Alfalfa (Medicago sativa) is a perennial forage legume that is widely distributed all over the world; therefore, it has an extremely complex genetic background. Though population structure and phylogenetic studies have been conducted on a large group of alfalfa nuclear genomes, information about the chloroplast genomes is still lacking. Chloroplast genomes are generally considered to be conservative and play an important role in population diversity analysis and species adaptation in plants. Here, 231 complete alfalfa chloroplast genomes were successfully assembled from 359 alfalfa resequencing data, on the basis of which the alfalfa chloroplast pan-genome was constructed. We investigated the genetic variations of the alfalfa chloroplast genome through comparative genomic, genetic diversity, phylogenetic, population genetic structure, and haplotype analysis. Meanwhile, the expression of alfalfa chloroplast genes under cold stress was explored through transcriptome analysis. As a result, chloroplast genomes of 231 alfalfa lack an IR region, and the size of the chloroplast genome ranges from 125,192 bp to 126,105 bp. Using population structure, haplotypes, and construction of a phylogenetic tree, it was found that alfalfa populations could be divided into four groups, and multiple highly variable regions were found in the alfalfa chloroplast genome. Transcriptome analysis showed that tRNA genes were significantly up-regulated in the cold-sensitive varieties, while rps7, rpl32, and ndhB were down-regulated, and the editing efficiency of ycf1, ycf2, and ndhF was decreased in the cold-tolerant varieties, which may be due to the fact that chloroplasts store nutrients through photosynthesis to resist cold. The huge number of genetic variants in this study provide powerful resources for molecular markers.

White clover (Trifolium repens L.) is an allopolyploid plant and an excellent perennial legume forage. However, white clover is subjected to various stresses during its growth, with cold stress being one of the major limiting factors affecting its growth and development. Beta-amylase (BAM) is an important starch-hydrolyzing enzyme that plays a significant role in starch degradation and responses to environmental stress. In this study, 21 members of the BAM gene family were identified in the white clover genome. A phylogenetic analysis using BAMs from Arabidopsis divided TrBAMs into four groups based on sequence similarity. Through analysis of conserved motifs, gene duplication, synteny analysis, and cis-acting elements, a deeper understanding of the structure and evolution of TrBAMs in white clover was gained. Additionally, a gene regulatory network (GRN) containing TrBAMs was constructed; gene ontology (GO) annotation analysis revealed close interactions between TrBAMs and AMY (α-amylase) and DPE (4-alpha-glucanotransferase). To determine the function of TrBAMs under various tissues and stresses, RNA-seq datasets were analyzed, showing that most TrBAMs were significantly upregulated in response to biotic and abiotic stresses and the highest expression in leaves. These results were validated through qRT-PCR experiments, indicating their involvement in multiple gene regulatory pathways responding to cold stress. This study provides new insights into the structure, evolution, and function of the white clover BAM gene family, laying the foundation for further exploration of the functional mechanisms through which TrBAMs respond to cold stress.

E3 ubiquitin ligases known as plant U-box (PUB) proteins regulate a variety of aspects of plant growth, development, and stress response. However, the functions and characteristics of the PUB gene family in alfalfa remain unclear. This work involved a genome-wide examination of the alfalfa U-box E3 ubiquitin ligase gene. In total, 210 members were identified and divided into five categories according to their homology with the members of the U-box gene family in Arabidopsis thaliana. The phylogenetic analysis, conserved motifs, chromosomal localization, promoters, and regulatory networks of this gene were investigated. Chromosomal localization and covariance analyses indicated that the MsPUB genes expanded MsPUB gene family members through gene duplication events during evolution. MsPUB genes may be involved in the light response, phytohormone response, growth, and development of several biological activities, according to cis-acting element analysis of promoters. In addition, transcriptome analysis and expression analysis by qRT-PCR indicated that most MsPUB genes were significantly upregulated under cold stress, drought stress, and salt stress treatments. Among them, MsPUBS106 and MsPUBS185 were significantly and positively correlated with cold resistance in alfalfa. MsPUBS110, MsPUBS067, MsPUBS111 and MsPUB155 were comprehensively involved in drought stress, low temperature, and salt stress resistance. All things considered, these discoveries offer fresh perspectives on the composition, development, and roles of the PUB gene family in alfalfa. They also provide theoretical guidance for further investigations into the mechanisms regulating the development, evolution, and stress tolerance of MsPUB.

Medicago sativa L. (alfalfa) is a major forage crop due to its high yield and stress resilience. However, its growth and productivity are often compromised by abiotic stresses, including cold, drought, and salinity. The plant defensin (PDF) gene family plays a crucial role in resistance to abiotic stress. In this study, a total of 11 MsPDF gene family members were identified in the alfalfa genome and classified into three groups. Phylogenetic and conserved motif analyses revealed that the MsPDF genes are highly conserved. Promoter analysis, gene regulatory network analysis (GRN), and gene ontology (GO)-enrichment analyses were used to infer the potential functions of MsPDF genes. The results showed that the gene actively responds to abiotic stress, participates in phytohormonal responses, and regulates plant growth and development through gene interactions. Transcriptome and qRT-PCR analyses showed that most of the MsPDF genes were significantly up-regulated under cold, drought, and salinity stresses. Among them, the MsPDF03 exhibited superior performance under cold stress. The MsPDF04, MsPDF08, and MsPDF09 genes were able to respond positively to drought and salt stresses. Finally, the monomeric, dimeric, and tetrameric structures of the proteins encoded by the MsPDF genes were predicted using AlphaFold 2 software. This study lays the foundation for the identification and evolutionary relationship analysis of the MsPDF gene family, and provides a new reference for subsequent research on abiotic stress resistance.

Marigold (Tagetes erecta L.), a crop of significant medicinal, ornamental, and economic value, faces severe industrialization challenges due to weed-induced yield losses (up to 60%). This study aims to identify safe and highly efficient herbicides for marigold, assess their effects on dominant weeds and crop safety, and provide a practical basis for large-scale cultivation. We evaluated 11 pre-emergence herbicides, 13 post-emergence herbicides, and agronomic practices (plastic mulch) through three field trials to optimize weed control, crop safety, and productivity. In Experiment 1, pre-emergence applications of pendimethalin (35% SC) and oxyfluorfen (240 g/L EC) under plastic mulch suppressed 85–99% of grass and broad-leaved weeds, elevating marigold yield to 1655.6 kg/667 m² and increasing lutein content by 10.7% compared to controls, with no phytotoxicity to subsequent wheat (Triticum aestivum L.)or broad beans (Vicia faba L.). Experiment 2 demonstrated that post-cultivation soil treatment with metolachlor · oxyfluorfen · pendimethalin (50% EC) enhanced weed suppression (47.8–53.6%) and yield (3.4% increase) while ensuring crop safety. Experiment 3 revealed that the post-emergence herbicides haloxyfop-P-methyl (108 g/L EC) and fomesafen (250 g/L SL) achieved over 92% reduction in grass weed biomass and over 75% reduction in broadleaf weed density, respectively, alongside a 6.1% yield improvement. Therefore, region-specific strategies are recommended based on local agronomic conditions: high-value production zones should adopt integrated systems combining plastic mulch with pre-emergence herbicides; arid lands with extended crop rotation intervals require pre-emergence herbicides after intertillage and earthing-up; labor-abundant regions can rotate targeted post-emergence herbicides to delay resistance evolution. This study provides data-driven optimization strategies for comprehensive weed management in marigold fields, offering practical solutions to enhance industrial productivity and ecological sustainability.