Appropriate root system architecture (RSA) can improve alfalfa yield, yet its genetic basis remains largely unexplored. Here, we evaluated six RSA traits in 171 alfalfa genotypes under controlled greenhouse conditions. We also analyzed five yield-related traits in normal and drought stress environments, finding a significant correlation (0.50) between root dry weight (RDW) and alfalfa dry weight under normal conditions (N_DW). A genome-wide association study (GWAS) was performed using 1,303,374 single nucleotide polymorphisms (SNPs) to explore the relationships between RSA traits. Sixty significant SNPs (-log₁₀(P) ≥ 5) were identified, with genes within 50 kb upstream and downstream ranges primarily enriched in GO terms related to root development, hormone synthesis and signaling, as well as morphological development. Further analysis identified 19 high-confidence candidate genes, including AUXIN RESPONSE FACTORs (ARFs), LATERAL ORGAN BOUNDARIES-DOMAIN (LBD), and WUSCHEL-RELATED HOMEOBOX (WOX). We verified that the forage dry weight under both normal and drought conditions exhibited significant differences among materials with different numbers of favourable haplotypes. Alfalfa containing more favourable haplotypes exhibited higher forage yields, whereas favourable haplotypes were not subjected to human selection during alfalfa breeding. Genomic prediction (GP) utilized SNPs from GWAS and machine learning for each RSA trait, achieving prediction accuracies ranging from 0.70 for secondary root position (SRP) to 0.80 for root length (RL), indicating robust predictive capability across the assessed traits. These findings provide new insights into the genetic underpinnings of root development in alfalfa, potentially informing future breeding strategies aimed at improving yield.

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.

Soil salinization poses a major threat to plant growth and ecosystem sustainability. Zoysia japonica, a salt-tolerant turfgrass, shows promise for saline–alkali soil remediation, yet its metabolic adaptation mechanisms remain poorly understood. Here, we applied non-targeted liquid chromatography/mass spectrometry (LC/MS) metabolomics to compare the responses of salt-tolerant (accession 68) and salt-sensitive (accession 9) genotypes of Z. japonica under salt stress. The sensitive genotype exhibited stronger metabolic disruption, with 843 differentially accumulated metabolites (largely down-regulated), compared with 595 in the tolerant genotype (predominantly up-regulated). We identified a coordinated tolerance mechanism primarily centered on lipid remodeling and energy maintenance. The tolerant genotype enhanced membrane stability through the accumulation of saturated glycerophospholipids and an increased phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio, while maintaining phosphatidic acid (PA) homeostasis which may facilitate SOS-dependent Na⁺ efflux. It also mitigated oxidative damage by stabilizing diacylglycerol (DAG), thereby potentially limiting protein kinase C (PKC) overactivation. Furthermore, sustained cardiolipin and riboflavin metabolism supported mitochondrial energy production in the tolerant genotype. Together, these findings provide new insights into the early metabolic basis of salt tolerance in Z. japonica, suggesting a potential crucial role for PA-mediated regulation of SOS-dependent sodium sequestration during the initial phase of stress, and implying potential targets for breeding stress-resilient turfgrasses.