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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 β-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., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Melilotus is one of the most important legume plants, in part due to its production of coumarin, but the genetic diversity among the 18 species remains unclear. In the present study, the analysis of genetic diversity of the Melilotus species was performed with simple sequence repeat (SSR) markers. From the PCR amplification, we selected 18 out of 70 primers that were used in previous studies. Three hundred twenty-six sampled individuals were assayed to study the genetic diversity and polymorphisms based on the SSR markers. All analyzed markers were polymorphic, and 287 alleles were identified, with 15.94 alleles per locus detected. The polymorphism information content ( PIC ) values ranged from 0.71 to 0.93, with an average of 0.87, which indicates that the markers were highly informative. Based on the unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis, we found that the 18 species were divided into two clusters. M. italicus and M.​ speciosus from cluster A and M. indicus and M. segetalis from cluster B were closely related. Population structure analysis suggested that the optimum number of groups was three. From the analysis of molecular variance, 17.79% of the variance was due to species differences, 31.61% of the variance was due to differences among populations within species and the remaining 50.60% was due to differences within populations. The results of the present study showed that these SSR markers will benefit the Melilotus research community for genetics and breeding. Furthermore, this study also established the foundation for future breeding programs, genetic improvement and coumarin production in the Melilotus species. [ABSTRACT FROM AUTHOR]

Abstract: Annual pasture legumes with high tolerance of combined salinity and waterlogging are needed for saline areas in many rain-fed agricultural regions. Melilotus siculus is an annual legume from saline marshy areas of the Mediterranean with potential use in saline pastures and, based on descriptions of its native habitat, was hypothesised to tolerate combined salinity and waterlogging. Experiments compared M. siculus to Trifolium michelianum and Medicago polymorpha, other annual pasture legumes with reported salt or waterlogging tolerances, with 0–450mM NaCl in hydroponics or in sand culture. Tolerance to combined salinity and waterlogging was also evaluated at 150, 400 and 550mM NaCl in a stagnant deoxygenated nutrient solution. M. siculus was substantially more salt tolerant than the other two species. At 450mM NaCl, shoot DM of M. siculus was 30% of control, compared with only 15% for the other species. M. siculus was also the most tolerant species to combined salinity and stagnant treatment, and produced new leaves even after 14d in stagnant nutrient solution with 550mM NaCl (∼ sea water salinity). In comparison, T. michelianum and M. polymorpha only survived up to 5d in stagnant solution with 400mM NaCl. Even at only 150mM NaCl in stagnant solution, shoot DM was just 30% of control for T. michelianum and M. polymorpha, compared to 60% for M. siculus. Tolerance to combined salinity and waterlogging was associated with higher root porosity and regulation of shoot Na+ and Cl−, with the more tolerant M. siculus having similar shoot Na+ and Cl− concentrations for both aerated-saline and stagnant-saline treatments. In summary, M. siculus is a pasture legume that can grow at high salinity (up to 550mM) even when in combination with waterlogging, as root porosity and associated O2 transport presumably enables continued regulation by the roots of Na+ and Cl− entry under these dual stress conditions. [Copyright &y& Elsevier]

This paper describes the range in coumarin concentrations at 90% flowering stage in 149 accessions belonging to 15 Melilotus species grown in a genetic resources field characterisation experiment in Adelaide, South Australia, and determines coumarin concentrations in Melilotus species grown at two contrasting field sites in South Australia (Kybybolite and Keith). In the genetic resources characterisation experiment, the mean coumarin content ranged from 0.06 to 0.753% of dry matter. M. segetalis, M. dentatus, M. sulcatus, M. siculus and M. infestus recorded low (0.06 to 0.113%) coumarin levels while moderate levels (0.332-0.753%) were registered in the other species. The coumarin levels at Kybybolite and Keith sites ranged from 0.095 to 0.943% amongst M. indicus accessions compared to M. siculus (0.007-0.100%) and M. sulcatus (0.007-0.810%). No coumarin was detected in a M. segetalis accession at these two sites. Accessions of M. siculus and an accession of M. segetalis (SA 36979) and of M. sulcatus (SA 40019) could be potential candidates as pasture legumes that can combine tolerance of both salinity and waterlogging, and acceptably low concentrations of coumarin. [ABSTRACT FROM AUTHOR]

Abstract: Previously unexploited legume species may offer utilization potential where environmental stresses constrain the use of more conventional forage crops. Melilotus indicus (L.) All., Yellow sweet clover, occurs as a weed in different habitats in Egypt. It grows in moderately saline areas, where traditional forage legumes cannot be cultivated. Our extensive field studies have recorded the species in many different habitats ranging from healthy agricultural lands to abandoned saline areas. The studied plants maintained high nodulation capacity (68 – 95%) and nitrogenase activities (about 1.81μmolC2H4 plant−1 h−1) in different habitats. Greenhouse experiments demonstrated that seed germination was maintained at 80% when growing on substrats containing 200mM NaCl and that 25% of the germination capability was preserved when 300mM NaCl was added to the growth medium. The growth rate of seedlings was not significantly affected by 200mM NaCl but was reduced by 30% under 300mM NaCl. It is supposed that M. indicus uses a salt inclusion mechanism for maintaining growth under saline conditions, as it accumulated high amounts of Na+ and Cl− ions. Leaf succulence and indices of leaf water status did not differ among the salt treatments, whereas relative water content was reduced by only 3% and water content at saturation increased by about 14% at high salt concentrations in the growing medium. Our results suggest recommending the cultivation of M. indicus in salt-affected soils, which are widespread and pose a problem for the farmers of Egypt and other countries in the world''s arid belt. [Copyright &y& Elsevier]

In the montane grasslands of Rocky Mountain National Park, Colorado, non-native plant species are affecting the distribution and cover of native plant species. Two exotic nitrogen-fixing Eurasian sweetclover species (Melilotus officinalis and M. alba) occur in patches that have less available nitrogen (N) and a species composition different from surrounding grassland patches. I tested whether burning sweetclover-invaded patches would increase native grass and perennial diversity and available soil nitrogen at this elevation. I burned 24 sweetclover-invaded and noninvaded field plots with a propane torch in May 1999. Burning increased species richness and ground cover. Specifically, one year later native, grass, and perennial species increased in previously invaded patches and sweetclover or other exotic species associated with sweetclover did not increase. Soil ammonium (NH4+) levels increased immediately after the burn in May, and nitrate (NO3-) levels increased later in July and August. New species appeared only in burned plots. While other methods to reduce sweetclover might remove resources, fire increases light, space, and nutrient availability for native species that, in turn, suppress the exotic species. [ABSTRACT FROM AUTHOR]

Four different low molecular weight (LMW) RNA profiles, designated I–IV, among 179 isolates from Medicago, Melilotus and Trigonella species growing in a field site in Northern Spain were identified. From sequence analysis of the 16S rRNA, atpD and recA genes as well as DNA–DNA hybridization analysis with representatives of each LMW RNA profile it was evident that isolates with LMW RNA profiles I and II belonged to Sinorhizobium meliloti and those displaying profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA electrophoretic mobility profiles were found within each of these two species. Collectively, LMW RNA profiles I and II (identified as S. meliloti) were predominant in Melilotus alba, Melilotus officinalis and Medicago sativa. Profiles III and IV (identified as S. medicae) were predominant in Melilotus parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella foenum-graecum. All the four LMW RNA profiles were identified among isolates from Trigonella monspelliaca nodules. These results revealed a different specificity by the hosts of the alfalfa cross-inoculation group towards the two bacterial species found in this study. [ABSTRACT FROM AUTHOR]

We compared the native bees visiting the flowers of three species of invasive plants, saltcedar (Tamarix spp.) and white and yellow sweet clover (Melilotus albus, M. officinalis), with those visiting seven concurrently blooming native plant species in mid-summer at three sites in Capitol Reef National Park, Utah. Overall, as many total species of bees visited the flowers of the three invasive plant species as visited the seven natives. On average, invasive species were visited by twice as many bee species as were natives. With a single exception, visitors of invasives were generalist bees, rather than specialists. Colletes petalostemonis, the only native legume specialist recorded, was an abundant forager on the flowers of both species of Melilotus, demonstrating that at least some specialist bees will move to invasive plants that are closely related to their usual hosts. Species abundant on the flowers of invasives tended to collect both pollen and nectar, suggesting that bees are using pollen of Tamarix and Melilotus to provision their offspring. We argue that invasives with entomophilous flowers are unlikely to either facilitate the reproduction of uncommon native plants or consistently compete with them for pollinators. Rather, they are likely, overtime, to selectively increase the carrying capacity and population size of native bees, specifically generalists, and specialists of closely related plant species. [ABSTRACT FROM AUTHOR]

The aim of this work was to examine variation for activity of symbiosis with Rhizobium meliloti in alfalfa, sweet clover and fenugreek. Seed specimens were obtained from the collection of the All-Union Research Institute for Plant Breeding and Rhizobium strains from the All-Union Research Institute for Agricultural Microbiology, both in Leningrad. Sterile methods of vegetative culture were employed. Statistical analysis of data on inter- and intracultivar variation allow one to suppose that in alfalfa and sweet clover, nitrogenase activity and symbiotic efficiency (ability of plants to increase biomass after inoculation) are controlled by different groups of genes. The latter symbiotic property is more strictly controlled by host genotype than the former. Fenugreek appeared to be more variable in its capacity for symbiosis than alfalfa and sweet clover. Differences between genotypes in symbiotic activity correlate with division into subspecies and are inherited in self-fertile progeny of fenugreek plants. Differences between R. meliloti strains for the efficiency of symbiosis with heterogenic natural fenugreek population are expressed to a lesser degree than with plants of more homogenic cultivar populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]