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To predict the role of the MsICE gene family in the response to abiotic stress, in this study, bioinformatics analysis and real-time fluorescence quantitative PCR were performed. Alfalfa (Medicago sativa) is one of the most economically valuable crops globally. Inducer of CBF expression (ICE), which is part of the basic helix–loop–helix (bHLH) transcription factor (TF) family, acts as a key regulator of cold tolerance. Despite this, there is little information available about ICE genes in alfalfa. Therefore, we studied the function of ICE TFs in alfalfa. We identified 11 MsICE genes from the alfalfa genome and classified them into two groups. Analysis of the protein motif and gene structure revealed relatively high conservation among subgroups of the tightly clustered MsICE genes. Through synteny analysis, we detected duplication events in the MsICE gene family, suggesting that the ICE gene family was formed through fragment duplications. All the MsICE proteins were located in the nucleus according to subcellular localization predictions. The promoter cis-regulatory elements of MsICE genes are largely involved in light (Box 4), hormone (ABRE), and stress (MYB) responses. The MsICE01/MsICE07/MsICE09/MsICE10/MsICE11 genes contained MYB- and MYC-binding motifs, indicating an association with abiotic stress. The specific expression patterns of MsICE genes in leaves were revealed by examining their expression patterns in different tissues. These findings suggest that these genes may sense external environmental changes through leaves. Abiotic stress can cause striking upregulation of MsICE07 (PCA score: −4.03) and MsICE10 (PCA score: −4.05) expression. In this study, candidate genes associated with cold stress were identified, and subsequent molecular biological analyses allowed elucidation of the biological functions of these genes in alfalfa. This research provides a theoretical foundation for enhancing alfalfa yield and quality under cold conditions.

Waxy maize (Zea mays L. sinensis Kulesh) is highly regarded for its high nutritional content and unique taste. Although the stalks and leaves contain high carbohydrate levels after ear harvesting, inadequate crude protein (CP) limits the utilization and promotion of waxy maize silage in animal husbandry. In this study, waxy maize and fodder soybeans were mixed for sowing in different proportions [1:0 (CK), 1:1 (A1), 1:2 (A2), 1:3 (A3), and 1:4 (A4)] to investigate the effects of different mixing ratios on the growth of the waxy maize, the chemical indices, fermentation quality, and the microbial community of the mixed silage after ear harvesting. The mixed planting of waxy maize and fodder soybeans in different proportions had no effect on the yield and quality of the waxy maize ears and increased the aboveground biomass after ear harvesting. After ear harvesting, the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents significantly decreased, and the CP content and relative feeding value (RFV) gradually increased in the mixed silage. The pH of the treatments was lower than 4.2 except for A4, and the lowest ammonia nitrogen (AN) concentration was observed in A3. With increasing proportions of fodder soybeans, the abundance of beneficial bacteria increased and that of harmful bacteria decreased; Firmicutes and Lactobacillus were the dominant phylum and genus, respectively, and both increased gradually. Redundancy analysis (RDA) revealed that the fermentation indices affecting the microbial community composition in the silage were inconsistent among the different mixed sowing combinations. The Mantel test showed that the composition of the microbial communities in the treatments was significantly correlated with the ADF, water-soluble carbohydrate (WSC), and propionic acid (PA) contents. Comprehensive analysis revealed that the optimal mixed sowing ratio of waxy maize to fodder soybeans was 1:3, and waxy maize and fodder soybeans silage can increase the utilization of aboveground biomass and improve the fermentation quality and feeding quality of silage by changing the microbial community. These findings lay a certain theoretical foundation for improving the utilization of waxy maize.

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT–PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses.