Alfalfa (Medicago sativa L.) is the most important legume forage crop worldwide with high nutritional value and yield. For a long time, the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agronomic traits. We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4, a cultivar widely cultivated in China, and a comprehensive database of genomic variations based on resequencing of 220 germplasms. Approximate 2.74 Gb contigs (N50 of 2.06 Mb), accounting for 88.39% of the estimated genome, were assembled, and 2.56 Gb contigs were anchored to 32 pseudo-chromosomes. A total of 34,922 allelic genes were identified from the allele-aware genome. We observed the expansion of gene families, especially those related to the nitrogen metabolism, and the increase of repetitive elements including transposable elements, which probably resulted in the increase of Zhongmu-4 genome compared with Medicago truncatula. Population structure analysis revealed that the accessions from Asia and South America had relatively lower genetic diversity than those from Europe, suggesting that geography may influence alfalfa genetic divergence during local adaption. Genome-wide association studies identified 101 single nucleotide polymorphisms (SNPs) associated with 27 agronomic traits. Two candidate genes were predicted to be correlated with fall dormancy and salt response. We believe that the allele-aware chromosome-level genome sequence of Zhongmu-4 combined with the resequencing data of the diverse alfalfa germplasms will facilitate genetic research and genomics-assisted breeding in variety improvement of alfalfa.

Background: Leaf size affects crop canopy morphology and photosynthetic efficiency, which can influence forage yield and quality. It is of great significance to mine the key genes controlling leaf development for breeding new alfalfa varieties. In this study, we mapped leaf length (LL), leaf width (LW), and leaf area (LA) in an F1 mapping population derived from a cultivar named ZhongmuNo.1 with larger leaf area and a landrace named Cangzhou with smaller leaf area. Results: This study showed that the larger LW was more conducive to increasing LA. A total of 24 significant quantitative trait loci (QTL) associated with leaf size were identified on both the paternal and maternal linkage maps. Among them, nine QTL explained about 11.50–22.45% phenotypic variation. RNA-seq analysis identified 2,443 leaf-specific genes and 3,770 differentially expressed genes. Combining QTL mapping, RNA-seq alalysis, and qRT-PCR, we identified seven candidate genes associated with leaf development in five major QTL regions. Conclusion: Our study will provide a theoretical basis for marker-assisted breeding and lay a foundation for further revealing molecular mechanism of leaf development in alfalfa.

Alfalfa growth and production in China are negatively impacted by high salt concentrations in soils, especially in regions with limited water supplies. Few reliable genetic markers are currently available for salt tolerance selection. As a result, molecular breeding strategies targeting alfalfa are hindered. Therefore, with the continuous increase in soil salinity in agricultural lands, it is indispensable that a salt-tolerant variety of alfalfa is produced. We collected 220 alfalfa varieties around the world for resequencing and performed genome-wide association studies (GWASs). Alfalfa seeds were germinated in saline water with different concentrations of NaCl, and the phenotypic differences in several key root traits were recorded. In the phenotypic analysis, the breeding status and geographical origin strongly affected the salt tolerance of alfalfa. Forty-nine markers were significantly associated with salt tolerance, and 103 candidate genes were identified based on linkage disequilibrium. A total of 2712 differentially expressed genes were upregulated and 3570 were downregulated based on transcriptomic analyses. Some candidate genes that affected root development in the seed germination stage were identified through the combination of GWASs and transcriptome analyses. These genes could be used for molecular breeding strategies to increase alfalfa’s salt tolerance and for further research on salt tolerance in general.

Leaf size significantly impacts photosynthetic capacity and forage yield in alfalfa, a major legume forage crop. Therefore, elucidating the genetic factors governing leaf development is critical for breeding improved alfalfa varieties. In this study, a genome-wide association analysis (GWAS) was performed to dissect the genetic architecture of leaf length (LL) and leaf width (LW) using 220 alfalfa accessions phenotyped over three years. Substantial variation for both traits was observed across environments, with coefficients of variation ranging from 10.09–16.53%. GWAS identified 26 significant SNPs associated with leaf morphology spread across seven chromosomes. Each SNP accounts for 9.7–15.6% of the phenotypic variance. Haplotype analyses confirmed positive correlations between the number of superior alleles and both LL and LW. BLAST searches revealed six candidate genes involved in leaf development within 20 kb flanking regions of significant SNPs. Our results provide novel marker-trait associations and candidate loci to facilitate molecular breeding efforts to optimize leaf size and improve productivity in alfalfa. This study establishes a foundation for integrating favorable alleles into future alfalfa varieties.

Alfalfa (Medicago sativa L.) is a high-nutritive-value forage crop that provides livestock with abundant protein and essential nutrients. Breeding elite cultivars with superior quality has become a major goal in modern alfalfa improvement. This study systematically evaluated 12 quality-related traits under field conditions using a diverse panel of 176 alfalfa accessions and investigated the genetic basis underlying these traits. Phenotypic analysis revealed variability across all traits, with coefficients of variation ranging from 2.56% to 15.72%. Based on multi-trait clustering analysis, 16 accessions with overall superior quality were identified. Genome-wide association studies (GWAS) detected 45 significant single nucleotide polymorphisms (SNPs) and 12 structural variants (SVs). Within the associated genomic regions, eight candidate genes were prioritized. RT-qPCR validation indicated that three of these genes (Msa.H.0301430, Msa.H.0290550, and Msa.H.0313490) negatively regulate quality traits, while one gene (Msa.H.0479570) acts as a positive regulator. Haplotype analysis further revealed a positive correlation between the number of favorable haplotypes and phenotypic performance. Genomic prediction (GP) achieved accuracies ranging from 0.71 to 0.86 for the traits when incorporating the top 5000 SNPs identified from GWAS. This study provides valuable insights into the genetic architecture of quality-related traits in alfalfa and lays a solid foundation for future molecular design breeding.

Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn²⁺-dependent enzyme that plays a significant character in the plant’s response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa MsFtsH8 by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both MsFtsH8-overexpressing Arabidopsis and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H₂O₂ accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of MsFtsH8 enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that MsFtsH8 mitigates oxidative damage caused by drought by modulating the plant’s antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering.