Background Flowering time is a pivotal agronomic trait that determines forage yield, quality, and environmental adaptability in alfalfa ( Medicago sativa ). The FT-INTERACTING PROTEIN (FTIP) family, a subgroup of multiple C2 domain and transmembrane region proteins (MCTPs), plays an essential role in florigen transport and flowering control across plant species. However, comprehensive characterization of FTIP family genes in alfalfa ( MsFTIPs ) has not been reported to date. Results In this study, 23 MsFTIPs from the alfalfa genome were identified and systematically analyzed their physicochemical properties, phylogenetic relationships, chromosomal localization, gene structures, conserved motifs, cis-acting regulatory elements, and expression patterns. We further performed functional characterization of MsFTIP1 , a homolog of AtFTIP1 . Subcellular localization assays demonstrated that MsFTIP1 was targeted to the endoplasmic reticulum (ER). Heterologous overexpression of MsFTIP1 in Arabidopsis resulted in significant early flowering under long-day conditions. Real-time quantitative PCR (RT-qPCR) further revealed that transcript levels of the key flowering-promoting genes AtSOC1 , AtAP1 , AtFT and AtLFY were significantly upregulated in transgenic lines. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation imaging (LCI) assays confirmed the direct physical interaction between MsFTIP1 and the florigen protein MsFT. Conclusion In our study, we identified 23 distinct FTIP genes in alfalfa, which were classified into six clades. We systematically characterized multiple gene-level features, encompassing physiochemical properties, phylogenetic phylogeny, exon-intron organization, conserved protein motifs, chromosomal distribution, gene duplication, cis-acting regulatory elements, protein tertiary structures, tissue-specific expression profiling, and transcriptional responses to drought, salinity and cold stresses. Furthermore, we had revealed the conserved function of MsFTIP1 in facilitating flowering by interacting with MsFT. Our findings provide a solid foundation for elucidating the molecular mechanisms underlying flowering-time regulation and supporting molecular breeding in alfalfa.

Reconstruction of a robust phylogeny of Medicago using genome-scale nuclear data from the Angiosperms353 probe set sheds light on the origin of the genus and provides evidence that pod morphological traits acted as key innovations, playing a central role in the adaptive radiation of Medicago.

In eukaryotes, XERODERMA PIGMENTOSUM GROUP D (XPD) is an integral subunit of the DNA repair/transcription complex TFIIH. In animals, XPD has been implicated in TFIIH-independent complexes regulating cell division, which, however, remains poorly understood in plants. Here, we identified XPD as a novel regulator of stomatal development in Arabidopsis. Its loss-of-function mutants exhibited increased stomatal precursor cells and formed stomatal clusters. Genetic analysis showed that XPD functions upstream of SPEECHLESS (SPCH) to control stomatal lineage entry, coordinates with MUTE to regulate meristemoid division and works together with FLP and FAMA to restrict GMC division. In a search of XPD interactors, we identified CDKA;1, which serves as both an essential cyclin-dependent kinase and a key SPCH activator. Consistently, xpd mutants exhibited enhanced stomatal lineage cell divisions and elevated SPCH protein levels. Furthermore, XPD acts upstream of CDKA;1, as expression of the dominant-negative CDKA;1.N146 allele significantly suppressed the excessive cell division and stomatal development defects in xpd plants. Our data highlight the precise regulation of stomatal development by XPD, expanding its critical TFIIH-independent roles in plant cell division and fate specification.

Abstract Native grasses possess extensive ecological adaptability, such as cold, drought, and salt tolerance, and tolerance to poor soil conditions. They play a crucial role in ensuring food security and ecological security. However, their development and utilization are still limited by lack of information on agronomic cultivation methods, low seed production, and difficulty in commercial production. This review summarizes the core requirements for native grass breeding and practical solutions. A “six‐step” strategy for native grass breeding is proposed: (1) establish a basic agronomic cultivation system; (2) develop economically feasible seed production technologies to ensure farmers’ access to seeds; (3) systematically collect and evaluate germplasm resources in the target environment; (4) promote superior germplasm lines and achieve large‐scale seed production; (5) initiate selection/breeding plans for key traits such as seed yield, seedling vitality, and multi‐trait productivity; and (6) on this basis, strategically integrate advanced technologies such as genetic markers, gene editing, and omics analysis to accelerate the process of precision breeding. This progressive strategy emphasizes laying a solid foundation for breeding before applying newly emerging technologies to cultivate native grass varieties with commercial value.

Alfalfa (Medicago sativa L.), a vital leguminous forage crop, faces growth and distribution constraints due to abiotic stresses, such as drought. Pathogenesis-related protein 1 (PR-1) is a major class of plant defense proteins involved in responses to environmental stress. However, the distribution and functional roles of the PR-1 gene family in alfalfa under drought stress remain largely unexplored. Nineteen MsPR-1 genes were identified in the alfalfa genome through systematic screening, with phylogenetic clustering dividing these genes into three evolutionarily distinct clades. Expression profiling highlighted MsPR-1-12 as a candidate gene for drought tolerance. Subcellular localization indicated that MsPR-1-12 is localized to the nucleus membranes and cellular membranes. Furthermore, reverse transcription and quantitative PCR(RT-qPCR) analysis demonstrated a significant upregulation of MsPR-1-12 under drought stress. Heterologous expression of MsPR-1-12 in yeast conferred enhanced drought resistance. These findings provide novel insights into the MsPR-1 gene family in alfalfa and offer valuable genetic resources for the development of drought-tolerant alfalfa varieties through genetic engineering.

Plant immune receptor proteins, including pattern recognition receptors and nucleotide-binding domains and leucine-rich repeat sequence receptors (NLRs), are essential for various aspects of plant growth and development, including microbial sensing and immune responses. However, a comprehensive and systematic understanding of immune-related gene families in alfalfa plants is lacking. This study aimed to identify and characterize these genes in alfalfa to fill this knowledge gap. We identified 736 receptor-like kinase (RLK), 407 receptor-like protein (RLP), and 870 NLR genes in the alfalfa genome and analyzed their structural and genetic variation. Phylogenetic analysis classified RLKs, RLPs, and NLRs into 22, 21, and 3 subgroups, respectively. Chromosomal location and synteny analyses revealed that single-gene duplication events contributed to the evolution of these genes. Expression profiling, promoter cis-acting elements, and associated network analyses demonstrated their critical roles in plant growth and stress prevention. Specifically, the alfalfa RLK gene (MsRLK725) was selected for functional validation due to its numerous genetic variants and significant upregulation under biotic stress. Overexpression of MsRLK725 in Nicotiana benthamiana enhanced resistance to Rhizoctonia solani and Phoma medicaginis, and the hairy root transient transformation system improved alfalfa's salt tolerance. Our findings offer valuable insights for future research on using immune-related genes in alfalfa, highlighting their potential in molecular breeding for stress tolerance and disease resistance.

BACKGROUND:As the primary organ of the male reproductive system, the testis facilitates spermatogenesis and androgen secretion. Due to the complexity of spermatogenesis, elucidating cellular heterogeneity and gene expression dynamics within the porcine testis is critical for advancing reproductive biology. Nevertheless, the cellular composition and regulatory mechanisms of porcine testes remain insufficiently characterized. In this study, we applied integrated long-read (Nanopore) and short-read (Illumina) scRNA-seq to Baoshan pig testes, establishing a comprehensive transcriptional profile to delineate cellular heterogeneity and molecular regulation. RESULTS:Through systematic analysis of testicular architecture and the temporal progression of spermatogenesis, we characterized 11,520 single cells and 23,402 genes, delineating germ cell developmental stages: proliferative-phase spermatogonia (SPG), early-stage spermatocytes (Early SPC) and late-stage spermatocytes (Late SPC) during meiosis, and spermiogenic-phase round spermatids (RS) followed by elongating/elongated spermatids (ES), culminating in mature spermatozoa (Sperm). We further identified nine distinct testicular cell types, with germ cells spanning all developmental stages and somatic components comprising Sertoli cells, macrophages, and peritubular myoid cells as microenvironmental constituents, revealing the cellular heterogeneity of testicular tissue and dynamic characteristics of spermatogenesis. We obtained the dynamic expression changes of 16 vital marker genes during spermatogenesis and performed immunofluorescence validation on 7 marker genes. Gene ontology analysis revealed that germ cells at various stages were involved in specific biological processes, while cell communication networks highlighted eight pivotal signaling pathways, including MIF, NRG, WNT, VEGF, BMP, CCL, PARs, and ENHO pathways. Long-read sequencing further captured the full integrity and diversity of RNA transcripts, identifying 60% of the novel annotated isoforms and revealing that FSM isoforms exhibited longer transcript lengths, longer coding sequences, longer open reading frames, and a great number of exons, suggesting the complexity of isoforms within the testicular microenvironment. CONCLUSIONS:Our results provide insight into the cellular heterogeneity, intercellular communication, and gene expression/transcript diversity in porcine testes, and offer a valuable resource for understanding the molecular mechanisms of porcine spermatogenesis.

Vicia sativa (common vetch, n=6) is an annual leguminous plant with high drought tolerance and high grain protein content. Historically and still today, vetches are important for nitrogen fixation to maintain cereal yields and feeding grazing livestock. In this study, we re-sequenced the genomes of 279 common vetch accessions from a wide geographic range covering western Eurasia and North Africa to construct a comprehensive nucleotide variation map. Population structure analyses indicate that the Middle Eastern region was the centre of origin of common vetch, which then entered Europe in two distinct waves. One wave propagated by humans through Turkey into the Carpathian Basin before spreading throughout Europe via “Danubian” and “Mediterranean” routes. Demographic inferences revealed a significant bottleneck for all common vetch groups initiated during the Last Glacial Maximum, followed by an expansion that coincides with the onset of the warm Holocene epoch and the Neolithic Revolution. Interestingly, we identified selective sweeps for the flowering time regulators VsSOC1 and VsFTb2 in northern latitude populations suggesting that both genes are important regulators of latitudinal adaptation. Overall, we provide valuable genomic resources for conservation and breeding programs to optimize food production in the face of population growth and reduced agricultural resources. ### Competing Interest Statement The authors have declared no competing interest. Australian Research Council, LP200200957 Leading Scientist Project of Gansu Province, 23ZDKA013 Gansu Provincial Science and Technology Major Projects, 22ZD6NA007

Wild perennial sister species Medicago archiducis-nicolai (rhizomatous/alpine) and M. ruthenica (non-rhizomatous/xeric) constitute vital genetic resources for forage improvement. To decode the genomic basis of their contrasting trait and habitat adaptation, we generated chromosome-scale genome assemblies, resequenced 128 individuals, profiled transcriptomes under cold/heat stress, and functionally validated causal alleles. We demonstrate that structural variations (SVs)-particularly gene duplications-are primary drivers of rhizome formation and alpine/xeric adaptation. Further, pervasive presence-absence SVs (PAVs) in noncoding regulatory regions underpin divergent allele-specific expression governing rhizome development and stress responses. Crucially, these regulatory PAVs induce contrasting expression patterns during trait development and stress adaptation. Our findings reveal a dual mechanism whereby coding and regulatory SVs convergently orchestrate phenotypic innovation and ecological specialization in sister species, offering valuable genomic resources for legume evolution studies and alfalfa breeding.

Forage crops not only provide food for livestock to meet the growing demands of the global population but are also essential for sustainable agricultural systems by rehabilitating infertile and marginal lands. A wide diversity of plant species is cultivated as forage crops, many of which possess complex genetic backgrounds, making them more challenging to improve compared to model plants and staple crops. Recent advancements in molecular biology, sequencing technologies, and genomic analysis tools have opened new avenues for the improvement of forage crops. This review provides a comprehensive examination of modern forage breeding, covering the primary types of forage, their characteristics, key functional genes utilized for enhancing forage traits, advanced breeding technologies, and the challenges and future directions in this field. By integrating the latest research and technological developments, this review aims to contribute to the advancement of forage breeding strategies that can meet the increasing global demand for sustainable and high-quality forage resources.

The common vetch (Vicia sativa L.) is a self-pollinated annual forage legume that is widely distributed worldwide. It has wide adaptability and high nutritional value and is commonly used as an important protein source for livestock feed. However, pod shattering seriously limits the yield of common vetch. To clarify the mechanism of pod shattering in common vetch, the pod walls of three shattering-resistant (SR) accessions (B65, B135, and B392) and three shattering-susceptible (SS) accessions (L33, L170, and L461) were selected for transcriptome sequencing. A total of 17,190 differentially expressed genes (DEGs) were identified in the pod wall of B135 and L461 common vetch at 5, 10, 15, 20, and 25 days after anthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that “phenylpropanoid biosynthesis” was the most significantly enriched pathway, and 40 structural genes associated with lignin biosynthesis were identified and differentially expressed in B135 and L461 common vetch. We analysed the DEGs in the pod wall of three SR and three SS accessions at 15 days after anthesis, and most of the DEGs were consistent with the significant enrichment pathways identified in B135 and L461 common vetch. The total lignin content of SR accessions was significantly lower than the SS accessions. The present study lays a foundation for understanding the molecular regulatory mechanism of pod shattering related to lignin biosynthesis in common vetch and provides reference functional genes for breeders to further cultivate shattering-resistant common vetch varieties.

The GRAS gene family encodes a group of plant-specific transcription factors essential for regulating plant growth, development and stress responses. While the GRAS gene family has been extensively studied in various plant species, a comprehensive characterization of the GRAS gene family in Medicago ruthenica has not yet been conducted. In this study, a total of 62 MrGRAS gene family members were identified through a comprehensive whole-genome analysis of M. ruthenica, and phylogenetic analysis categorized these 62 genes into 13 distinct groups. Gene structure and conserved domain analysis showed that MrGRAS genes from the same evolutionary branch share similar exon–intron architecture and conserved motifs. A large number of hormone-responsive, growth and development and stress-responsive cis-regulatory elements were detected in the upstream sequences of MrGRAS genes. RT-qPCR analysis showed that drought stress significantly induced the expression of nine selected MrGRAS genes. Overall, this study analyzed the phylogenetic relationships, conserved domains, cis-regulatory elements and expression patterns of the GRAS gene family in M. ruthenica, filling the gap in the identification of the MrGRAS gene family and laying the foundation for functional analysis of the MrGRAS gene family.

Common vetch (Vicia sativa L.) is one of the most economically important annual pasture legumes worldwide. Many factors affect the yield of common vetch; however, the genetic architecture and gene functions associated with common vetch yield have not been explored. On the basis of 115.53 million single-nucleotide polymorphisms (SNPs) and 18.55 million insertions-deletions (InDels) identified in 222 diverse common vetch accessions, we performed a comprehensive genome-wide association study (GWAS) on the 4-year results for six yield-related agronomic traits (water loss rate, dry weight, lodging index, stem thickness, absolute height and natural height). In total, 2864 SNPs and 481 InDels were respectively identified to be significantly associated with these six traits. Furthermore, a water loss rate-related candidate gene (Vs-pyruvate decarboxylase 2 [VsPDC2]) was functionally characterised and demonstrated to be a key regulator of the water loss rate in the heterologous species Arabidopsis thaliana. This study is the first to use GWAS to investigate the genetic architecture and key regulatory genes associated with drought tolerance and yield in common vetch, thereby providing valuable insights for common vetch breeding and future research.

Late embryogenesis abundant (LEA) proteins are pivotal mediators of plant abiotic stress adaptation, but their functional roles and regulatory mechanisms in forage crops like Medicago sativa remain unclear. Through a genome-wide analysis of the LEA_3 subgroup in alfalfa, we identified MsLEA_3–6, whose expression is significantly induced by drought. To elucidate its role in drought tolerance, we conducted a comprehensive molecular and physiological characterization of MsLEA_3–6. Phylogenetic analysis placed MsLEA_3–6 within a conserved clade of drought-responsive LEA proteins. Its promoter harbored stress-related cis-elements, and its expression was upregulated under drought and salinity. Subcellular localization revealed plasma membrane association, while β-glucuronidase GUS staining confirmed ubiquitous expression in the roots, stems, leaves, flowers, and seeds of transgenic Arabidopsis. Overexpression of MsLEA_3–6 enhanced drought tolerance in Arabidopsis (improved root growth and germination under mannitol) and alfalfa (reduced H2O2 and malondialdehyde accumulation, elevated proline/soluble sugars, and attenuated oxidative stress via ROS scavenging). A soil moisture monitoring system demonstrated that MsLEA_3–6 overexpression led to improved photosynthetic performance under drought conditions. Furthermore, the expression of stress/ABA-responsive genes (ABI5, ABF3, NCED5, and NCED9) was altered in the transgenic lines compared with those in the wild-type plants under both normal and drought conditions. Dual-luciferase assays identified MsWRKY71 (MS.gene023126) as a direct binder of the MsLEA_3–6 promoter, forming a stress-responsive regulatory module. Our findings identify MsLEA_3–6 as a functionally conserved, drought-responsive LEA protein that enhances osmotic adjustment and oxidative stress mitigation in transgenic plants, likely through coordinated regulation of ABA signaling and MsWRKY71-mediated transcriptional activation. While the findings support the functional relevance of MsLEA_3–6 under controlled conditions, further studies are needed to elucidate the underlying molecular mechanisms and to validate its utility in improving drought tolerance under field environments.

Germplasm resources within the Medicago genus are highly regarded for their value as forage crops and their critical roles in nitrogen cycling, ecosystem restoration, and soil structure improvement. Therefore, understanding the diversity of seed morphology in this genus is essential for advancing its development and utilization. This study analyzed seed samples from 587 germplasm accessions representing 77 species within Medicago genus, as well as 32 accessions from 21 species within the closely related genus Trigonella. A statistical analysis was conducted on twelve traits, including seven quantitative traits-straight length (SL), straight width (SW), width-to-length ratio (WL), perimeter (PE), radicle length (RL), hilum length (HL), and 100-seed weight (SY)-and five qualitative traits, including seed coat condition, radicle characteristics, seed size, shape, and color. The results revealed that: (1) there was significant diversity (P < 0.05) in SL, SW, WL, PE, RL, HL, and SY across Medicago species; (2) principal component analysis of the 587 Medicago accessions identified SL, SW, PE, HL, RL, and SY as the primary contributors to morphological diversity; and (3) high-resolution images of seeds from various accessions were captured for future research. This study provides a solid foundation for the establishment of seed banks and the enhancement of germplasm resources through the systematic analysis of these morphological traits.

Severe drought stress can significantly reduce alfalfa production, and the phytohormone abscisic acid plays a central role in responses to abiotic stress. As abscisic acid receptors, the PYRABACTIN RESISTANCE 1/PYR1-LIKE/ABA-binding REGULATORY COMPONENT OF ABA RECEPTOR (PYR1/PYL/RCAR) family constitutes a critical component of the ABA signaling pathway, mediating adaptive responses and protecting plants under drought conditions. In this study, nine MsPYL genes were identified in alfalfa, and their expression levels were found to be significantly upregulated in response to ABA and drought stress. The overexpression of MsPYL genes in both Arabidopsis thaliana and alfalfa significantly improved drought tolerance. Among the MsPYL family genes, functional analysis of MsPYL6 and MsPYL9 revealed that both genes enhanced water-use efficiency by reducing leaf stomatal density, promoting stomatal closure, and decreasing transpiration rates when overexpressed. In contrast, the RNAi plants exhibited the opposite phenotypes, with increased stomatal density and higher transpiration rates. Furthermore, overexpression plants exhibited reduced malondialdehyde content and lower reactive oxygen species levels, indicating enhanced cellular stability under stress. Additionally, transcriptome analysis showed that drought-responsive genes related to antioxidant defense, stomatal regulation, and photosynthesis were more abundant in drought-treated OE plants and less abundant in RNAi plants. Interaction analysis revealed that all MsPYL proteins could interact with at least one MsPP2CA, indicating a conserved interaction pattern in ABA signaling. These findings confirm that MsPYLs play a crucial role in the ABA signaling pathway by modulating the expression of downstream drought-tolerant genes, thereby enabling alfalfa to effectively respond to drought stress conditions.

The homeodomain-leucine zipper (HD-Zip) transcription factors are plant-specific proteins that play pivotal roles in regulating various biological processes. Here, we conducted a comprehensive genome-wide analysis of the HD-Zip transcription factor family in alfalfa (Medicago sativa L.), identifying a total of 40 MsHDZ genes. Phylogenetic analysis classified the MsHDZ genes into four subfamilies (I-IV). Gene structure and conserved protein motif analyses revealed a high degree of structural conservation and functional diversity among the MsHDZs. Chromosomal mapping showed that MsHDZ genes are unevenly distributed across the alfalfa genome. Expression profiling indicated that MsHDZ genes exhibit tissue-specific expression patterns and are significantly induced under drought and salt stresses. Notably, the overexpressing MsHDZ18 enhanced drought tolerance in transgenic plants, suggesting its critical role in abiotic stress responses. These findings provide valuable insights into the functional roles of HD-Zip transcription factors in alfalfa.

Alfalfa (Medicago sativa L.) is a prominent and distinct species within the pasture germplasm innovation industry. However, drought poses a substantial constraint on the yield and distribution of alfalfa by adversely affecting its growth. Although lineage-specific genes are instrumental in modulating plant responses to stress, their role in mediating alfalfa's tolerance to drought stress has yet to be elucidated. In this study, a total of 199 alfalfa-specific genes (ASGs) and 3,054 legume-specific genes (LSGs) were identified in alfalfa. Compared with evolutionarily conserved genes, ASGs have shorter sequence length and fewer or no intron. Many alfalfa ASGs can be induced by various abiotic stresses, and the capability of MsASG166 to enhance drought resistance has been substantiated through transgenic research in both yeast and Arabidopsis thaliana. The RNA-Seq and WGCNA analyses revealed that DREB2A and MADS are pivotal genes in the molecular mechanisms through which MsASG166 positively modulates plant drought resistance. This study marks the first identification of lineage-specific genes in alfalfa and an examination of the molecular roles of the MsASG166 gene in drought stress responses. The findings offer valuable genetic resources for the development of novel, genetically engineered alfalfa germplasm with enhanced drought tolerance.

Ground cover management (GCM) is an important practice for sustainable agricultural development, which has been garnering popularity in numerous orchards worldwide. It can effectively improve soil health, reduce soil erosion, and enhance carbon sequestration. However, the effects of GCM on soil moisture content (SMC) dynamics vary with management and environmental factors, and it remains unclear how these factors affect GCMinduced SMC. In addition, it remains unknown how SMC and influencing factors such as climatic, edaphic, and agronomic factors are inter-related in responding to mulching practices. Therefore, we performed a metaanalysis to identify the effect of these factors on SMC accumulation and elucidate the potential mechanisms involved under different environmental and management conditions. Results revealed that GCM practices significantly enhanced SMC (9.2%) compared with the effects of traditional clean tillage. The maximum benefits of SMC were observed when the average annual temperature was smaller than 8 celcius, initial soil organic carbon was 20 g kg-1, and tree age was greater than 20 yr. Further analysis using a random forest model revealed that sampling time, tree age, and soil depth were the predominant drivers of SMC change. Overall, these findings highlight the critical importance of GCM in increasing SMC in orchards worldwide. In addition, the results show that the corresponding changes are controlled by specific climatic, soil, and management factors. These findings provide insights and guidance that can assist practitioners and policy-makers to establish and manage sitespecific GCM practices to maximize SMC benefits and promote sustainable agricultural development.