Cultivated Alfalfa Research Articles

Oligo-FISH barcode chromosome identification system provides novel insights into the natural chromosome aberrations propensity in the autotetraploid cultivated alfalfa

Oligo-FISH barcode chromosome identification system provides novel insights into the natural chromosome aberrations propensity in the autotetraploid cultivated alfalfa

Genome-wide identification of JAZ gene family members in autotetraploid cultivated alfalfa (Medicago sativa subsp. sativa) and expression analysis under salt stress

BackgroundJasmonate ZIM-domain (JAZ) proteins, which act as negative regulators in the jasmonic acid (JA) signalling pathway, have significant implications for plant development and response to abiotic stress.ResultsThrough a comprehensive genome-wide analysis, a total of 20 members of the JAZ gene family specific to alfalfa were identified in its genome. Phylogenetic analysis divided these 20 MsJAZ genes into five subgroups. Gene structure analysis, protein motif analysis, and 3D protein structure analysis revealed that alfalfa JAZ genes in the same evolutionary branch share similar exon‒intron, motif, and 3D structure compositions. Eight segmental duplication events were identified among these 20 MsJAZ genes through collinearity analysis. Among the 32 chromosomes of the autotetraploid cultivated alfalfa, there were 20 MsJAZ genes distributed on 17 chromosomes. Extensive stress-related cis-acting elements were detected in the upstream sequences of MsJAZ genes, suggesting that their response to stress has an underlying function. Furthermore, the expression levels of MsJAZ genes were examined across various tissues and under the influence of salt stress conditions, revealing tissue-specific expression and regulation by salt stress. Through RT‒qPCR experiments, it was discovered that the relative expression levels of these six MsJAZ genes increased under salt stress.ConclusionsIn summary, our study represents the first comprehensive identification and analysis of the JAZ gene family in alfalfa. These results provide important information for exploring the mechanism of JAZ genes in alfalfa salt tolerance and identifying candidate genes for improving the salt tolerance of autotetraploid cultivated alfalfa via genetic engineering in the future.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit’s efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Genetic diversity, population structure, and taxonomic confirmation in annual medic (Medicago spp.) collections from Crimea, Ukraine.

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and species-representative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species.

Characterization and expression profiles of WUSCHEL-related homeobox (WOX) gene family in cultivated alfalfa (Medicago sativa L.)

The WUSCHEL-related homeobox (WOX) family members are plant-specific transcriptional factors, which function in meristem maintenance, embryogenesis, lateral organ development, as well as abiotic stress tolerance. In this study, 14 MsWOX transcription factors were identified and comprehensively analyzed in the cultivated alfalfa cv. Zhongmu No.1. Overall, 14 putative MsWOX members containing conserved structural regions were clustered into three clades according to phylogenetic analysis. Specific expression patterns of MsWOXs in different tissues at different levels indicated that the MsWOX genes play various roles in alfalfa. MsWUS, MsWOX3, MsWOX9, and MsWOX13-1 from the three subclades were localized in the nucleus, among which, MsWUS and MsWOX13-1 exhibited strong self-activations in yeast. In addition, various cis-acting elements related to hormone responses, plant growth, and stress responses were identified in the 3.0 kb promoter regions of MsWOXs. Expression detection of separated shoots and roots under hormones including auxin, cytokinin, GA, and ABA, as well as drought and cold stresses, showed that MsWOX genes respond to different hormones and abiotic stress treatments. Furthermore, transcript abundance of MsWOX3, and MsWOX13-2 were significantly increased after rhizobia inoculation. This study presented comprehensive data on MsWOX transcription factors and provided valuable insights into further studies of their roles in developmental processes and abiotic stress responses in alfalfa.

Genome-wide identification and expression analysis of the Auxin-Response factor (ARF) gene family in Medicago sativa under abiotic stress

BackgroundAlfalfa (Medicago sativa) is the most widely planted legume forage and one of the most economically valuable crops in the world. The periodic changes in its growth and development and abiotic stress determine its yield and economic benefits. Auxin controls many aspects of alfalfa growth by regulating gene expression, including organ differentiation and stress response. Auxin response factors (ARF) are transcription factors that play an essential role in auxin signal transduction and regulate the expression of auxin-responsive genes. However, the function of ARF transcription factors is unclear in autotetraploid-cultivated alfalfa.ResultA total of 81 ARF were identified in the alfalfa genome in this study. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, identifying that ARF genes are mainly involved in transcriptional regulation and plant hormone signal transduction pathways. Phylogenetic analysis showed that MsARF was divided into four clades: I, II, III, and IV, each containing 52, 13, 7, and 9 genes, respectively. The promoter region of the MsARF gene contained stress-related elements, such as ABRE, TC-rich repeats, MBS, LTR. Proteins encoded by 50 ARF genes were localized in the nucleus without guide peptides, signal peptides, or transmembrane structures, indicating that most MsARF genes are not secreted or transported but only function in the nucleus. Protein structure analysis revealed that the secondary and tertiary structures of the 81 MsARF genes varied. Chromosomal localization analysis showed 81 MsARF genes were unevenly distributed on 25 chromosomes, with the highest distribution on chromosome 5. Furthermore, 14 segmental duplications and two sets of tandem repeats were identified. Expression analysis indicated that the MsARF was differentially expressed in different tissues and under various abiotic stressors. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression profiles of 23 MsARF genes were specific to abiotic stresses such as drought, salt, high temperature, and low temperature, as well as tissue-specific and closely related to the duration of stress.ConclusionThis study identified MsARF in the cultivated alfalfa genome based on the autotetraploid level, which GO, KEGG analysis, phylogenetic analysis, sequence characteristics, and expression pattern analysis further confirmed. Together, these findings provide clues for further investigation of MsARF functional verification and molecular breeding of alfalfa. This study provides a novel approach to systematically identify and characterize ARF transcription factors in autotetraploid cultivated alfalfa, revealing 23 MsARF genes significantly involved in response to various stresses.

Genome-wide identification of the GRAS transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under drought stress

GRAS transcription factors are one of the most important plant-specific gene families and play diverse roles in plant development and abiotic stress. A total of 55 MsGRAS genes were obtained from the autotetraploid cultivated alfalfa genome of XinJiangDaYe. Phylogenetic analysis demonstrated that the MsGRAS genes were classified into 10 subgroups, including HAM, LAS, SCL4/7, DLT, SCR, SCL3, DELLA, PAT1, SHR, and LISCL. Gene structure analysis showed that 92.7% of MsGRAS genes lacked introns. Many hormone- and stress-related cis-regulatory elements have been identified in the promoter regions of MsGRAS genes. A total of 55 MsGRAS genes were unevenly mapped on 32 alfalfa chromosomes, except for MsGRAS53 and MsGRAS55. Gene duplication analysis demonstrated that segmental duplication was the key driving force for the expansion of this gene family during evolution. Expression analysis of 55 MsGRAS genes in various tissues and under drought stress showed that they were differentially expressed. RT-qPCR analysis revealed that the expression of eight selected MsGRAS genes was significantly induced by drought stress. Collectively, our study is the first to investigate GRAS genes in autotetraploid cultivated alfalfa, and these findings will further enrich our knowledge regarding GRAS transcription factor genes and establish a foundation for enhancing drought stress tolerance by genetic engineering in autotetraploid cultivated alfalfa.

Influence of sowing time on the yield of fodder grasses

Cultivated alfalfa is a species native to the Middle East and the most abundant food species in the world. In much of the dry steppe zone of Western Kazakhstan, it is the most important legume fodder crop grown for hay, dehydrated feed, pellets and silage. Alfalfa is also particularly important for restoring soil fertility: without the cost of nitrogen, residues increase soil organic matter, the root system mobilizes nutrients deep in the soil profile and soil, improves structure, water permeability and water retention. In addition, alfalfa requires only a small amount of herbicides and pesticides and no N-fertilization. This is consistent with EU policy and public concern about the environmental impact of agricultural activities. Alfalfa hay fields have also been shown to be highly beneficial for enriching biodiversity. Its high protein content meets the demands of the feed market, especially after the problems caused by the use of concentrates of animal origin, currently banned in the EU. The process of growth and development of plants is directly related to the biological characteristics of their development. All available phenological observations of the growth and development of crops provide an assessment of the conditions that ensure their productivity. The peculiarities of the growth and development of plants, caused by the deficiencies of at least one of the conditions of their vital activity, lead to a delay, and sometimes to a cessation of their growth. In this regard, the intensity of growth, and with this the level of their fodder significance, can serve as indicators of their provision with the necessary living conditions.

Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa.

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Total Saponin Content (TSC) of different alfalfa (Medicago sativa L.) cultivars cultivated in field experiment

Saponins are secondary metabolites produced by various plants. These compounds have important role in the defence system. The word saponin refers to a group of different chemical compounds. Basically, sugar conjugates of triterpenoids or steroids are called saponins. Triterpene-type saponins are more specific among dicotyledonous plants, while steroid-type saponins are more characteristic of plants belonging to the monocotyledonous taxonomic group. Alfalfa is a large-scale cultivated and foraged fodder plant in Hungary. In the defence mechanism of alfalfa, saponins also play an important role. However, large amount of saponins can be toxic in animal fodder, especially in the poultry farming and piggery. As a dicotyledonous plant, the alfalfa saponins are mainly triterpenoid type. In our study we measure the total triterpenoid saponin content and leaf stem ratio of field cultivated alfalfa cultivars. Samples were collected from a randomized block design experiment, planted in the Demonstration Garden and Arboretum of Institutes of Agricultural Research and Educational Farm, in Debrecen, in 2018. Three different cultivar were investigated, and the samples were collected three different times of the growing season at growing stage of early flowering, for three years (2018-2020.). There was no difference in the TSC of examined varieties on the average of all measured data, although the sampling time showed significant effect on TSC. Our results attract attention to the fact that ageing of alfalfa stands can cause increase in TSC.

Genome-wide analysis of the GlutathioneS-Transferase family in wild Medicago ruthenica and drought-tolerant breeding application ofMruGSTU39 gene in cultivated alfalfa.

Transformation of MruGSTU39 in M. ruthenica and alfalfa enhanced growth and survival of transgenic plants by up-regulating GST and glutathione peroxidase activity to detoxify ROS under drought stress. Glutathione S-transferases (GSTs) are ubiquitous supergenefamily which play crucial roles in detoxification of reactive oxygenspecies (ROS). Despite studies on GSTs, few studies have focused on them in perennial, wild plant species with high tolerance to environmental stress. Here, we identified 66 MruGST genes from the genome of Medicago ruthenica, a perennial legume species native to temperate grasslands with high tolerance to environmental stress. These genes were divided into eight classes based on their conserved domains, phylogenetic tree and gene structure, with the tau class being the most numerous. Duplication analysis revealed that GST family in M. ruthenica was expanded by segmental and tandem duplication. Several drought-responsive MruGSTs were identified by transcriptomic analyses. Of them, expression of MruGSTU39 was up-regulated much more in a tolerant accession by drought stress. Transformation of MruGSTU39 in M. ruthenica and alfalfa (Medicago sativa) enhanced growth and survival of transgenic seedlings than their wild-type counterparts under drought. We demonstrated that MruGSTU39 can detoxify ROS to reduce its damage to membrane by up-regulating activities of GST and glutathione peroxidase. Our findings provide full-scaleknowledge on GST family in the wild legume M. ruthenica with high tolerance to drought, and highlight improvement tolerance of legume forages to drought using genomic information of M. ruthenica.

Genome-wide identification of the MADS-box transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under abiotic stress

BackgroundAlfalfa, the “queen of forage”, is the most extensively cultivated forage legume in the world. The development and yield of alfalfa are seriously limited by abiotic stress. MADS-box transcription factors are one of the largest gene families and play a pivotal role in plant development and abiotic stress. However, little is known regarding the MADS-box transcription factors in autotetraploid cultivated alfalfa.ResultsIn the present study, we identified 120 MsMADS-box genes in the alfalfa genome. Phylogenetic analysis indicated that 75 type-I MsMADS-box genes were classified into the Mα, Mβ, and Mγ subgroups, and 45 type-II MsMADS-box genes were classified into 11 subgroups. The promoter region of MsMADS-box genes containing several hormone and stress related elements. Chromosomal location analysis revealed that 117 MsMADS-box genes were unevenly distributed on 32 chromosomes, and the remaining three genes were located on unmapped scaffolds. A total of nine pairs of segmental duplications and four groups of tandem duplications were found. Expression analysis showed that MsMADS-box genes were differentially expressed in various tissues and under abiotic stresses. qRT-PCR analysis revealed that the expression profiles of eight selected MsMADS-box genes were distinct under various stresses.ConclusionsIn this study, MsMADS-box genes were identified in the cultivated alfalfa genome based on autotetraploid level, and further confirmed by Gene Ontology (GO) analysis, phylogenetic analysis, sequence features and expression analysis. Taken together, these findings will provide clues for further study of MsMADS-box functions and alfalfa molecular breeding.Our study is the first to systematically identify and characterize the MADS-box transcription factors in autotetraploid cultivated alfalfa (Medicago sativa L.), and eight MsMADS-box genes were significantly involved in response to various stresses.

Phenotypic Diversity and Productivity of Medicago sativa Subspecies from Drought-Prone Environments in Mediterranean Type Climates.

The phenotypic diversity and productivity of a diverse alfalfa (M. sativa subspp.) panel of cultivars, landraces and wild relatives with putative drought tolerance were evaluated in two Mediterranean environments (central Chile and Southern Australia). In Chile, 70 accessions were evaluated in rainfed conditions and in Australia 30 accessions under rainfed and irrigated conditions, during three growing seasons. Large phenotypic variation was observed among and within subspecies for NDVI, stem length, intercepted PAR and forage yield. Principal component analysis indicated that the first two principal components (PC) accounted for 84.2% of total variance; fall dormancy, taxa, and breeding status were closely related to the agronomical performance of alfalfa accessions. Forage yield varied largely among accessions across years and locations. A linear relationship was found between annual forage yield and annual water added to the experiments (R2 = 0.60, p < 0.001). The GxE analysis for forage yield allowed the detection of the highest yielding accessions for each of the two mega-environments identified. The accessions CTA002 and CTA003 showed greater forage yield in both Chile and Australia environments. It is concluded that new breeding lines derived from crosses between cultivated alfalfa (M. sativa subsp. sativa) and wild relatives belonging to the primary (M. sativa subsp. falcata) and tertiary (M. arborea) gene pool, achieve outstanding agronomical performance in drought-prone environments.

Genomic analysis of Medicago ruthenica provides insights into its tolerance to abiotic stress and demographic history.

Medicago ruthenica has been recently cultivated as a new forage crop and has been recognized as a source of genes to improve abiotic stress tolerance in cultivated alfalfa because of its remarkable tolerance to drought, salinity-alkalinity, and cold and snowy winters. Here, we reveal a chromosome-scale genome sequence of M.ruthenica based on Illumina, PacBio, and Hi-C data. The assembled genome consists of 903.56Mb with 50,268 annotated protein-coding genes, which is larger and contains relatively more genes than Medicago truncatula (420Mb and 44,623 genes) and Medicago sativa spp. caerulea (793Mb and 47,202 genes). All three species shared the ancestral Papilionoideae whole-genome duplication event before their divergence. The more recent expansion of repetitive elements compared to that in the other two species was determined to have contributed greatly to the larger genome size of M.ruthenica. We further found that multiple gene and transcription factor families (e.g., SOS homologous genes, NAC, C2H2, and CAMTA) have expanded in M.ruthenica, which might have led to its enhanced tolerance to abiotic stress. In addition, M.ruthenica harbors more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed two genetic lineages, reflecting the west and east of its geographical distribution, respectively. The two lineages probably diverged during the last glaciation and survived in multiple refugia at the last glacial maximum, followed by recent expansion. Our genomic data provide a genetic basis for further molecular breeding research on M.ruthenica and alfalfa.

СТРУКТУРА МИКОКОМПЛЕКСОВ ФИЛЛОПЛАНЫ БОБОВЫХ КУЛЬТУР

The importance of legumes as a component of biological crop rotations dictates the need to study the species composition of microscopic fungi in the phylloplan and to determine the presence of phytopathogenic, toxigenic, allergenic and opportunistic species in mycocomplexes. The aim was to study the structure of mycocomplexes of the phylloplan of cultivated peanuts, cowpea, sowing vetch, varicoloured clover, creeping clover, alfalfa, and hornbeam. The isolation of fungi from the phylloplan was carried out by washing from the leaf surface in 4 replicates. The prepared samples were transferred to Petri dishes with Saburo media. The experiment was repeated 3-5 times. The cultures were incubated in a thermostat at 25 ± 2 ºС for 5-14 days. The grown colonies were seeded into a test tube on an oblique nutrient medium. From the leaf surface of the studied legumes, 17 genera were identified, represented by 31 species of fungi, which are grouped according to the frequency of occurrence of species in the complex and the proportion in the group. A high proportion of A. alternata fungi was characteristic of the mycocomplexes of leaves of the common vetch, varicoloured elm, creeping clover, and alfalfa. F. sporotrichioides was found on creeping clover and alfalfa, F. moniliforme was found on horned clover, and both species were found on radiant cowpea. The content of toxigenic, opportunistic and allergenic species of fungi is least of all revealed under annual species: cultivated peanuts and radiant cowpea. The species composition of the phylloplan of leguminous crops should be taken into account in the development of plant protection measures and taking into account their correct placement in crop rotations.

The Chromosome-Level Genome Sequence of the Autotetraploid Alfalfa and Resequencing of Core Germplasms Provide Genomic Resources for Alfalfa Research

Alfalfa (Medicago sativa) is one of the most important forage crops in the world; however, its molecular genetics and breeding research are hindered due to the lack of a high-quality reference genome. Here, we report a de novo assembled 816-Mb high-quality, chromosome-level haploid genome sequence for ‘Zhongmu No.1’ alfalfa, a heterozygous autotetraploid. The contig N50 is 3.92 Mb, and 49 165 genes are annotated in the genome. The alfalfa genome is estimated to have diverged from M. truncatula approximately 8 million years ago. Genomic population analysis of 162 alfalfa accessions revealed high genetic diversity, weak population structure, and extensive gene flow from wild to cultivated alfalfa. Genome-wide association studies identified many candidate genes associated with important agronomic traits. Furthermore, we showed that MsFTa2, a Flowering Locus T homolog, whose expression is upregulated in salt-resistant germplasms, may be associated with fall dormancy and salt resistance. Taken together, these genomic resources will facilitate alfalfa genetic research and agronomic improvement.

Trace organic contaminants in field-scale cultivated alfalfa, soil, and pore water after 10 years of irrigation with reclaimed wastewater

Several studies have assessed the presence of trace organic contaminants (TrOCs) in reclaimed wastewater and their uptake into crops but there are limited data from field-scale studies. In this research, we studied the fate of 12 TrOCs in wastewater samples, 11 in plants and in soil pore water, and 10 in soil at an agricultural research farm, which was irrigated with reclaimed wastewater for more than 10 years. First, we examined the presence of the compounds in the primary, secondary, and treated effluent of a regional water reclamation facility which provides the irrigation water. The agricultural field produced alfalfa, and we measured the contaminants in the alfalfa, soil, and pore water. Reclaimed wastewater contained 11 of 12 TrOCs, with concentrations ranging from 26 ± 9 ng/L (DEET) to 1539 ± 1899 ng/L (trimethoprim). Atrazine was not detected. Pore water collected during non-irrigating periods contained all analyzed TrOCs, from 3 ± 0.1 ng/L (diphenhydramine) to 343 ± 360 ng/L (primidone). TrOCs measured in pore water samples indicated that they may leach to shallow groundwater even when fields are not being irrigated. Soils from 0 to 60 cm contained nine TrOCs from below the reporting limit (fluoxetine) to 329 ng/g (carbamazepine). Atrazine was not detected. TrOC concentrations decreased with increasing soil depth, except carbamazepine, which was more recalcitrant. Nine TrOCs were present in the shoots and leaves of alfalfa grown in the reclaimed wastewater irrigated plots, at concentrations ranging from <1 ng/g (diphenhydramine and fluoxetine) to 49 ng/g dry weight (carbamazepine). Atrazine and caffeine were not detected. We estimated that a maximum of 14% of DEET from the reclaimed wastewater was accumulated in alfalfa. Overall, despite some accumulation of TrOCs in the soil, alfalfa uptake was limited, which results in low exposure to foraging animals.

Uticaj faze košenja i setvene norme na prinos i kvalitet lucerke

Cultivated alfalfa (Medicago sativa L.) is a highly productive forage crop with great economic potential. Our objectives were to investigate the effects and interactions of environment, harvest maturity stage, seeding rate and cultivars on alfalfa dry matter yield and quality. The field experiment was carried out over 2010-2012 at the Experimental Field of Institute of Field and Vegetable Crops in Novi Sad, at two locations: (I) Cenej, and (II) Rimski Sancevi, which were characterized by a contrasting soil condition. The experimental treatments included two seeding rates of 8 and 16 kg ha-1, three different harvest maturity stages of alfalfa, and four alfalfa cultivars included as a subplot in every treatment. Harvesting alfalfa five times within a year (cutting in the beginning of flowering) in the second and third year of stand life is the most efficient harvest regime that allows full exploitation of cultivar genetic potential and environmental conditions. There was no difference in yields between harvests at early and full flowering stage (15.9 t ha-1). Data related to forage quality components show increase of nutritive value when harvesting alfalfa in earlier maturity stage. Maturity stage in the moment of harvesting significantly affected all quality parameters. The results obtained suggest that in a temperate climate of Southeast Europe there is little justification for planting higher seeding rates of alfalfa varieties under good establishment practices. Increasing seeding rates above 16 kg ha-1 does not provide a long-term boon in alfalfa production.

Linkage Disequilibrium (LD) Analysis in Alfalfa (Medicago sativa L.) Populations Spreading in Different Geographies of the World

Cultivated alfalfa (Medicago sativa L.) was derived from Medicago sativa species complex as a result of breeding efforts. New efforts have focused on to determine the DNA polymorphisms based on molecular markers and to link these polymorphisms with related phenotype recently. Especially, the relationships between genotypes and phenotypes are evaluated as Linkage Analysis and Association Mapping Studies. On the basis of information, in this study, Linkage Disequilibrium (LD) analysis was also performed using 31 SSR markers for the 70 populations. For the LD analysis, 23 of the 31 markers identified the physical distances on the 8 chromosomes of the alfalfa. Distances of 103 loci on 8 chromosomes were determined based on Medicago truncatula genome. A significant (p&amp;lt;0,0001) LD value was not observed in these populations for the evaluated markers.

Multiple Regulatory Networks Are Activated during Cold Stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.