Mingjiu Wang

Agropyron belongs to Triticeae of the Poaceae family; its plants are rich in nutrients and have good palatability, serving as important forage grasses in the temperate zone of the Northern Hemisphere. The accurate identification of Agropyron germplasm resources is essential for breeding. However, the morphological variation of Agropyron is complex, making identification on the basis of traditional morphological characteristics difficult. Molecular markers are effective tools for the rapid and accurate identification of many forage plants. Therefore, in this study, second-generation high-throughput sequencing, chloroplast genome assembly, chloroplast genome comparison, and phylogenetic analysis were conducted on 48 Agropyron samples from 5 species and 2 varieties. Using next-generation sequencing reads, the chloroplast genome was assembled and annotated. Analyses of repetitive sequences, codon usage bias, and nucleotide diversity revealed high conservation: the genome length ranged from 135,321 to 135,564 bp, the GC content was stable at 38%, and all the genomes encoded 132 consistent genes. Simple repeat sequences (SSRs) were mainly of the A/T type (21–28), and direct repeats were predominant in long repeat sequences (29–31). Each sample harbored 29–34 tandem repeats, with Agropyron mongolicum Keng (0506) showing the maximum (34). The long repeat sequence included 13 palindromic and 29–31 forward repeats in most samples, with no reverse or complementary repeats detected. The codon endings were predominantly A/U (RSCU > 1 codons accounted for 48.4%). Nucleotide diversity analysis revealed 6 hypervariable regions (e.g., psbA and rpl32), but the hypervariable regions did not contain enough informative sites to distinguish the five species of Agropyron. In the phylogenetic tree, all Agropyron samples formed a highly supported monophyletic clade (bootstrap = 100), but the intragenus relationships among the five species and two varieties remained unresolved, most likely due to insufficient phylogenetically informative sites because there were only 178 phylogenetically informative sites in the 135,978 chloroplast effective sites (0.13%). We also did not find species-specific chloroplast genome SNPs or indels. PCA revealed that different species could not be separated on the first two principal components. STRUCTURE analysis based on Angiosperms353 markers showed that K = 2 was the optimal grouping result, but the genetic grouping did not match the taxonomy of Agropyron, which was consistent with the results of analysis based on the chloroplast genome. Our study comprehensively revealed the chloroplast genome information of Agropyron plants and its consistency across the genus.

Background: Alfalfa (Medicago sativa) is a high-quality legume forage. Phosphorus (P) is essential for plant productivity. Approximately half of global arable soils exhibit available-P deficiency, constraining plants growth and yield. This study aimed to explore how hormones mediate the responses of alfalfa to phosphate starvation. Methods: We studied the morphological and physiological changes alfalfa (Zhongmu No.3) seedlings treated with auxin, ethylene and gibberellin under conditions of phosphate deficiency. 20-day-old alfalfa seedlings were exposed to varying concentrations of hormones in Hoagland solution. Changes in root morphology, photosynthesis, physiological characters and the levels of phosphate starvation induced (PSI) genes expression were monitored. Result: IAA stimulated lateral-root growth (1.16-fold vs. control), up-regulated the organic phosphate transporter (PHT1-4) and purple acid phosphatase (PAP23) and inhibited primary-root elongation. Ethylene promoted the growth of root hairs and 10 μM aminocyclopropane-1-carboxylic acid (ACC) increased the elongation of roots and their surface area.1 μM ACC boosted shoot and root acid phosphatase activity and up-regulated 1-aminocyclopropane-1-carboxylate oxidase (ACO), whereas 5 μM gibberellin A3 (GA3) promoted stem and leaf growth, increasing stem diameter by 16.10% and leaf area by 36.90%, while elevating sulfoquinovosyl diacylglycerol (SQD2) content and DELLA expression. There was a strong negative correlation (-0.79) between the plant height and root-to-shoot ratio and a positive correlation (0.62) between the number of lateral roots and the activity of acid phosphatase (ACP) in the roots. The most effective concentrations of hormones was 1 μM ACC, which enhanced the plant height, stem diameter, lateral root formation, chlorophyll biosynthesis and nitrogen content and induced the levels of expression of PSI and key genes in hormone signaling.

Fertilization is a critical measure for vegetation restoration and ecological reconstruction in degraded grasslands. However, little is known about the long-term effects of different combinations of nitrogen (N), phosphorus (P), potassium (K) on plant and microbial communities in degraded grasslands. This study conducted a four-year (2017–2020) N, P, K addition experiment in the Khorchin Grassland, a degraded typical grassland located in Zhalute Banner, Tongliao City, Inner Mongolia, to investigate the effects of fertilization treatment on plant functional groups and microbial communities after grazing exclusion. Our results showed that the addition of P, NP, and NPK compound fertilizers significantly increased aboveground biomass of the plant community, which is mainly related to the improvement of nutrient availability to promote the growth of specific plant functional groups, especially annual and biennial plants and perennial bunchgrasses. However, the addition of N, P, and NP fertilizers significantly reduced the species diversity of the plant community. At the same time, the addition of N, P, and NP fertilizers and the application of N and NP significantly reduced fungal species diversity but had no significant effect on soil bacteria. Our study provides new insights into the relationships between different types of fertilization and plant community productivity and biodiversity in degraded grasslands over four years of fertilization, which is critical for evaluating the effect of fertilization on the restoration of degraded grassland.

Introduction: Caucasian clover (Trifolium ambiguum Bieb.), a perennial legume forage grass, exhibits strong adaptability and resistance to adverse conditions. Owing to its rhizome specificity, it cannot nodulate or fix nitrogen outside of its original location, which limits its promotion and cultivation. The phenomenon of spontaneous nodulation of Caucasian clover was observed for the first time in Inner Mongolia, and investigation of its nodulation and nitrogen fixation mechanisms assists in the subsequent promotion of Caucasian clover cultivation from a theoretical perspective.Methods: In this study, rhizobia extracted from Caucasian clover were inoculated into the field, and the nodulation part of the root system was investigated during the regreening stage of the second year using transcriptomics and proteomics techniques.Results and Discussion: The study identified 70,280 differentially expressed genes (DEGs) and 770 differentially expressed proteins (DEPs) in total, among which carbonic anhydrase, cyanate lyase, phenylalanine, caffeate/5-hydroxyferulate 3-O-methyltransferase, caffeoyl CoA 3-O methyltransferase, chalcone synthase, and chalcone isomerase may have been the main factors affecting the nodulation and nitrogen fixation of Caucasian clover. This study theoretically contributes to the future genetic validation and selection of Caucasian clover varieties with a strong ability to nodulate and fix nitrogen.

Background: In order to explore the causes of the trait occurrence of multi-leaf clover, the above-ground organs of multi-leaf clover and standard clover were extracted for transcriptome sequencing analysis. Methods: Take the aerial organs (stem and leaf) of standard clover and multi-leaf clover, wipe them clean, put them into cryogenic tubes and store them in liquid nitrogen, bring them back to the laboratory and store them in ultra-low temperature refrigerator for sequencing. Result: The analysis identified 1,985 differentially expressed genes (DEGs), including 794 upregulated and 1,191 downregulated genes, primarily enriched in protein phosphorylation, defense response, nuclear localization, cytosolic activity, oxidoreductase activity and protein binding processes. We found 68 and 121 genes uniquely expressed in polyphyllous and standard clover, respectively. KEGG enrichment analysis revealed a uniquely expressed gene (c68409.graph_c0) in polyphyllous clover involved in DNA replication, nucleotide excision repair, homologous recombination and mismatch repair pathways during meiosis. These findings provide a theoretical foundation for understanding the molecular mechanisms of leafy trait expression in crosses between Mengnong clover No.1 and white clover, supporting future research on stable multi-leaf trait inheritance.

Isoflavones are important secondary metabolites in leguminous plants with significant physiological functions and economic value. However, the genetic variation, transcriptional regulation, and metabolic pathways governing isoflavone biosynthesis in Trifolium pratense remain largely unexplored. In this study, we systematically analyzed 500 accessions of T. pratense for isoflavone content and performed RNA-seq-based transcriptomic profiling to investigate the molecular mechanisms underlying isoflavone biosynthesis. Cluster analysis revealed significant genetic variation, with distinct transcriptional profiles between high- (H1, H2, H3) and low-isoflavone (L1, L2, L3) groups. GO and KEGG pathway enrichment analyses identified key metabolic pathways, including phenylpropanoid metabolism, flavonoid biosynthesis, carbohydrate metabolism, and hormone signaling, which play crucial roles in isoflavone regulation. Weighted gene co-expression network analysis (WGCNA) identified three key gene modules—MEblue, MEturquoise, and MEyellow—strongly correlated with isoflavone content. The MEturquoise and MEyellow modules were upregulated in high-isoflavone groups and were enriched in phenylpropanoid biosynthesis, lipid metabolism, and transcriptional regulation, suggesting that these pathways actively promote isoflavone accumulation. Conversely, the MEblue module, highly expressed in low-isoflavone groups, was enriched in sugar metabolism and MAPK signaling, indicating a potential metabolic flux shift away from secondary metabolism. Moreover, key rate-limiting enzymes (PAL, C4H, 4CL, CHS, and IFS) exhibited higher expression in high-isoflavone groups, highlighting their importance in precursor supply and enzymatic catalysis. Additionally, transcription factors such as MYB, WRKY, and NAC were identified as potential regulators of isoflavone biosynthesis, indicating a complex interplay between hormonal, circadian, and environmental signals. This study provides a comprehensive molecular framework for understanding isoflavone biosynthesis in T. pratense and identifies key regulatory genes and metabolic pathways that could be targeted for genetic improvement, metabolic engineering, and molecular breeding. The findings offer valuable insights into enhancing isoflavone production in legumes for agricultural, nutritional, and pharmaceutical applications.

The study of soil environment in drainage fields is important for environmental management and ecological restoration, and there is currently a knowledge gap in understanding the impact of soil microbial communities in the Shengli coalfield drainage fields and the corresponding ecological effects. To investigate the changes in rhizosphere soil microbial communities of different dominant plants after years of restoration, this study examines the improvement effects of different dominant plants on the soil environment. This study is based on high-throughput sequencing to restore the slope of coal mine spoil after 15 years as the sampling site. The rhizosphere soil of five dominant plants was selected for microbial community analysis, and functional prediction of the microbial community was conducted. The dominant plants selected included Erect Milkvetch (Astragalus adsurgens), Lemongrass (Caragana korshinskii), Alfalfa (Medicago sativa), Phyllanthus pinnatifida (Elymus dahuricus), and Brassica Rapa (Brassica campestris). The results showed that after 15 years of restoration, the soil physicochemical properties in the Phyllanthus pinnatifida group were better than those in the other groups overall, but some of them were inferior to those in the lemon-stripped mallard group. Abundant saprophytic fungal communities were found in different dominant plant groups, mainly belonging to the phyla Ascomycota and Basidiomycota, resulting in significantly higher organic matter content in the dominant plant groups compared to the CK group. The bacterial communities were dominated by the phyla Actinobacteriota, Proteobacteria, Chloroflexi, and Firmicutes. Among these microbial phyla, the Phyllanthus pinnatifida group had higher abundance, which is beneficial for vegetation colonization. Redundancy analysis showed that soil pH was significantly correlated with microbial communities. Organic matter content and pH are the main factors influencing the composition of soil microbial communities, significantly affecting the composition of microorganisms in different groups. After years of restoration, the environment of the Shengli Coalfield's spoil heap has been greatly improved. The planting of various beneficial plants has resulted in significant improvements to the soil microbial community and physicochemical properties, with Phyllanthus pinnatifida having the most positive impact. This lays the foundation for the subsequent restoration of the slope of the spoil heap.

Background: Saline-alkali soil seriously restricts plant growth. Caucasian clover (Trifolium ambiguum Bieb.) is a high-quality leguminous forage with strong adaptability and stress resistance. However, current research on Caucasian clover under saline-alkali stress remains limited. The purpose of this study is to use NaHCO3 solution to carry out alkali stress on Caucasian clover plantsand to understand the changes of plant growth and physiological characteristics under stress, which is of great significance for its popularization and cultivation in alkaline soil. Methods: To study the effects of alkaline stress on the growth and physiological characteristics of Caucasian clover, plants were subjected to varying concentrations (0, 50, 100, 150, 200 mmol/L) of NaHCO3 in pot experiments. Post-stress, parameters such as dry weight, root-to-shoot ratio, main root length, chlorophyll, malondialdehyde (MDA) contentand the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were measured. Result: This study demonstrated that alkaline stress significantly affected the growth and physiological characteristics of Caucasian clover. With increasing alkaline stress, the aboveground and underground dry weights, as well as the main root length, decreased significantly. Photosynthesis was inhibited, with chlorophyll content increasing under low alkaline stress but decreasing sharply at higher concentrations. The activities of antioxidant enzymes (SOD, POD, CAT) increased significantly under low stress but gradually decreased under higher stress levels. Meanwhile, MDA content increased significantly under high alkaline stress. Correlation analysis showed a strong positive correlation between SOD activity and CAT activity, as well as between aboveground dry weight and main root length (P less than 0.01), while chlorophyll content was negatively correlated with MDA content (P less than 0.05).

Caucasian clover (Trifolium ambiguum M.Bieb.) is a perennial legume known for its exceptional cold tolerance, commonly used in agriculture and ecosystems in cold climates. Given the impact of climate change, enhancing the cold adaptation of Caucasian clover is crucial for sustaining agricultural productivity. This study employs metabolomics, transcriptomics, and Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the molecular mechanisms of Caucasian clover’s response to low-temperature stress. Metabolomic analysis showed that low-temperature stress triggered the accumulation of fatty acids, amino acids, and antioxidants, which are critical for maintaining membrane stability and antioxidant capacity, thus protecting the plant from oxidative damage. Transcriptomic analysis revealed significant upregulation of genes involved in cold adaptation, particularly those related to antioxidant defense, membrane lipid repair, and signal transduction, including genes in the ABA signaling pathway and antioxidant enzymes, thereby improving cold tolerance. WGCNA identified gene modules closely linked to cold adaptation, especially those involved in antioxidant defense, fatty acid metabolism, signal transduction, and membrane repair. These modules function synergistically, with coordinated gene expression enhancing cold resistance. This study also investigated the isoflavonoid biosynthesis pathway under low-temperature stress, highlighting its role in enhancing antioxidant capacity and cold tolerance. Low-temperature stress induced upregulation of key enzyme genes, such as Isoflavone Synthase (IFS) and Isoflavone-7-O-Glucosyltransferase (IF7GT), promoting antioxidant metabolite accumulation and further enhancing the plant’s cold adaptation. Overall, this study offers novel molecular insights into the cold tolerance mechanisms of Caucasian clover and provides valuable theoretical support for breeding cold-resistant crops in cold climates.

The rapid increase in fertilizer use has led to the degradation of soil quality, nutrient imbalances, reduced biodiversity, and soil compaction. To address these challenges, hybrid soybeans with efficient biological nitrogen fixation capabilities and broad environmental adaptability were selected as green manure to reduce fertilizer application, thereby improving soil fertility and structure. This study utilized the varieties “Forage Soybean S001” (S001), “Neinong S002 Forage Soybean” (S002), “Mengnong S003 Forage Soybean” (S003), “Mengnong S004 Forage Soybean” (S004), “Mengnong S005 Forage Soybean” (S005), and “Mengnong S006 Forage Soybean” (S006) as green manure materials. The clean tillage (CK) treatment served as the control, ensuring a residue-free soil surface while maintaining consistent practices in soil preparation, irrigation, and field management across all treatments. Field planting of green manure and subsequent crops was conducted at the M-Grass Ecology and Environment (Group) Company’s experimental site in Hohhot in early May of 2023 and 2024. The plots each measured 20 m², with three replications arranged in a randomized block design. A combination of field experiments and laboratory analyses was utilized to investigate the effects of incorporating various hybrid soybean varieties as green manure on soil nutrient levels, soil enzyme activity, soil microbial communities, and the subsequent growth of oats. The results indicated that incorporating various hybrid soybean varieties as green manure into the soil significantly improved soil nutrient levels and enzyme activity. The diversity and richness of soil bacterial communities increased significantly, accompanied by alterations in community structure and composition. These changes enhanced soil fertility and optimized the microbial community structure, promoting the growth of subsequent crops. Among all the treatments, S001 and S004 were particularly effective in enhancing the soil environment, indicating their potential as superior green manure resources for broader application.