A comparative plastome approach enhances the assessment of genetic variation in the Melilotus genus.

BACKGROUND: Melilotus, a member of the Fabaceae family, is a pivotal forage crop that is extensively cultivated in livestock regions globally due to its notable productivity and ability to withstand abiotic stress. However, the genetic attributes of the chloroplast genome and the evolutionary connections among different Melilotus species remain unresolved. RESULTS: In this study, we compiled the chloroplast genomes of 18 Melilotus species and performed a comprehensive comparative analysis. Through the examination of protein-coding genes, we successfully established a robust phylogenetic tree for these species. This conclusion is further supported by the phylogeny derived from single-nucleotide polymorphisms (SNPs) across the entire chloroplast genome. Notably, our findings revealed that M. infestus, M. siculus, M. sulcatus, and M. speciosus formed a distinct subgroup within the phylogenetic tree. Additionally, the chloroplast genomes of these four species exhibit two shared inversions. Moreover, inverted repeats were observed to have reemerged in six species within the IRLC. The distribution patterns of single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within protein-coding genes indicated that ycf1 and ycf2 accumulated nonconservative alterations during evolutionary development. Furthermore, an examination of the evolutionary rate of protein-coding genes revealed that rps18, rps7, and rpl16 underwent positive selection specifically in Melilotus. CONCLUSIONS: We present a comparative analysis of the complete chloroplast genomes of Melilotus species. This study represents the most thorough and detailed exploration of the evolution and variability within the genus Melilotus to date. Our study provides valuable chloroplast genomic information for improving phylogenetic reconstructions and making biogeographic inferences about Melilotus and other Papilionoideae species.

Chromosome-scale assembly and analysis of Melilotus officinalis genome for SSR development and nodulation genes analysis.

Melilotus officinalis is an important legume crop with forage and Chinese medicinal value. The unknown genome of M. officinalis restricted the domestication and utilization of the species and its germplasm resource diversity. A chromosome-scale assembly of the M. officinalis genome was assembled and analysed. The 976.27 Mb of genome was divided into eight chromosomes covering 99.16% of the whole genome. A total of 50022 genes were predicted in the genome. M. officinalis and Melilotus albus shared a common ancestor 0.5-5.65 million years ago (MYA). A genome-wide doubling event occurred 68.93 MYA according to the synonymous nucleotide-substitution values. A total of 552102 tandem repeats were predicted, and 46004 SSR primers of TRs with 10 or more base pairs were developed and designed. The elucidation of the M. officinalis genome provides a compelling model system for studying the genetic, evolutionary and biosynthesis of this legume.

Genome-Wide Identification of the GRAS Family Genes in Melilotus albus and Expression Analysis under Various Tissues and Abiotic Stresses.

The GRAS gene family is a plant-specific family of transcription factors, which play an important role in many metabolic pathways, such as plant growth and development and stress response. However, there is no report on the comprehensive study of the GRAS gene family of Melilotus albus. Here, we identified 55 MaGRAS genes, which were classified into 8 subfamilies by phylogenetic analysis, and unevenly distributed on 8 chromosomes. The structural analysis indicated that 87% of MaGRAS genes have no intron, which is highly conservative in different species. MaGRAS proteins of the same subfamily have similar protein motifs, which are the source of functional differences of different genomes. Transcriptome and qRT-PCR data were combined to determine the expression of 12 MaGRAS genes in 6 tissues, including flower, seed, leaf, stem, root and nodule, which indicated the possible roles in plant growth and development. Five and seven MaGRAS genes were upregulated under ABA, drought, and salt stress treatments in the roots and shoots, respectively, indicating that they play vital roles in the response to ABA and abiotic stresses in M. albus. Furthermore, in yeast heterologous expression, MaGRAS12, MaGRAS34 and MaGRAS33 can enhance the drought or salt tolerance of yeast cells. Taken together, these results provide basic information for understanding the underlying molecular mechanisms of GRAS proteins and valuable information for further studies on the growth, development and stress responses of GRAS proteins in M. albus.

Genome and systems biology of Melilotus albus provides insights into coumarins biosynthesis.

Melilotus species are used as green manure and rotation crops worldwide and contain abundant pharmacologically active coumarins. However, there is a paucity of information on its genome and coumarin production and function. Here, we reported a chromosome-scale assembly of Melilotus albus genome with 1.04 Gb in eight chromosomes, containing 71.42% repetitive elements. Long terminal repeat retrotransposon bursts coincided with declining of population sizes during the Quaternary glaciation. Resequencing of 94 accessions enabled insights into genetic diversity, population structure, and introgression. Melilotus officinalis had relatively larger genetic diversity than that of M. albus. The introgression existed between M. officinalis group and M. albus group, and gene flows was from M. albus to M. officinalis. Selection sweep analysis identified candidate genes associated with flower colour and coumarin biosynthesis. Combining genomics, BSA, transcriptomics, metabolomics, and biochemistry, we identified a ß-glucosidase (BGLU) gene cluster contributing to coumarin biosynthesis. MaBGLU1 function was verified by overexpression in M. albus, heterologous expression in Escherichia coli, and substrate feeding, revealing its role in scopoletin (coumarin derivative) production and showing that nonsynonymous variation drives BGLU enzyme activity divergence in Melilotus. Our work will accelerate the understanding of biologically active coumarins and their biosynthetic pathways, and contribute to genomics-enabled Melilotus breeding.

Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance.

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein-protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.

Genome-Wide Analysis of the UDP-Glycosyltransferase Family Reveals Its Roles in Coumarin Biosynthesis and Abiotic Stress in Melilotus albus.

Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Genetic diversity, phylogenetic structure and development of core collections in Melilotus accessions from a Chinese gene bank.

Melilotus is an important forage legume, with high values as feed and medicine, and widely used as green manure, honey plant, and wildlife habitat enhancer. The genetic diversity, structure and subdivision of this forage crop remain unclear, and plant genetic resources are the basis of biodiversity and ecosystem diversity and have attracted increasing attention. In this study, the whole collection of 573 accessions from the National Gene Bank of Forage Germplasm (NGBFG, China) and 48 accessions from the National Plant Germplasm System (NPGS, USA) in genus Melilotus were measured with respect to five seed characters: seed length, width, width-to-length ratio, circumference and 100-seed weight. Shannon' genetic diversity index (H') and phenotypic differentiation (Pst) were calculated to better describe the genetic diversity. The ITS and matK sequences were used to construct phylogenetic trees and study the genetic relationships within genus Melilotu. Based on seed morphology and molecular marker data, we preliminarily developed core collections and the sampling rates of M. albus and M. officinalis were determined to be 15% and 25%, respectively. The results obtained here provide preliminary sorting and supplemental information for the Melilotus collections in NGBFG, China, and establish a reference for further genetic breeding and other related projects.

Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus.

BACKGROUND: A popular strategy to study alternative splicing in non-model organisms starts from sequencing the entire transcriptome, then assembling the reads by using de novo transcriptome assembly algorithms to obtain predicted transcripts. A similarity search algorithm is then applied to a related organism to infer possible function of these predicted transcripts. While some of these predictions may be inaccurate and transcripts with low coverage are often missed, we observe that it is possible to obtain a more complete set of transcripts to facilitate possible functional assignments by starting the search from the intermediate de Bruijn graph that contains all branching possibilities. RESULTS: We develop an algorithm to extract similar transcripts in a related organism by starting the search from the de Bruijn graph that represents the transcriptome instead of from predicted transcripts. We show that our algorithm is able to recover more similar transcripts than existing algorithms, with large improvements in obtaining longer transcripts and a finer resolution of isoforms. We apply our algorithm to study salt and waterlogging tolerance in two Melilotus species by constructing new RNA-Seq libraries. CONCLUSIONS: We have developed an algorithm to identify paths in the de Bruijn graph that correspond to similar transcripts in a related organism directly. Our strategy bypasses the transcript prediction step in RNA-Seq data and makes use of support from evolutionary information.

Tolerance to partial and complete submergence in the forage legume Melilotus siculus: an evaluation of 15 accessions for petiole hyponastic response and gas-filled spaces, leaf hydrophobicity and gas films, and root phellem.

Background and Aims: Submergence is a severe stress for most plants. Melilotus siculus is a waterlogging- (i.e. root zone hypoxia) tolerant annual forage legume, but data were lacking for the effects of partial and full submergence of the shoots. The aim was to compare the tolerance to partial and full submergence of 15 M. siculus accessions and to assess variation in traits possibly contributing to tolerance. Recovery ability post-submergence was also evaluated. Methods: A factorial experiment imposed treatments of water level [aerated root zone with shoots in air as controls, stagnant root zone with shoots in air, stagnant root zone with partial (75 %) or full shoot submergence] on 15 accessions, for 7 d on 4-week-old plants in a 20/15 °C day/night phytotron. Measurements included: shoot and root growth, hyponastic petiole responses, petiole gas-filled spaces, leaflet sugars, leaflet surface hydrophobicity, leaflet gas film thickness and phellem area near the base of the main root. Recovery following full submergence was also assessed. Key Results: Accessions differed in shoot and root growth during partial and full shoot submergence. Traits differing among accessions and associated with tolerance were leaflet gas film thickness upon submergence, gas-filled spaces in petioles and phellem tissue area near the base of the main root. All accessions were able to re-orientate petioles towards the vertical under both partial and full submergence. Petiole extension rates were maintained during partial submergence, but decreased during full submergence. Leaflet sugars accumulated during partial submergence, but were depleted during full submergence. Growth resumption after full submergence differed among accessions and was positively correlated with the number of green leaves retained at desubmergence. Conclusions: Melilotus siculus is able to tolerate partial and full submergence of at least 7 d. Leaflet surface hydrophobicity and associated gas film retention, petiole gas-filled porosity and root phellem abundance are important traits contributing to tolerance. Post-submergence recovery growth differs among accessions. The ability to retain green leaves is essential to succeed during recovery.

Analysis of miRNAs and their target genes in five Melilotus albus NILs with different coumarin content.

MicroRNAs (miRNAs) exhibit diverse and important roles in regulation of various biological processes at the post-transcriptional level in plants. In this study, Melilotus albus miRNA and their target genes were elucidated from five M. albus near-isogenic lines which differ in coumarin content to construct small RNA libraries through high-throughput sequencing. A total of 417 known miRNAs and 76 novel miRNAs were identified in M. albus. In addition, 4155 different target genes for 114 known miRNA families and 14 target genes for 2 novel miRNAs were identified in M. albus. Moreover, mtr-miR5248 and mtr-miR7701-5p target c35498_g3 and gma-miR396a-3p target c37211_g1 involved in coumarin biosynthesis were identified by using the differential expression of the miRNAs and their target genes correlation analysis. The abundance of miRNAs and potential target genes were validated by qRT-PCR analysis. We also found that there were both positive and negative expression changing patterns between miRNAs and their related target genes. Our first and preliminary study of miRNAs will contribute to our understanding of the functions and molecular regulatory mechanisms of miRNAs and their target genes, and provide information on regulating the complex coumarin pathway in M. albus for future research.

Coumarin Content, Morphological Variation, and Molecular Phylogenetics of Melilotus.

Melilotus albus and Melilotus officinalis are widely used in forage production and herbal medicine due to the biological activity of their coumarins, which have many biological and pharmacological activities, including anti-HIV and anti-tumor effects. To comprehensively evaluate M. albus and M. officinalis coumarin content (Cou), morphological variation, and molecular phylogeny, we examined the Cou, five morphological traits and the molecular characterization based on the trnL-F spacer and internal transcribed spacer (ITS) regions of 93 accessions. Significant (p < 0.05) variation was observed in the Cou and all five morphological traits in both species. Analysis of population differentiation (Pst) of the phenotypic traits showed that powdery mildew resistance (PMR) had the greatest Pst, meaning that this trait demonstrated the largest genetic differentiation among the accessions. The Pst values of dry matter yield (DMY) and Cou were relatively high. Biplot analysis identified accessions with higher DMY and higher and lower Cou. Analysis of molecular sequence variation identified seven haplotypes of the trnL-F spacer and 13 haplotypes of the ITS region. Based on haplotype and sequence analyses, the genetic variation of M. officinalis was higher than that of M. albus. Additionally, ITS sequence analysis showed that the variation among accessions was larger than that among species across three geographical areas: Asia, Europe, and North America. Similarly, variation among accessions for both the trnL-F and ITS sequences were larger than the differences between the geographical areas. Our results indicate that there has been considerable gene flow between the two Melilotus species. Our characterization of Cou and the morphological and genetic variations of these two Melilotus species may provide useful insights into germplasm improvement to enhance DMY and Cou.

Genetic variation and diversity in 199 Melilotus accessions based on a combination of 5 DNA sequences.

Melilotus is an important genus of legume plants and an herbage with excellent nitrogen fixation; it can tolerate extreme environmental conditions and possesses important medicinal value. However, there is limited genetic information about the genus; thus, we analysed four chloroplast loci (rbcL, matK, psbA-trnH and trnL-F) and one nuclear region (ITS) to determine the genetic diversity of 199 accessions from 18 Melilotus species. The rbcL and matK sequences were highly conserved, whereas the trnL-F and ITS sequences contained variable and parsimony-informative sites. In our analyses of the single and combined regions, we calculated the pairwise distance, haplotype and nucleotide diversity and gaps and then constructed phylogenetic trees to assess the genetic diversity, and our results revealed significant variations among the different accessions. The genetic distance values were between zero and nine, and based on the combined regions, the highest frequency value was approximately four. Melilotus showed high haplotype and nucleotide diversity, particularly in the ITS sequences, with values of 0.86 and 0.0087, respectively. The single ITS sequence, psbA-trnH, and the combined matK+rbcL+trnL-F (MRT) and matK+rbcL+psbA-trnH+trnL-F+ITS (MRPTI) regions showed interspecific variation in the gap analysis. Phylogenetic trees calculated using ITS, psbA-trnH and MRPTI sequences indicated distinct genetic relationship in 18 species, and these species could be divided into two groups. By determining the genetic diversity of plants, we can evaluate the genetic relationships among species and accessions, providing a basis for preserving and utilizing the genetic resources of Melilotus.

Cross-species transferability of EST-SSR markers developed from the transcriptome of Melilotus and their application to population genetics research.

Melilotus is one of the most important legume forages, but the lack of molecular markers has limited the development and utilization of Melilotus germplasm resources. In the present study, 151 M clean reads were generated from various genotypes of Melilotus albus using Illumina sequencing. A total of 19,263 potential EST-SSRs were identified from 104,358 unigene sequences. Moreover, 18,182 primer pairs were successfully designed, and 550 primer pairs were selected using criteria of base repeat type, fragment length and annealing temperature. In addition, 550 primer pairs were screened by using PCR amplification products and used to assess polymorphisms in 15 M. albus accessions. A total of 114 primer pairs were detected as being highly polymorphic, and the average polymorphism information content (PIC) value was 0.79. Furthermore, those 114 polymorphic primer pairs were used to evaluate the transferability to 18 species of the genus Melilotus, and 70 EST-SSR markers were found to be transferable among the 18 Melilotus species. According to the UPGMA dendrogram and STRUCTURE analysis, the 18 Melilotus species were classified into three clusters. This study offers a valuable resource for the genetic diversity and molecular assisted breeding of germplasm resources in the genus Melilotus.

Potential DNA barcodes for Melilotus species based on five single loci and their combinations.

Melilotus, an annual or biennial herb, belongs to the tribe Trifolieae (Leguminosae) and consists of 19 species. As an important green manure crop, diverse Melilotus species have different values as feed and medicine. To identify different Melilotus species, we examined the efficiency of five candidate regions as barcodes, including the internal transcribed spacer (ITS) and two chloroplast loci, rbcL and matK, and two non-coding loci, trnH-psbA and trnL-F. In total, 198 individuals from 98 accessions representing 18 Melilotus species were sequenced for these five potential barcodes. Based on inter-specific divergence, we analysed sequences and confirmed that each candidate barcode was able to identify some of the 18 species. The resolution of a single barcode and its combinations ranged from 33.33% to 88.89%. Analysis of pairwise distances showed that matK+rbcL+trnL-F+trnH-psbA+ITS (MRTPI) had the greatest value and rbcL the least. Barcode gap values and similarity value analyses confirmed these trends. The results indicated that an ITS region, successfully identifying 13 of 18 species, was the most appropriate single barcode and that the combination of all five potential barcodes identified 16 of the 18 species. We conclude that MRTPI is the most effective tool for Melilotus species identification. Taking full advantage of the barcode system, a clear taxonomic relationship can be applied to identify Melilotus species and enhance their practical production.

Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content.

Coumarin and its derivatives are widely used as fragrances in industrial products and have medical value. The goal of the present study was to discover genes and pathways related to coumarin biosynthesis in Melilotus albus using transcriptome analysis. The genes of five M. albus near-isogenic lines (NILs) that had different coumarin content and ß-glucosidase activity according to the investigation of pedigree were quantified and then analysed by RNA-Seq. Using transcriptome analysis, differentially expressed genes (DEGs) were identified in two pairwise comparisons that differed in coumarin content as well as in two pairwise comparisons that differed in ß-glucosidase activity. Gene expression pattern analysis suggested similar transcriptional trends in the genotypes with the same coumarin levels. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database of DEGs was used to identify functional pathways associated with coumarin biosynthesis. We identified 111 unigenes, with several DEGs among them possibly being related to coumarin synthesis pathways. Unigenes encoding a hexokinase, an abscisic acid receptor, a phenylalanine ammonia-lyase (PAL) and two peroxidases particularly showed correspondence with the coumarin content of different genotypes. These results will contribute to a better understanding of the coumarin biosynthesis in M. albus.

Interspecific Phylogenic Relationships within Genus Melilotus Based on Nuclear and Chloroplast DNA.

Melilotus comprises 19 species, while the phylogenetic relationships between species remain unclear. In the present work, three chloroplast genes, rbcL, matK, trnL-F, and one nuclear region, ITS (internal transcribed spacer) belonging to 48 populations of 18 species of Melilotus were sequenced and phylogenetic trees were constructed to study their interspecific relationships. Based on the phylogenetic tree generated in this study using rbcL analysis, the Melilotus genus is clearly monophyletic in the legume family. Both Bayesian and maximum-parsimony approaches were used to analyze the data. The nrDNA ITS provided more informative characteristics (9.8%) than cpDNA (3.0%). Melilotus contains two closely related groups, clade I and clade II. M. spicatus, M. indicus and M. segetalis have a close relationship. M. infestus, M. siculus and M. sulcatus are closely related. The comparing between molecular phylogeny and flower color classification in Melilotus showed that the flower color is not much informative for phylogenetics of this genus.

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera.

BACKGROUND AND AIMS: Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history. METHODS: Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes. KEY RESULTS: The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes. CONCLUSIONS: The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Effect of salt stress on the expression of NHX-type ion transporters in Medicago intertexta and Melilotus indicus plants.

Medicago intertexta and Melilotus indicus, two wild leguminous herbs with different tolerance to salinity were investigated for NaCl-induced changes in the expression level of some Na(+) transporters. M. indicus plants grew well at NaCl concentration from 0 to 400 mM, whereas growth of M. intertexta plants was severely inhibited at NaCl concentrations higher than 100 mM. In M. intertexta, increasing NaCl in the growth media caused a strong increase in Na(+) content concomitant with a decrease in K(+) content in leaves and, above all, roots. In comparison, M. indicus plants cultivated in the presence of NaCl accumulated much less Na(+) in leaves and roots and no differences in K(+) content among plants grown in nutrient solution containing 100-400 mM NaCl were detected. The expression levels of four genes coding for NHX-type Na(+)/H(+) antiporters in the above two wild legumes were studied in plants cultivated under the different NaCl concentrations. Expression levels of the genes were higher in M. intertexta as compared with M. indicus plants. In M. intertexta, salt treatments increased MtNHX1, MtNHX3 and MtNHX4 transcript levels in leaves and roots. However, in M. indicus NaCl treatments only induced the expression of MtNHX1 in roots. Our data suggest that two different mechanisms, Na(+) avoidance or accumulation into cellular compartments, are developed by the two wild legumes to cope with salt stress, and that expression of NHX antiporters is linked to the accumulator phenotype.