of plant height in adaptation to these regions remains unclear. Here, through a genome-wide association study, we identify a plant height regulating gene on chromosome 13 (PH13) encoding a WD40 protein with three main haplotypes in natural populations. We find that an insertion of a Ty1/Copia-like retrotransposon in the haplotype 3 leads to a truncated PH13H3 with reduced interaction with GmCOP1s, resulting in accumulation of STF1/2, and reduced plant height. In addition, PH13H3 allele has been strongly selected for genetic improvement at high latitudes. Deletion of both PH13 and its paralogue PHP can prevent shade-induced ESE and allow high-density planting. This study provides insights into the mechanism of shade-resistance and offers potential solutions for breeding high-yielding soybean cultivar for high-latitude regions.

RNA-Seq and Comparative Transcriptomic Analyses of Asian Soybean Rust Resistant and Susceptible Soybean Genotypes Provide Insights into Identifying Disease Resistance Genes.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most destructive foliar diseases that affect soybeans. Developing resistant cultivars is the most cost-effective, environmentally friendly, and easy strategy for controlling the disease. However, the current understanding of the mechanisms underlying soybean resistance to P. pachyrhizi remains limited, which poses a significant challenge in devising effective control strategies. In this study, comparative transcriptomic profiling using one resistant genotype and one susceptible genotype was performed under infected and control conditions to understand the regulatory network operating between soybean and P. pachyrhizi. RNA-Seq analysis identified a total of 6540 differentially expressed genes (DEGs), which were shared by all four genotypes. The DEGs are involved in defense responses, stress responses, stimulus responses, flavonoid metabolism, and biosynthesis after infection with P. pachyrhizi. A total of 25,377 genes were divided into 33 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with pathogen defense. The DEGs were mainly enriched in RNA processing, plant-type hypersensitive response, negative regulation of cell growth, and a programmed cell death process. In conclusion, these results will provide an important resource for mining resistant genes to P. pachyrhizi infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.

Genome-Wide Identification of GmSPS Gene Family in Soybean and Expression Analysis in Response to Cold Stress.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.

Heterologous expression of the Glycine soja Kunitz-type protease inhibitor GsKTI improves resistance to drought stress and Helicoverpa armigera in transgenic Arabidopsis lines.

Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The aim of this study was to identify and functionally characterize a Kunitz-like protease inhibitor, GsKTI, from the wild soybean (Glycine soja) variety ED059. Expression patterns suggest that drought stress and insect herbivory may induce GsKTI transcript levels. Transgenic Arabidopsis lines overexpressing GsKTI have been shown to exhibit enhanced drought tolerance by regulating the ABA signaling pathway and increasing xylem cell number. Transgenic Arabidopsis leaves overexpressing GsKTI interfered with insect digestion and thus had a negative effect on the growth of Helicoverpa armigera. It is concluded that GsKTI increases resistance to drought stress and insect attack in transgenic Arabidopsis lines.

Identification of Quantitative Trait Locus and Candidate Genes for Drought Tolerance in a Soybean Recombinant Inbred Line Population.

With global warming and regional decreases in precipitation, drought has become a problem worldwide. As the number of arid regions in the world is increasing, drought has become a major factor leading to significant crop yield reductions and food crises. Soybean is a crop that is relatively sensitive to drought. It is also a crop that requires more water during growth and development. The aim of this study was to identify the quantitative trait locus (QTL) that affects drought tolerance in soybean by using a recombinant inbred line (RIL) population from a cross between the drought-tolerant cultivar 'Jindou21' and the drought-sensitive cultivar 'Zhongdou33'. Nine agronomic and physiological traits were identified under drought and well-watered conditions. Genetic maps were constructed with 923,420 polymorphic single nucleotide polymorphism (SNP) markers distributed on 20 chromosomes at an average genetic distance of 0.57 centimorgan (cM) between markers. A total of five QTLs with a logarithm of odds (LOD) value of 4.035-8.681 were identified on five chromosomes. Under well-watered conditions and drought-stress conditions, one QTL related to the main stem node number was located on chromosome 16, accounting for 17.177% of the phenotypic variation. Nine candidate genes for drought resistance were screened from this QTL, namely Glyma.16G036700, Glyma.16G036400, Glyma.16G036600, Glyma.16G036800, Glyma.13G312700, Glyma.13G312800, Glyma.16G042900, Glyma.16G043200, and Glyma.15G100700. These genes were annotated as NAC transport factor, GATA transport factor, and BTB/POZ-MATH proteins. This result can be used for molecular marker-assisted selection and provide a reference for breeding for drought tolerance in soybean.

CRISPR/Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis.

Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (Glycine max). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticide use and reduce yield loss. The marker gene for QTL-M, Glyma.07g110300 (LOC100775351) that encodes a UDP-glycosyltransferase (UGT) is the major determinant of resistance against leaf-chewing insects in soybean; it exhibits a loss of function in insect-resistant soybean germplasms. In this study, Agrobacterium-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate Glyma.07g110300-gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the GmUGT coding region, which resulted in an enhanced resistance to Helicoverpa armigera and Spodoptera litura. Additionally, overexpressing GmUGT produced soybean varieties that were more sensitive to H. armigera and S. litura. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that GmUGT is involved in imparting resistance to leaf-chewing insects by altering the flavonoid content and expression patterns of genes related to flavonoid biosynthesis and defense. Furthermore, ectopic expression of the GmUGT gene in the ugt72b1 mutant of Arabidopsis substantially rescued the phenotype of H. armigera resistance in the atugt72b1 mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the UGT genes.

Identification of the Important Genes of Bradyrhizobium diazoefficiens 113-2 Involved in Soybean Nodule Development and Senescence.

Legume nodule development and senescence directly affect nitrogen fixation efficiency and involve a programmed series of molecular events. These molecular events are carried out synchronously by legumes and rhizobia. The characteristics and molecular mechanisms of nitrogen fixation at soybean important developmental stages play critical roles in soybean cultivation and fertilizer application. Although the gene expression of soybean were analyzed in nodules at five important soybean developmental stages, information on the expression of rhizobial genes in these nodule samples is limited. In the present study, we investigated the expression of Bradyrhizobium diazoefficiens 113-2 genes in the nodule samples from five developmental stages of soybean (Branching stage, flowering stage, fruiting stage, pod stage and harvest stage). Similar gene expression patterns of B. diazoefficiens 113-2 were existed during optimal symbiotic functioning, while different expression patterns were found among early nodule development, nitrogen fixation progress and nodule senescence. Besides, we identified 164 important different expression genes (DEGs) associated with nodule development and senescence. These DEGs included those encoding nod, nif, fix proteins and T3SS secretion system-related proteins, as well as proteins involved in nitrogen metabolism, ABC transporters and two-component system pathways. Gene Ontology, KEGG pathway and homology analysis of the identified DEGs revealed that most of these DEGs are uncharacterized genes associated with nodule development and senescence, and they are not core genes among the rhizobia genomes. Our results provide new clues for the understanding of the genetic determinants of soil rhizobia in nodule development and senescence, and supply theoretical basis for the creation of high efficiency soybean cultivation technology.

Genome-Wide Identification and Characterization of Soybean GmLOR Gene Family and Expression Analysis in Response to Abiotic Stresses.

The LOR (LURP-one related) family genes encode proteins containing a conserved LOR domain. Several members of the LOR family genes are required for defense against Hyaloperonospora parasitica (Hpa) in Arabidopsis. However, there are few reports of LOR genes in response to abiotic stresses in plants. In this study, a genome-wide survey and expression levels in response to abiotic stresses of 36 LOR genes from Glycine max were conducted. The results indicated that the GmLOR gene family was divided into eight subgroups, distributed on 14 chromosomes. A majority of members contained three extremely conservative motifs. There were four pairs of tandem duplicated GmLORs and nineteen pairs of segmental duplicated genes identified, which led to the expansion of the number of GmLOR genes. The expansion patterns of the GmLOR family were mainly segmental duplication. A heatmap of soybean LOR family genes showed that 36 GmLOR genes exhibited various expression patterns in different tissues. The cis-acting elements in promoter regions of GmLORs include abiotic stress-responsive elements, such as dehydration-responsive elements and drought-inducible elements. Real-time quantitative PCR was used to detect the expression level of GmLOR genes, and most of them were expressed in the leaf or root except that GmLOR6 was induced by osmotic and salt stresses. Moreover, GmLOR4/10/14/19 were significantly upregulated after PEG and salt treatments, indicating important roles in the improvement of plant tolerance to abiotic stress. Overall, our study provides a foundation for future investigations of GmLOR gene functions in soybean.

Genome-Wide Investigation and Expression Profiling Under Abiotic Stresses of a Soybean Unknown Function (DUF21) and Cystathionine-ß-Synthase (CBS) Domain-Containing Protein Family.

Cystathionine-ß-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon-intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family.

Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway.

MYB transcription factors (TFs) have been reported to regulate the biosynthesis of secondary metabolites, as well as to mediate plant adaption to abiotic stresses, including drought. However, the roles of MYB TFs in regulating plant architecture and yield potential remain poorly understood. Here, we studied the roles of the dehydration-inducible GmMYB14 gene in regulating plant architecture, high-density yield and drought tolerance through the brassinosteroid (BR) pathway in soybean. GmMYB14 was shown to localize to nucleus and has a transactivation activity. Stable GmMYB14-overexpressing (GmMYB14-OX) transgenic soybean plants displayed a semi-dwarfism and compact plant architecture associated with decreased cell size, resulting in a decrease in plant height, internode length, leaf area, leaf petiole length and leaf petiole angle, and improved yield in high density under field conditions. Results of the transcriptome sequencing suggested the involvement of BRs in regulating GmMYB14-OX plant architecture. Indeed, GmMYB14-OX plants showed reduced endogenous BR contents, while exogenous application of brassinolide could partly rescue the phenotype of GmMYB14-OX plants. Furthermore, GmMYB14 was shown to directly bind to the promoter of GmBEN1 and up-regulate its expression, leading to reduced BR content in GmMYB14-OX plants. GmMYB14-OX plants also displayed improved drought tolerance under field conditions. GmBEN1 expression was also up-regulated in the leaves of GmMYB14-OX plants under polyethylene glycol treatment, indicating that the GmBEN1-mediated reduction in BR level under stress also contributed to drought/osmotic stress tolerance of the transgenic plants. Our findings provided a strategy for stably increasing high-density yield and drought tolerance in soybean using a single TF-encoding gene.

Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis.

BACKGROUND: Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. RESULTS: In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. CONCLUSIONS: Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding.

KEY MESSAGE: Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and several important drought-tolerant genotypes, traits, SNPs and genes were possibly useful for soybean genetic breeding. Drought is an adverse environmental factor affecting crops growth, development, and yield. Promising genotypes and genes with improved tolerance to drought are probably effective ways to alleviate the situation. In this study, our main task was to determine drought tolerance level of 136 soybean genotypes, the correlations among physiological and agronomic traits under drought, and drought-tolerant single nucleotide polymorphism (SNPs) and genes. In this study, twenty-six varieties were identified as excellent tolerant genotypes to stress among which S14, S93 and S135 with high drought-tolerant index (DTI > 1.3) and yield (Y > 300 kg). Fourteen varieties were identified as drought-sensitive genotypes, such as S25, S45 and S58, with low drought-tolerant index (DTI < 0.5). 422 SNPs and 302 genes correlated with seed number per plant (SNPP), maturity (M), number of seeds per pod (NSPP), node number of main stem (NNMS), Stem diameter (SD) and pull stem (PS) were detected under well-watered and drought conditions by genome-wide association study (GWAS). Among them, we found SNPs (Chr 3:1758920-1958934) between drought-tolerant and sensitive genotypes. 13 genes (Glyma.03G017800, Glyma.03G018000, Glyma.03G018200, Glyma.03G018400, Glyma.03G018500, Glyma.03G018600, Glyma.03G018700, Glyma.03G018800, Glyma.03G018900, Glyma.03G019000, Glyma.03G019100, Glyma.03G019200, Glyma.03G019300) correlated with NNMS were detected. By qRT-PCR, the expression level of Glyma.03G018000 and Glyma.03G018900 in drought-tolerant varieties was significantly increased, but low or no expression in sensitive varieties under drought stress. This study provides important drought-tolerant genotypes, traits, SNPs and potential genes, possibly useful for soybean genetic breeding.

Genome-Wide Analysis of the WOX Gene Family and Function Exploration of GmWOX18 in Soybean.

WUSCHEL-related homeobox (WOX) is a family of transcription factors that are unique to plants and is characterized by the presence of a homeodomain. The WOX transcription factor plays an important role in regulating plant growth and development and the response to abiotic stress. Soybean is one of the most important oil crops worldwide. In this study, based on the available genome data of soybean, the WOX gene family was identified by bioinformatics analysis. The chromosome distribution, gene and protein structures, phylogenetic relationship and gene expression patterns of this family were comprehensively compared. The results showed that a total of 33 putative WOX genes in the soybean genome were found and then designated as GmWOX1- GmWOX33, which were distributed across 19 chromosomes except chromosome 16. Multiple sequence analysis of the GmWOX gene family revealed a highly conserved homeodomain. Phylogenetic tree analysis showed that 33 WOX genes could be divided into three major clades (modern/WUS, intermediate and ancient) in soybean. Of these 33 WOX genes, some showed differential expression patterns in the tested tissues (leaves, pods, unopen and open flowers, nodules, seed, roots, root hairs, stems, shoot apical meristems and shoot tips). In addition, the expression profile and qRT-PCR analysis showed that most of the GmWOX genes responded to different abiotic stress treatments (cold and drought). According to the expression pattern of GmWOX genes in the high regeneration capacity soybean material P3, overexpression of GmWOX18 was selected for function analysis. The overexpression of GmWOX18 increased the regeneration ability of clustered buds. The results will provide valuable information for further studies on the roles of WOX genes in regulating soybean growth, development and responses to abiotic stress, as well as a basis for the functional identification and analysis of WOX genes in soybean.

CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.

BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

GmSYP24, a putative syntaxin gene, confers osmotic/drought, salt stress tolerances and ABA signal pathway.

As major environment factors, drought or high salinity affect crop growth, development and yield. Transgenic approach is an effective way to improve abiotic stress tolerance of crops. In this study, we comparatively analyzed gene structures, genome location, and the evolution of syntaxin proteins containing late embryogenesis abundant (LEA2) domain. GmSYP24 was identified as a dehydration-responsive gene. Our study showed that the GmSYP24 protein was located on the cell membrane. The overexpression of GmSYP24 (GmSYP24ox) in soybean and heteroexpression of GmSYP24 (GmSYP24hx) in Arabidopsis exhibited insensitivity to osmotic/drought and high salinity. However, wild type soybean, Arabidopsis, and the mutant of GmSYP24 homologous gene of Arabidopsis were sensitive to the stresses. Under the abiotic stresses, transgenic soybean plants had greater water content and higher activities of POD, SOD compared with non-transgenic controls. And the leaf stomatal density and opening were reduced in transgenic Arabidopsis. The sensitivity to ABA was decreased during seed germination of GmSYP24ox and GmSYP24hx. GmSYP24hx induced up-regulation of ABA-responsive genes. GmSYP24ox alters the expression of some aquaporins under osmotic/drought, salt, or ABA treatment. These results demonstrated that GmSYP24 played an important role in osmotic/drought or salt tolerance in ABA signal pathway.

De novo reconstruction of human adipose transcriptome reveals conserved lncRNAs as regulators of brown adipogenesis.

Obesity has emerged as an alarming health crisis due to its association with metabolic risk factors such as diabetes, dyslipidemia, and hypertension. Recent work has demonstrated the multifaceted roles of lncRNAs in regulating mouse adipose development, but their implication in human adipocytes remains largely unknown. Here we present a catalog of 3149 adipose active lncRNAs, of which 909 are specifically detected in brown adipose tissue (BAT) by performing deep RNA-seq on adult subcutaneous, omental white adipose tissue and fetal BATs. A total of 169 conserved human lncRNAs show positive correlation with their nearby mRNAs, and knockdown assay supports a role of lncRNAs in regulating their nearby mRNAs. The knockdown of one of those, lnc-dPrdm16, impairs brown adipocyte differentiation in vitro and a significant reduction of BAT-selective markers in in vivo. Together, our work provides a comprehensive human adipose catalog built from diverse fat depots and establishes a roadmap to facilitate the discovery of functional lncRNAs in adipocyte development.

Genome-Wide Identification and Classification of Soybean C2H2 Zinc Finger Proteins and Their Expression Analysis in Legume-Rhizobium Symbiosis.

Root nodule symbiosis (RNS) is one of the most productive and economical systems for nitrogen fixation, and previous studies have shown that several nodule-specific C2H2-zinc finger proteins (ZFPs) play important roles in symbiosis establishment and nodule function. However, C2H2-ZFPs are the most widespread ZFPs in eukaryotes, and a great variation of structure and function exist among the family members. It remains largely unclear whether or not special types of C2H2-ZF genes participate in symbiosis, especially in soybean. In the present study, we performed a genome-wide survey of soybean C2H2-ZF genes, and 321 soybean C2H2-ZF genes were identified and classified into 11 clearly distinguishable subsets (Gm-t1-SF, Gm-t2-SF, Gm-1i-Q-SF, Gm-1i-M-SF, Gm-1i-Z-SF, Gm-1i-D-SF, Gm-2i-Q-SF, Gm-2i-M-SF, Gm-2i-Mix-SF, Gm-3i-SF, and Gm-4i-SF) based on the arrangements, numbers, and types of C2H2-ZF domains. Phylogenetic and gene ontology analyses were carried out to assess the conserved sequence and GO function among these subsets, and the results showed that the classification of soybean C2H2-ZFPs was reasonable. The expression profile of soybean C2H2-ZFPs in multiple tissues showed that nearly half of soybean C2H2-ZFPs within different subsets had expressions in nodules, including a clustering branch consisting of 11 Gm-1i-Q-SF genes specifically expressed in symbiotic-relative tissues. RNA-Seq was used to identify symbiosis-related soybean C2H2-ZFPs, and the expression pattern of the soybean C2H2-ZFPs in roots and nodules at different development stages showed that soybean C2H2-ZFPs mainly played roles in nodule development or nodule function rather than nodulation signal transduction, and nearly half of these genes had high expressions and/or different expression patterns during soybean nodule development, especially for the six clustering branches of genes consisting of different subsets of C2H2-ZFPs. Furthermore, the selected symbiosis-related soybean C2H2-ZFPs might function in legume-rhizobium symbiosis through regulating or interacting with other key proteins. Taken together, our findings provided useful information for the study on classification and conservative function of C2H2-ZFPs, and offered solid evidence for investigation of rhizobium symbiosis-related C2H2-ZFPs in soybean or other legumes.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.