Seed germination and seedling growth represent two vulnerable stages in a plant life cycle and it is important to determine how they will be affected by environmental changes associated with climate change. This study examines the impact of salinity levels (0, 50, 100, and 150 mM) at each of three temperatures (15, 25, and 35˚C) on seed germination and radicle growth for three dormant and three non-dormant varieties of conventional, glyphosate resistant, or reduced lignin alfalfa. The genetic basis and phenotypic plasticity for seed germination and seedling growth in response to salinity and temperature were determined. Both traits were phenotypically plastic with respect to temperature and salinity. Seed germination was 87.0% ± 1.7 at 25˚C, 0mM (no salinity), but decreased to 33.9% ± 3.9 germination at 35˚C and 150mM. Radicle length went from 36.7 ± 1.5 mm after four days at 25˚C, 0mM, to 10.5 ± 0.7 mm at 35˚C, 150mM. The phenotypic response brought seed germination and radicle length away from their optimum, but we detected standing genetic variation for both seed germination and radicle length. Selection to increase both traits at high temperature and salinity would facilitate alfalfa establishment under climate change.

Salt stress is a major limiting factor for alfalfa yield due to its relatively low salt tolerance. High soil salinity adversely impacts alfalfa growth and development, leading to reduced yield. Melatonin (MT) is known to play a significant role in enhancing plant resistance to abiotic stresses. However, the mechanisms underlying melatonin-mediated abiotic stress responses, particularly salt stress, are not well understood. To address this gap, we conducted a study focusing on germinating alfalfa seeds under salt stress. Physiological indexes and transcriptomics analyses were carried out on the germinated seeds to investigate the effects of melatonin on alfalfa seedlings under salt stress conditions. Our results revealed that the application of melatonin led to an increase in shoot length and fresh weight of alfalfa seedlings under salt stress. Moreover, key physiological indexes such as peroxidase (POD) activity and glutathione (GSH) content were enhanced, while levels of malondialdehyde and superoxide anions decreased. Transcriptomic analysis identified a total of 2,131 differentially expressed genes in the salt-treated group, with 726 up-regulated and 1,405 down-regulated genes, while the MT-treated group showed 2896 differentially expressed genes, of which 1,097 were up-regulated and 1,799 were down-regulated. Further, KEGG enrichment analysis highlighted the enrichment of differentially expressed genes in pathways including flavonoid biosynthesis, ABC transporter, glutathione metabolism, and the MAPK signaling pathway, with these pathways more significantly enriched in the MT-treated group. These findings collectively suggest that melatonin plays a crucial role in alfalfa's response to salt stress and offer new insights into the genomic mechanisms underlying melatonin-mediated salt tolerance in alfalfa.

The present study has been one of the first attempts to thoroughly examine the effects of different kefir sources on fermentation characteristics, aerobic stability, and microbial communities of alfalfa silages. The effects of commercial kefir (CK) and homemade kefir culture (HK) applied with four different application doses (untreated control (CON), 5.0, 5.7, and 6.0 log cfu g-1) on wilted alfalfa and stored at an ambient temperature of 25-30 °C. After 45 days ensiling, fermentation characteristics and aerobic stability of silages were measured and bacterial diversity was investigated by 16S ribosomal RNA gene sequencing using GenomeLab™ GeXP platform. Both CK and HK accelerate more lactic acid production and reduced ammonia nitrogen concentration. Factor analysis of kefir sources suggest that the addition of kefir improves the aerobic stability of silages even the initial water soluble carbohydrate (WSC) content is inadequate via its antimicrobial effect on yeast and mould formation. Enterococcus faecium, Pediococcus pentosaceous, and Lactobacillus brevis were dominant bacterial species among the treated groups at silo opening while Lactobacillus plantarum and Lactobacillus brevis became dominant bacterial species after 7 days of aerobic exposure. In conclusion, application of kefir on alfalfa silages improves fermentation quality and aerobic stability even with low WSC content.

Alfalfa, an important legume forage, is an ideal crop for sustainable agriculture and a potential bioenergy plant. Drought, one of the most common environmental stresses, substantially affects plants’ growth, development and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of 4 samples, each with 3 biological replicates. We identified 348 known miRNAs, belonging to 80 miRNA families, from the 12 libraries and 281 novel miRNAs using Mireap software. 18 known miRNAs in roots and 12 known miRNAs in leaves were screened out as drought-responsive miRNAs. Except for miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs were different between roots and leaves in alfalfa. This is the first study discovering miR157a, miR1507, miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 are drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

Salt stress is one of the major abiotic stresses that lead to crop yield reduction worldwide. Melatonin (MT), an endogenous hormone synthesized in plants, plays an important role in responding to abiotic stimuli such as drought, salinity, heat, cold, etc. However, the molecular mechanisms of MT-mediated abiotic stresses remain to be better understood. In order to explore the key genes and pathways of melatonin in alleviating salt stress, physiological and biochemical indicators as well as transcriptomics profile were conducted in alfalfa seedlings. The results showed that the germination rate, bud length and fresh weight of alfalfa seedlings added with melatonin were higher than those treated with salt alone. In addition, Malondialdehyde (MDA) and Superoxide anion (O2-) contents also displayed a decreasing trend. The activity and content of Superoxide dismutase (SOD)、peroxidase (POD) and Glutathione (GSH) increased to varying degrees. In order to further reveal the molecular mechanism of MT-mediated salt tolerance of alfalfa, transcriptome analysis was performed on alfalfa seedlings. 2,181 differentially expressed genes (DEGs) were obtained in the salt-treated group, including 780 upregulated genes and 1,401 down-regulated genes, while 4,422 DEGs were obtained in the MT group, among which 1,438 were upregulated and 2,984 were downregulated. By using GO functional annotation and KEGG pathway enrichment, we found that DEGs were mainly enriched in the biosynthesis of flavonoids, isoflavones, plant hormones, Glutathione (GSH), soluble sugars and other substances, as well as in the ABC transporter and MAPK signaling pathways. Among them, the DEGs in the MT group was more enriched in these pathways, suggesting that MT can combat salt stress by regulating the expression of genes related to phytohormone and antioxidant capacity, which has important reference value for the breeding of salt-tolerant alfalfa and other crops.

Alfalfa primarily produces seeds through cross-pollination among different individuals. Self-pollination results in severe inbreeding depression, such as weak seedlings and termination of growth. Identifying the genetic loci associated with vigorous plant growth could enable the elimination of deleterious alleles and eventually develop inbred alfalfa lines for hybrid production. In this study, 215 alfalfa accessions were self-pollinated for three generations. Within accessions, pairs of weak and strong plants were sampled and bulked for exome sequencing. We extracted individual DNA from 534 plants that included parental clones, strong and weak pairs of plants, and plants selected based on the number of seeds produced. Among them, we formed 42 pools, including 16 with strong plants and 17 with weak plants, 3 top-seeded plants, 3 low-seeded plants, and 3 no-seeded plants. Along with 79 individuals, these pools were sequenced in the target regions covering 112,626 contigs across the entire alfalfa genome. From the 121 samples, (42+79) genotyped, 13.2 million SNPs including indels were generated. After filtering for MAF (>5%), depth (<20X), and no missing genotype and removing indels, a total of 588,136 SNPs were obtained which were used for final analysis. A genome-wide association study was performed and identified 11 genetic loci associated with alfalfa plant growth vigor. The GO analysis generated 12 significantly enriched GO terms. The associated SNPs were on, and near genes involved in stress response, defense responses against pathogens, and plant reproduction. These identified SNPs benefit the development of alfalfa inbred lines by purging the deleterious alleles and biomass improvement through marker-assisted selection.

Alfalfa is an important forage crop around the world. LncRNAs are considered to be a class of functional biomacromolecules, while little is known about lncRNAs in alfalfa. In this study, RNAs from different tissues of alfalfa were sequenced and anlyzed with full-length transcriptome sequencing technology, yielding the full-length transcripts dataset of alfalfa. Based on sequencing and public RNA-seq data, lncRNAs of alfalfa were predicted genome-wide by CPC2 and PLEK. The results showed most lncRNAs shared low sequence conservation with those in other plant species, part of which seems originate from plastid genome. We also identified 88563 lncRNAs, approximately 99.8% of total lncRNAs, with possibility of coding small ORFs using two prediction tools. Our research generated the biggest sequence set of alfalfa lncRNAs, and revealed some plastid originated lncRNAs with high sequence conservation.

Plant breeding aims to develop elite crop varieties appropriate for various environments with higher quality and quantity of production. Researchers use quantitative trait loci (QTL) mapping and association studies to identify regions in the genome responsible for the variation of the quantitative traits of interest. However, mapped regions do not always translate to functional proteins, which makes it challenging to identify genes associated with traits of interest. The biological functions of proteins are strongly dependent on their 3D structure. Alternatively, if proteins can be directly linked with the phenotypes, the effect of mutations on phenotypic changes can be assessed. Innovation of deep learning models in biology opens new avenues of exploration. AlphaFold is an AI system that predicts the 3D structure of a protein from its amino acid sequence with near experimental accuracy and was used in this study. Point mutations with a significant influence on the 3D structure of a protein can capture the effect on phenotypes through association study, and this provides insights into the regions that are of functional importance. In the current study, 534 plants were selected based on plant vigor, and 154 missense variants that change amino acid sequences, including 5 significant hits from previous study, were included. The changes in protein 3D structure were assessed by association with the phenotype. The analysis identified five significant associations, four of which were also identified in previous study of SNPs GWAS, however, a new fifth association was also identified which was annotated as disease resistance gene in Medicago truncatula. This study helps to associate SNPs that could be missed by GWAS due to stringent Bonferroni corrected p-values by providing a more robust filter for SNPs using features from predicted protein 3D structures.

Alfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies such as differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus, and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa. In the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in Arabidopsis thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physicochemical properties, motif analysis, and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that particular bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought, and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. These expression patterns were further verified by qRT-PCR. However, further functional characterization of bZIP transcription factors in alfalfa will help illuminate the role they play in stress tolerance mechanisms in legumes and facilitate the molecular breeding of stress tolerance in alfalfa.

A remote sensing-based evapotranspiration (ET) study was conducted over the Central Arizona Irrigation and Drainage District (CAIDD), an Arizona agricultural region. ET was assessed means for 137 wheat plots, 183 cotton plots, and 225 alfalfa plots. The remote sensing ET models were the Satellite-Based Energy Balance for Mapping Evapotranspiration with Internalized Calibration (METRIC), the Two Source Energy Balance (TSEB), and Vegetation Index ET for the US Southwest (VISW). Remote sensing data were principally Landsat 5, supplemented by Landsat 7, MODIS Terra, MODIS Aqua, and ASTER. The models produced similar daily ET for wheat, with 6–8 mm/d mid-season. For cotton and alfalfa daily ET showed greater differences, where TSEB produced largest daily ET, METRIC the least, and VISW in the midrange. Modeled cotton ET at mid-season ranged from 9.5 mm/d (TSEB), to 8 mm/d (VISW), and 6 mm/d (METRIC). For alfalfa ET, values at peak cover ranged from 8 mm/d (TSEB), 6 mm/d (VISW), and 5 mm/d (METRIC). Model bias ranged −10% to +18%. Relative to potential ET, FAO-56 ET, and USDA-SW gravimetric-ET, model variability ranged from negligible to 35% of annual crop water use. Model averaging was found a useful way to consider and reconcile all ET estimates.

In the context of market relations in agriculture in the Kyzylorda region, in order to meet the needs of animal husbandry in complete feed, it is necessary to create a solid forage base. The successful solution to the tasks of increasing the number of livestock and increasing the productivity of animal husbandry in the ecological conditions of the Aral Sea region largely depends on the timely procurement of feed and the transfer of feed production to an industrial basis. It is possible to increase the production of high-quality feeds through the introduction of high-yielding varieties, the use of new resource-saving cultivation and harvesting technologies, the optimal organization of cultivation production processes, and increasing the efficiency of the use of technical means. Of all the harvested types of feeds, alfalfa has the best effect on increasing milks yield and meat gains. The green mass of alfalfa is well-eaten by all farm animals. For example, when feeding cattle, its utilization rate is 92.6%. In terms of protein richness, alfalfa hay surpasses many other feeds, so 100 kg of alfalfa hay contains 11.6 kg of digestible protein, 1.77 kg of calcium, 0.22 kg of phosphorus, and 4.5 g of carotene. In addition, in the conditions of rice crop rotation, according to its biological characteristics, alfalfa (Medicago sativa L.) is considered indispensable in agrotechnical and reclamation relations, especially in saline soils. The economic value of alfalfa lies in its wide ecological plasticity, the ability to grow back quickly after mowing, and high productivity.

Background: Alfalfa, the most economically important forage legume worldwide, features modest genetic progress due to long selection cycles and large extent of non-additive genetic variance associated with its autotetraploid genome. Methods: To improve the efficiency of genomic selection in alfalfa, we explored the effects of genome parametrization (as autotetraploid and diploid dosage, plus allele ratios) and SNP marker subsetting (all available SNPs; only genic regions; only non-genic regions) on genomic regressions, together with various levels of filtering on reading depth and missing rates. We used Genotyping by Sequencing-generated data and focused on traits of different genetic complexity, i.e., dry biomass yield in moisture-favorable (FE) and drought stress (SE) environments, leaf size, and onset of flowering, assessed on 143 genotyped plants from a genetically-broad European reference population and their phenotyped half-sib progenies. Results: On average, allele ratio improved the predictive ability compared with other genome parametrizations (+7.9% vs. tetraploid dosage, +12.6% vs. diploid dosage), SNP subsetting offered an advantage compared with any tested subsettings (+3.7% vs. genic regions, +7.6% vs. non-genic regions). However, when focusing on specific traits, different combinations of genome parametrization and subsetting reached better performances. We also released Legpipe2, a SNP calling pipeline tailored for reduced representation (GBS, RAD) medium-sized genotyping experiments.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, incubation periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old plantlets raised in vitro. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage (76.2%) of GUS-positive alfalfa leaf explants was observed after 3 days of agroinfection at a bacterial concentration of 0.6, 2 days of preculture, 30 min of infection time, 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa.