Alfalfa (Medicago sativa L.), an important forage crop with high nutritional value and good palatability, plays a vital role in the development of animal husbandry in China. In Northeast China, there are vast areas of saline–alkali land that remain undeveloped. Given that alfalfa is a highly adaptable forage crop, exploring its salt tolerance at the molecular transcriptional level and identifying salt-tolerant genes has great significance for breeding salt-resistant alfalfa varieties. This also provides valuable genetic resources for better utilization of saline–alkali land. In this study, we conducted two rounds of screening on 41 alfalfa varieties and identified WL168 as a salt-sensitive variety and Longmu801 as a salt-tolerant variety. After 7 days of 300 mM salt stress, both varieties showed a decreasing trend in plant height, fresh weight, and dry weight over time, but Longmu801 demonstrated better water retention ability compared to WL168. Chlorophyll content also declined, but chlorophyll a and total chlorophyll levels in Longmu801 were higher than in WL168. Hydrogen peroxide and malondialdehyde levels increased overall, but Longmu801 had significantly lower levels than WL168 under prolonged stress. Both varieties showed increasing trends in soluble sugars, proline, and antioxidant enzymes (SOD, POD, CAT), with Longmu801 significantly outperforming WL168. This suggests that the two varieties share similar growth and physiological response mechanisms, with their differences primarily arising from variations in indicator levels. In the above, comparisons between varieties were conducted based on the relative values of the indicators in relation to their controls. Transcriptomic analysis revealed that under salt stress, Longmu801 had 16,485 differentially expressed genes (DEGs) relative to its control, while WL168 had 18,726 DEGs compared to its control. Among these, 2164 DEGs shared the same expression trend, with GO functions enriched in response to oxidative stress, nucleus, plasma membrane, and others. The KEGG pathways were enriched in phenylpropanoid biosynthesis, protein processing in the endoplasmic reticulum, starch and sucrose metabolism, and others. This suggests that alfalfa’s transcriptional response mechanism to salt stress involves these pathways. Additionally, the variety-specific DEGs were also enriched in the same KEGG pathways and GO functions, indicating that the differences between the two varieties stem from their unique stress-responsive DEGs, while their overall mechanisms for coping with stress remain similar. To further identify salt stress-related genes, this study conducted WGCNA analysis using 32,683 genes and physiological indicators. Six modules closely related to physiological traits were identified, and the top five genes ranked by degree in each module were selected as hub genes. Further analysis of these hub genes identified five genes directly related to salt stress: Msa085011, Msa0605650, Msa0397400, Msa1258740, and Msa0958830. Mantel test analysis revealed that these genes showed strong correlations with physiological indicators. This study will provide important insights for breeding salt-tolerant alfalfa varieties.

Selenium (Se) plays an important role in the growth of plants. Alfalfa (Medicago sativa L.) is a perennial legume forage crop with high nutritional value and Se-rich functions. Many studies have shown that selenium can promote alfalfa growth, but few have explored the molecular biology mechanisms behind this effect. In this study, alfalfa was divided into two groups. One group was sprayed with sodium selenite (Na₂SeO₃) and the other group was sprayed with distilled water as a control. This study determined the growth, reproductive traits, physiological changes, transcriptome and metabolome of both groups of alfalfa. We found that foliar spraying of 100 mg/L Na₂SeO₃ could significantly increase the growth rate, dry weight, total Se content, amount of pollen per flower, pollen viability, pod spirals, and seed number per pod of alfalfa plants. The level of chlorophyll, soluble protein, proline, and glutathione also increased dramatically in Na₂SeO₃-sprayed alfalfa seedlings. After transcriptome and metabolome analysis, a total of 614 differentially expressed genes (DEGs) and 1500 differentially expressed metabolites (DEMs), including 26 secondary differentially metabolites were identified. The DEGs were mainly enriched in MAPK signaling pathway, phenylpropanoid biosynthesis, isoflavonoid biosynthesis, cutin, suberine, and wax biosynthesis, and glycerolipid metabolism. The DEMs were mainly enriched in flavone and flavonol biosynthesis, carbon metabolism, glyoxylate and dicarboxylate metabolism, nitrogen metabolism, and phenylpropanoid biosynthesis. Integrative analysis of transcriptome and metabolome showed that the foliar spraying of Na₂SeO₃ mainly affects phenylpropanoid biosynthesis to promote alfalfa growth.

Leymus chinensis is a perennial forage grass that has good palatability, high yield and high feed value, but seed dormancy is a major problem limiting the widespread cultivation of L. chinensis. Here, we performed transcriptomic and metabolomic analysis of hulled and de-hulled seeds of L. chinensis treated with or without GA₃ to investigate the changes in gene and metabolites associated with dormancy release induced by GA₃. The germination test revealed that the optimum concentration of GA₃ for disruption of L. chinensis seed dormancy was 577 μM. A total of 4327 and 11,919 differentially expressed genes (DEGs) and 871 and 650 differentially abundant metabolites were identified in de-hulled and hulled seeds treated with GA₃, respectively, compared with seeds soaked in sterile water. Most of the DEGs were associated with starch and sucrose metabolism, protein processing in the endoplasmic reticulum, endocytosis and ribosomes. Furthermore, isoquinoline alkaloid biosynthesis, tyrosine metabolism, starch and sucrose metabolism, arginine and proline metabolism, and amino sugar and nucleotide sugar metabolism were significantly enriched pathways. Integrative analysis of the transcriptomic and metabolomic data revealed that starch and sucrose metabolism is one of the most important pathways that may play a key role in providing carbon skeletons and energy supply for the transition of L. chinensis seeds from a dormant state to germination by suppressing the expression of Cel61a, egID, cel1, tpsA, SPAC2E11.16c and TPP2, enhancing the expression of AMY1.1, AMY1.2, AMY1.6 and GLIP5, and inhibiting the synthesis of cellobiose, cellodextrin, and trehalose while promoting the hydrolysis of sucrose, starch, cellobiose, cellodextrin, and trehalose to glucose. This study identified several key genes and provided new insights into the molecular mechanism of seed dormancy release induced by GA₃ in L. chinensis. These putative genes will be valuable resources for improving the seed germination rate in future breeding studies.

Selenium (Se) is an essential trace element in living systems. In this study, we applied a spray of 50 mg/kg sodium selenite to alfalfa (Medicago sativa L.) at different stages of development (bud, initial bloom, and full bloom stages). After 0, 1, 3, 7, and 45 days of ensiling, we assessed the fermentation quality, chemical composition, and bacterial community of the alfalfa. Our findings indicated that the addition of Se led to an increase in the Se content and a decrease in the pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents. As ensiling fermentation progressed, the Se treatments increased the relative abundance of Lactobacillus, which was significantly greater than that in the control group (42.44% vs. 3.76%). In conclusion, the addition of sodium selenate to silage additive ensures the quality of the silage and reduces bacterial community diversity. This study provides valuable insights for the investigation of Se enrichment in alfalfa haylage silage.