Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

The long noncoding RNA LAL contributes to salinity tolerance by modulating LHCB1s' expression in Medicago truncatula.

Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s' expression in M. truncatula.

An effective electricity worker identification approach based on Yolov3-Arcface.

To address the issues of low efficiency and high complexity of detection models for electric power workers in distribution rooms, the electric power worker identification approach is proposed. The ArcFace loss function is used as the coordinate regression loss of the target box. According to the score, the template box with the highest score is selected for prediction, which speeds up the rate of convergence. Dimensional clustering is used to set template boxes for bounding box prediction. The experimental results show that the improved YOLOv3 is a high-performance and lightweight model. The electric power worker identification approach proposed in this paper has a high-speed recognition process, accurate recognition results. The effectiveness of the approach is verified with better detection performance and robustness.

Physiological and lipidomic response of exogenous choline chloride alleviating salt stress injury in Kentucky bluegrass (Poa pratensis).

Introduction: Choline participates in plant stress tolerance through glycine betaine (GB) and phospholipid metabolism. As a salt-sensitive turfgrass species, Kentucky bluegrass (Poa pratensis) is the main turfgrass species in cool-season areas. Methods: To improve salinity tolerance and investigate the effects of choline on the physiological and lipidomic responses of turfgrass plants under salinity stress conditions, exogenous choline chloride was applied to Kentucky bluegrass exposed to salt stress. Results: From physiological indicators, exogenous choline chloride could alleviate salt stress injury in Kentucky bluegrass. Lipid analysis showed that exogenous choline chloride under salt-stress conditions remodeled the content of phospholipids, glycolipids, and lysophospholipids. Monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine content were increased and phosphatidic acid content were decreased in plants after exogenous choline chloride under salt treatment. Plant leaf choline content increased, but GB was not detected in exogenous choline chloride treatment plants under nonstress or salt-stress conditions. Discussion: GB synthesis pathway related genes showed no clear change to choline chloride treatment, whereas cytidyldiphosphate-choline (CDP-choline) pathway genes were upregulated by choline chloride treatment. These results reveal that lipid remodeling through choline metabolism plays an important role in the salt tolerance mechanism of Kentucky bluegrass. Furthermore, the lipids selected in this study could serve as biomarkers for further improvement of salt-sensitive grass species.

The role of Class â ¡ KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXII) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (orthologue of Arabidopsis Class M KNOX) and NO APICAL MERISTEM (orthologue of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for MtKNOX4-mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula.

Mechanism of microbial regulation on methane metabolism in saline-alkali soils based on metagenomics analysis.

Saline-alkali soils constitute a globally important carbon pool that plays a critical role in soil carbon dioxide (CO2) and methane (CH4) fluxes. However, the relative importance of microorganisms in the regulation of CH4 emissions under elevated salinity remains unclear. Here, we report the composition of CH4 production and oxidation microbial communities under five different salinity levels in the Yellow River Delta, China. This study also obtained the gene number of microbial CH4 metabolism via testing the soil metagenomes, and further investigated the key soil factors to determine the regulation mechanism. Spearman correlation analysis showed that the soil electrical conductivity, salt content, and Na+, and SO42- concentrations showed significantly negative correlations with the CO2 and CH4 emission rates, while the NO2--N concentration and NO2-/NO3- ratio showed significantly positive correlations with the CO2 and CH4 emission rates. Metabolic pathway analysis showed that the mcrA gene for CH4 production was highest in low-salinity soils. By contrast, the relative abundances of the fwdA, ftr, mch, and mer genes related to the CO2 pathway increased significantly with rising salinity. Regarding CH4 oxidation processes, the relative abundances of the pmoA, mmoB, and mdh1 genes transferred from CH4 to formaldehyde decreased significantly from the control to the extreme-salinity plot. The greater abundance and rapid increase of methanotrophic bacteria compared with the lower abundance and slow increase in methanogenic archaea communities in saline-alkali soils may have increased CH4 oxidation and reduced CH4 production in this study. Only CO2 emissions positively affected CH4 emissions from low- to medium-salinity soils, while the diversities of CH4 production and oxidation jointly influenced CH4 emissions from medium- to extreme-salinity plots. Hence, future investigations will also explore more metabolic pathways for CH4 emissions from different types of saline-alkali lands and combine the key soil enzymes and regulated biotic or abiotic factors to enrich the CH4 metabolism pathway in saline-alkali soils.

Genome-Wide Identification and Characterization of the Msr Gene Family in Alfalfa under Abiotic Stress.

Alfalfa (Medicago sativa) is an important leguminous forage, known as the "The Queen of Forages". Abiotic stress seriously limits the growth and development of alfalfa, and improving the yield and quality has become an important research area. However, little is known about the Msr (methionine sulfoxide reductase) gene family in alfalfa. In this study, 15 Msr genes were identified through examining the genome of the alfalfa "Xinjiang DaYe". The MsMsr genes differ in gene structure and conserved protein motifs. Many cis-acting regulatory elements related to the stress response were found in the promoter regions of these genes. In addition, a transcriptional analysis and qRT-PCR (quantitative reverse transcription PCR) showed that MsMsr genes show expression changes in response to abiotic stress in various tissues. Overall, our results suggest that MsMsr genes play an important role in the response to abiotic stress for alfalfa.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Dissection of the response mechanism of alfalfa under phosphite stress based on metabolomic and transcriptomic data.

Phosphite, a reduced form of phosphate, inhibits the growth and even has toxic effect on plants. To learn more about the mechanism of alfalfa responses to phosphite, the morphological and physiological characteristics, and the metabolites and transcript levels were comprehensively analyzed following the exposure of alfalfa seedlings to phosphite and phosphate under greenhouse conditions. The results showed that phosphite inhibited seedling growth and photosynthesis. However, the absorption efficiency of phosphite was higher than that of phosphate in roots, which was supported by increased total phosphorus concentration of 16.29% and 52.30% on days 8 and 12. Moreover, phosphite stress affected the synthesis of lipids and carbohydrates, which were reflected in enhanced glycolipid and sulfolipid in roots and amylose in shoots. Phosphite stress resulted in a decrease in indole acetic acid (IAA) in the whole plant and zeatin in the shoots, which could enable alfalfa to adapt to the phosphite environment. Some genes involved in phosphate starvation response included SPX, phosphate response regulator2, and inorganic phosphate transporter 1-4 (PHT1;4) in roots were affected by phosphite stress. In addition, some genes that are involved in stress responses and DNA repair were induced by phosphite stress. These observations together suggest that alfalfa responds to phosphite stress by inhibiting growth, regulating the genes induced by phosphate starvation, improving oxidative protection, promoting DNA repair, and adjusting the IAA and zeatin signaling transductions. Our findings provide novel insights into the molecular response to phosphite stress in alfalfa.

Evaluation of the tolerance and forage quality of different ecotypes of seashore paspalum.

Seashore paspalum is a halophytic, warm-season grass with wide applications. It is noted for its superior salt tolerance in saline environments; however, the nutritive value of seashore paspalum and the effect of salinity remains to be determined. Therefore, this study aimed to evaluate the relationship between agronomic traits and forage quality and identified the effects of short-term high-salt stress (1 week, 700 mM NaCl) on the growth and forage nutritive value of 16 ecotypes of seashore paspalum. The salt and cold tolerances of the seashore paspalum ecotypes were assessed based on the survival rate following long-term high-salt stress (7 weeks, 700 mM NaCl) and exposure to natural low temperature stress. There were significant genetic (ecotype-specific) effects on plant height, leaf-stem ratio, and survival rate of seashore paspalum following salt or low temperature stress. Plant height was significantly negatively correlated with the leaf-stem ratio (r = -0.63, P<0.01), but the heights and leaf-stem ratios were not significantly correlated with the fresh weight (FW) and dry weight (DW) of the shoots. High salinity decreased the FW and DW of the shoots by 50.6% and 23.6%, respectively, on average. Seashore paspalum exhibited outstanding salt tolerance and forage quality at high salinity. The survival rate of the different ecotypes of seashore paspalum varied from 6.5% to 49.0% following treatment with 700 mM NaCl for 7 weeks. The crude protein (CP) content of the control and treatment groups (700 mM NaCl) was 17.4% and 19.3%, respectively, of the DW on average, and the CP content of most ecotypes was not significantly influenced by high salinity. The average ether extract (EE) content ranged from 4.6% to 4.4% of the DW under control and saline conditions, respectively, indicating that the influence was not significant. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of the control group were 57.4% and 29.8%, respectively, of the DW on average. Salt stress reduced the content of NDF and ADF to 50.2% and 25.9%, respectively, of the DW on average. Altogether, the results demonstrated that stress did not have any significant effects on the CP and EE content of most ecotypes, but reduced the NDF and ADF content and improved relative feed value (RFV). The results obtained herein support the notion that seashore paspalum is a good candidate for improving the forage potential of saline soils and can provide useful guidelines for livestock producers.

Genome-wide identification of growth-regulating factor transcription factor family related to leaf and stem development in alfalfa.

Growth-regulating factors (GRFs) play crucial roles in plant growth and stress response. To date, there have been no reports of the analysis and identification of the GRF transcription factor family in alfalfa. In this study, we identified 27 GRF family members from alfalfa (Medicago sativa L.) "Xinjiang Daye", and analyzed their physicochemical properties. Based on phylogenetic analysis, these MsGRFs were divided into five subgroups, each with a similar gene structure and conserved motifs. MsGRFs genes are distributed on 23 chromosomes, and all contain QLQ and WRC conserved domains. The results of the collinearity analysis showed that all MsGRFs are involved in gene duplication, including multiple whole-genome duplication or segmental duplication and a set of tandem duplication, indicating that large-scale duplication is important for the expansion of the GRF family in alfalfa. Several hormone-related and stress-related cis-acting elements have been found in the promoter regions of MsGRFs. Some MsGRFs were highly expressed in young leaves and stems, and their expression decreased during development. In addition, the leaf size of different varieties was found to vary, and MsGRF1 to 4, MsGRF18 to 20, and MsGRF22 to 23 were differentially expressed in large and small leaf alfalfa varieties, suggesting that they are critical in the regulation of leaf size. The results of this study can benefit further exploration of the regulatory functions of MsGRFs in growth and development, and can identify candidate genes that control leaf size development.

Effects of autotoxicity and allelopathy on seed germination and seedling growth in Medicago truncatula.

Autotoxicity is a form of intraspecific allelopathy, in which a plant species inhibits the establishment or growth of the same species through the release of toxic chemical compounds into the environment. The phenomenon of autotoxicity in crops is best traced in alfalfa (Medicago sativa). A close relative of alfalfa, M. truncatula, has been developed into an excellent model species for leguminous plants. However, it is not known whether M. truncatula has autotoxicity. In this study, M. truncatula root exudates showed a negative impact on the growth of M. truncatula seedlings, indicating autotoxicity. Detailed analyses with plant extracts from M. truncatula and alfalfa revealed varying degrees of suppression effects in the two species. The extracts negatively affected seed germination potential, germination rate, radicle length, hypocotyl length, synthetic allelopathic effect index, plant height, root growth, fresh weight, dry weight, net photosynthetic rate, transpiration rate, and stomatal conductance in both M. truncatula and alfalfa. The results demonstrated that autotoxicity and allelopathic effects exist in M. truncatula. This opens up a new way to use M. truncatula as a model species to carry out in-depth studies of autotoxicity and allelopathy to elucidate biochemical pathways of allelochemicals and molecular networks controlling biosynthesis of the chemicals.

Comprehensive transcriptomic and metabolomic profiling reveals the differences between alfalfa sprouts germinated with or without light exposure.

Alfalfa sprouts are among the most nutritionally rich foods, and light exposure is a critical factor in determining their biomass and quality. However, detailed metabolic and molecular differences between yellow and green alfalfa sprouts remain unclear. In this study, comprehensive metabolomic and transcriptomic analyses were integrated to evaluate the nutrient composition of alfalfa sprouts during germination with or without light exposure. Differentially expressed genes and differentially accumulated metabolites in green and yellow alfalfa sprouts were significantly enriched in secondary metabolic pathways, such as the isoflavonoid biosynthesis pathway. Green alfalfa sprouts contained a wide variety of lipids, flavonoids, phenolic acids, and terpenoids, among which the top three upregulated were calycosin, methyl gallate, and epicatechin 3-gallate, whereas yellow alfalfa sprouts contained relatively more isoquercitrin. These results provide new insights into the nutritional value and composition of alfalfa sprouts under different germination regimes.

The formation of stipule requires the coordinated actions of the legume orthologs of Arabidopsis BLADE-ON-PETIOLE and LEAFY.

Stipule morphology is a classical botanical key character used in plant identification. Stipules are considerably diverse in size, function and architecture, such as leaf-like stipules, spines or tendrils. However, the molecular mechanism that regulates stipule identity remains largely unknown. We isolated mutants with abnormal stipules. The mutated gene encodes the NODULE ROOT1 (MtNOOT1), which is the ortholog of BLADE-ON-PETIOLE (BOP) in Medicago truncatula. We also obtained mutants of MtNOOT2, the homolog of MtNOOT1, but they do not show obvious defects in stipules. The mtnoot1 mtnoot2 double mutant shows a higher proportion of transformation from stipules to leaflet-like stipules than the single mutants, suggesting that they redundantly determine stipule identity. Further investigations show that MtNOOTs control stipule initiation together with SINGLE LEAFLET1 (SGL1), which functions in development of lateral leaflets. Increasing SGL1 activity in mtnoot1 mtnoot2 is sufficient for the transformation of stipules to leaves. Moreover, MtNOOTs inhibit SGL1 expression during stipule development, which is probably conserved in legume species. Our study proposes a genetic regulatory model for stipule development, specifically with regard to the MtNOOTs-SGL1 module, which functions in two phases of stipule development, first in the control of stipule initiation and second in stipule patterning.

A High-Quality Genome Assembly of Sorghum dochna.

Sweet sorghum (Sorghum dochna) is a high-quality bio-energy crop that also serves as food for humans and animals. However, there is little information on the genomic characteristics of S. dochna. In this study, we presented a high-quality assembly of S. dochna with PacBio long reads, Illumina short reads, high-throughput chromosome capture technology (Hi-C) sequencing data, gene annotation, and a comparative genome analysis. The results showed that the genome of S. dochna was assembled to 777 Mb with a contig N50 of 553.47 kb and a scaffold N50 of 727.11 kb. In addition, the gene annotation predicted 37,971 genes and 39,937 transcripts in the genome of S. dochna. A Venn analysis revealed a set of 7,988 common gene annotations by integrating five databases. A Cafe software analysis showed that 191 gene families were significantly expanded, while 3,794 were significantly contracted in S. dochna. A GO enrichment analysis showed that the expanded gene families were primarily clustered in the metabolic process, DNA reconstruction, and DNA binding among others. The high-quality genome map constructed in this study provides a biological basis for the future analysis of the biological characteristics of S. dochna, which is crucial for its breeding.

Carex rigescens caffeic acid O-methyltransferase gene CrCOMT confer melatonin-mediated drought tolerance in transgenic tobacco.

Melatonin is an important, multifunctional protective agent against a variety of abiotic and biotic stressors in plants. Caffeic acid O-methyltransferase (COMT) catalyzes the last step of melatonin synthesis in plants and reportedly participates in the regulation of stress response and tolerance. However, few studies have reported its function in melatonin-mediated drought resistance. In this study, CrCOMT was identified and was strongly induced by drought stress in Carex rigescens. CrCOMT overexpression in transgenic tobacco increased tolerance to drought stress with high levels of seed germination, relative water content, and survival rates. CrCOMT overexpression in tobacco improved membrane stability, and plants exhibited lower relative electrolytic leakage and malondialdehyde content, as well as higher photochemical efficiency than the wildtype (WT) under drought stress. The transgenic plants also had higher levels of proline accumulation and antioxidant enzyme activity, which decreased oxidative stress damage due to reactive oxygen species (ROS) hyperaccumulation under drought stress. The transcription of drought stress response and ROS scavenging genes was significantly higher in the CrCOMT overexpression plants than in the WT plants. In addition, CrCOMT transgenic tobacco plants exhibited higher melatonin content under drought stress conditions. Exogenous melatonin was applied to C. rigescens under drought stress to confirm the function of melatonin in mediating drought tolerance; the relative water content and proline content were higher, and the relative electrolytic leakage was lower in melatonin-treated C. rigescens than in the untreated plants. In summary, these results show that CrCOMT plays a positive role in plant drought stress tolerance by regulating endogenous melatonin content.

A de novo assembled high-quality chromosome-scale Trifolium pratense genome and fine-scale phylogenetic analysis.

BACKGROUND: Red clover (Trifolium pratense L.) is a diploid perennial temperate legume with 14 chromosomes (2n = 14) native to Europe and West Asia, with high nutritional and economic value. It is a very important forage grass and is widely grown in marine climates, such as the United States and Sweden. Genetic research and molecular breeding are limited by the lack of high-quality reference genomes. In this study, we used Illumina, PacBio HiFi, and Hi-C to obtain a high-quality chromosome-scale red clover genome and used genome annotation results to analyze evolutionary relationships among related species. RESULTS: The red clover genome obtained by PacBio HiFi assembly sequencing was 423 M. The assembly quality was the highest among legume genome assemblies published to date. The contig N50 was 13 Mb, scaffold N50 was 55 Mb, and BUSCO completeness was 97.9%, accounting for 92.8% of the predicted genome. Genome annotation revealed 44,588 gene models with high confidence and 52.81% repetitive elements in red clover genome. Based on a comparison of genome annotation results, red clover was closely related to Trifolium medium and distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum among legumes. Analyses of gene family expansions and contractions and forward gene selection revealed gene families and genes related to environmental stress resistance and energy metabolism. CONCLUSIONS: We report a high-quality de novo genome assembly for the red clover at the chromosome level, with a substantial improvement in assembly quality over those of previously published red clover genomes. These annotated gene models can provide an important resource for molecular genetic breeding and legume evolution studies. Furthermore, we analyzed the evolutionary relationships among red clover and closely related species, providing a basis for evolutionary studies of clover leaf and legumes, genomics analyses of forage grass, the improvement of agronomic traits.

Metagenomic analysis reveals antibiotic resistance genes and virulence factors in the saline-alkali soils from the Yellow River Delta, China.

The propagation of antibiotic resistance genes (ARGs) and virulence factors (VFs) in the saline-alkali soils and associated environmental factors remains unknown. In this study, soil samples from the Yellow River Delta, China with four salinity gradients were characterized by their physiochemical properties, and shotgun metagenomic sequencing was used to identify the ARGs and VFs carried by microorganisms. Soil salinization significantly reduced the relative abundances of Solirubrobacterales, Propionibacteriales, and Micrococcales, and quorum sensing in microorganisms. The number of ARGs and VFs significantly decreased in medium and high saline-alkali soils as compared with that in non-saline-alkali soil, however, the ARGs of Bacitracin, and the VFs of iron uptake system, adherence, and stress protein increased significantly in saline-alkali soils. Spearman analysis showed that the ARGs of fluoroquinolone, tetracycline, aminoglycoside, beta-lactam, and tigecycline were positively correlated with soil pH. Similarly, we observed an increased contribution to the ARGs and VFs by taxa belonging to Solirubrobacterales and Gemmatimonadales, respectively. The control plot was mainly improved from saline-alkali land through application of animal manure, which tended to contain large amounts of ARGs and VFs in this study. Further studies are needed to observe ARGs and VFs in the saline-alkali land for multiple years and speculate the potential risks caused by varied ARGs and VFs to the soil ecosystem and human health.