The black honey bee genome: insights on specific structural elements and a first step towards pangenomes.

BACKGROUND: The honey bee reference genome, HAv3.1, was produced from a commercial line sample that was thought to have a largely dominant Apis mellifera ligustica genetic background. Apis mellifera mellifera, often referred to as the black bee, has a separate evolutionary history and is the original type in western and northern Europe. Growing interest in this subspecies for conservation and non-professional apicultural practices, together with the necessity of deciphering genome backgrounds in hybrids, triggered the necessity for a specific genome assembly. Moreover, having several high-quality genomes is becoming key for taking structural variations into account in pangenome analyses. RESULTS: Pacific Bioscience technology long reads were produced from a single haploid black bee drone. Scaffolding contigs into chromosomes was done using a high-density genetic map. This allowed for re-estimation of the recombination rate, which was over-estimated in some previous studies due to mis-assemblies, which resulted in spurious inversions in the older reference genomes. The sequence continuity obtained was very high and the only limit towards continuous chromosome-wide sequences seemed to be due to tandem repeat arrays that were usually longer than 10 kb and that belonged to two main families, the 371 and 91 bp repeats, causing problems in the assembly process due to high internal sequence similarity. Our assembly was used together with the reference genome to genotype two structural variants by a pangenome graph approach with Graphtyper2. Genotypes obtained were either correct or missing, when compared to an approach based on sequencing depth analysis, and genotyping rates were 89 and 76% for the two variants. CONCLUSIONS: Our new assembly for the Apis mellifera mellifera honey bee subspecies demonstrates the utility of multiple high-quality genomes for the genotyping of structural variants, with a test case on two insertions and deletions. It will therefore be an invaluable resource for future studies, for instance by including structural variants in GWAS. Having used a single haploid drone for sequencing allowed a refined analysis of very large tandem repeat arrays, raising the question of their function in the genome. High quality genome assemblies for multiple subspecies such as presented here, are crucial for emerging projects using pangenomes.

Detection of DNA methylation signatures through the lens of genomic imprinting.

Genomic imprinting represents an original model of epigenetic regulation resulting in a parent-of-origin expression. Despite the critical role of imprinted genes in mammalian growth, metabolism and neuronal function, there is no molecular tool specifically targeting them for a systematic evaluation. We show here that enzymatic methyl-seq consistently outperforms the bisulfite-based standard in capturing 165 candidate regions for genomic imprinting in the pig. This highlights the potential for a turnkey, fully customizable and reliable capture tool of genomic regions regulated by cytosine methylation in any population of interest. For the field of genomic imprinting, it opens up the possibility of detecting multilocus imprinting variations across the genome, with implications for basic research, agrigenomics and clinical practice.

Marinitoga aeolica sp. nov., a novel thermophilic anaerobic heterotroph isolated from a shallow hydrothermal field of Panarea Island in the Aeolian archipelago, Italy.

A novel thermophilic strain, designated BP5-C20AT, was isolated from the shallow hydrothermal field of the Panarea island in the Aeolian archipelago close to Sicily, Italy. Cells are motile rods surrounded with a 'toga', Gram-stain-negative and display a straight to curved morphology during the exponential phase. Strain BP5-C20AT is thermophilic (optimum 55 °C), moderately acidophilic (optimum pH 5.6) and halotolerant (optimum 25 g l-1 NaCl). It can use yeast extract, peptone and tryptone. It uses the following carbohydrates: cellobiose, fructose, glucose, maltose, starch, sucrose and xylan. Elemental sulphur is used as an electron acceptor and reduced to hydrogen sulphide. The predominant cellular fatty acid is C16 : 0. Phylogenetic analysis showed that strain BP5-C20AT shared 97.3 % 16S rRNA gene sequence identity with the closest related species Marinitoga lauensis LG1T. The complete genome of strain BP5-C20AT is 2.44 Mb in size with a G+C content of 27.3 mol%. The dDDH and ANI values between the genomes of strains BP5-C20AT and M. lauensis LG1T are 31.0 and 85.70% respectively. Finally, from its physiological, metabolic and genomic characteristics, strain BP5-C20AT (=DSM 112332T=JCM 39183 T) is proposed as representative of a novel species of the genus Marinitoga named Marinitoga aeolica sp. nov. and belonging to the order Petrotogales, in the phylum Thermotogota.

Comparative Genome Analysis of Enterococcus cecorum Reveals Intercontinental Spread of a Lineage of Clinical Poultry Isolates.

Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of E. cecorum cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of E. cecorum belong mainly to one phylogenetic clade. IMPORTANCE Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated E. cecorum isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of E. cecorum strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of E. cecorum-related diseases.

Thermospira aquatica gen. nov., sp. nov., a novel thermophilic spirochete isolated from a Tunisian hot spring, and description of the novel family Thermospiraceae.

A novel thermophilic, anaerobic bacterium, strain F1F22T, was isolated from hot spring water collected in northern Tunisia. The cells were non-motile, Gram-negative and helical with hooked ends, 0.5×10-32 µm in size. Growth of the strain was observed at 45-70 °C (optimum, 55 °C), in 0.0-1.0 % (w/v) NaCl (optimum without NaCl) and at pH 6.5-8.5 (optimum, pH 7.5). Yeast extract was required for growth, and the strain grew on glucose, sucrose and maltose. The major fatty acids were C16:0 (40.2 %), iso-C16: 0 (30.2 %) and C16 :0 DMA (14.5 %). The genome consisted of a circular chromosome (2.5 Mb) containing 2672 predicted protein-encoding genes with a G+C content of 43.15 mol %. Based on a comparative 16S rRNA gene sequence analysis, strain F1F22T formed a deeply branching lineage within the phylum Spirochaetota, class Spirochaetia, order Brevinematales, and had only low sequence similarity to other species of the phylum (lower than 83 %). Genome-based analysis of average nucleotide identity and digital DNA-DNA hybridization of strain F1F22T with Treponema caldarium DSM 7334T, Brevinema andersonii ATCC 43811T and Spirochaeta thermophila DSM 6578T showed values between 63.26 and 63.52 %, and between 20 and 25 %. Hence, we propose strain F1F22T as a representative of a novel family (Thermospiraceae fam. nov.), genus and species of Brevinematales: Thermospira aquatica gen. nov., sp. nov. (type strain F1F22T=JCM 31314T=DSM 101182T).

Methanobacterium alkalithermotolerans sp. nov., a novel alkaliphilic and hydrogen-utilizing methanogen isolated from an alkaline geothermal spring (La Crouen, New Caledonia).

An anaerobic, hydrogenotrophic methane-producing archaeon was isolated from an alkaline thermal spring (42 °C, pH 9.0) in New Caledonia. This methanogen, designated strain CANT, is alkaliphilic, thermotolerant, with Gram-positive staining non-motile cells. Strain CANT grows autotrophically using hydrogen exclusively as an energy source and carbon dioxide as the sole carbon source (without the requirement of yeast extract or other organic compounds). It grows at 20-45 °C (optimum, 45 °C) and pH 7.3-9.7 (optimum, pH 9.0). NaCl is not required for growth (optimum 0 %) but is tolerated up to 1.5 %. It resists novobiocin, streptomycin and vancomycin but is inhibited by ampicillin and penicillin, among other antibiotics. The genome consists of a circular chromosome (2.2 Mb) containing 2126 predicted protein-encoding genes with a G+C content of 36.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CANT is a member of the genus Methanobacterium, most closely related to the alkaliphilic Methanobacterium alcaliphilum WeN4T with 98.5 % 16S rRNA gene sequence identity. The genomes of strain CANT and M. alcaliphilum DSM 3459, sequenced in this study, share 71.6 % average nucleotide identity and 14.0 % digital DNA-DNA hybridization. Therefore, phylogenetic and physiological results indicate that strain CANT represents a novel species, for which the name Methanobacterium alkalithermotolerans sp. nov. is proposed, and strain CANT (=DSM 102889T= JCM 31304T) is assigned as the type strain.

Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity.

DNA methylations play an important role in the biology of bacteria. Often associated with restriction modification (RM) systems, they are important drivers of bacterial evolution interfering in horizontal gene transfer events by providing a defence against foreign DNA invasion or by favouring genetic transfer through production of recombinogenic DNA ends. Little is known regarding the methylome of the Mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, genome-wide detection of DNA methylations was conducted using single molecule real-time (SMRT) and bisulphite sequencing in several strains of Mycoplasma agalactiae, an important ruminant pathogen and a model organism. Combined with whole-genome analysis, this allowed the identification of 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenomic landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.

Complete Genome Sequence of Stenotrophomonas maltophilia 1800, a New Bacterial Strain with Potential for Bioremediation of Oil-Contaminated Environments.

Stenotrophomonas maltophilia strain 1800 was isolated from the effluent of an industrial oil refinery in Algeria. Its genome was sequenced using Illumina MiSeq (2 × 150-bp read pairs) and Oxford Nanopore (long reads) technologies and assembled using Unicycler. It is composed of one chromosome of 4.83 Mb.

Alkalicella caledoniensis gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from 'La Crouen' alkaline thermal spring, New Caledonia.

A novel anaerobic, alkaliphilic, mesophilic, Gram-stain-positive, endospore-forming bacterium was isolated from an alkaline thermal spring (42 °C, pH 9.0) in New Caledonia. This bacterium, designated strain LB2T, grew at 25-50 °C (optimum, 37 °C) and pH 8.2-10.8 (optimum, pH 9.5). Added NaCl was not required for growth (optimum, 0-1 %) but was tolerated up to 7 %. Strain LB2T utilized a limited range of substrates, such as peptone, pyruvate, yeast extract and xylose. End products detected from pyruvate fermentation were acetate and formate. Both ferric citrate and thiosulfate were used as electron acceptors. Elemental sulphur, nitrate, nitrite, fumarate, sulphate, sulfite and DMSO were not used as terminal electron acceptors. The two major cellular fatty acids were iso-C15 : 0 and C16 : 0. The genome consists of a circular chromosome (3.7 Mb) containing 3626 predicted protein-encoding genes with a G+C content of 36.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate is a member of the family Proteinivoraceae, order Clostridiales within the phylum Firmicutes. Strain LB2T was most closely related to the thermophilic Anaerobranca gottschalkii LBS3T (93.2 % 16S rRNA gene sequence identity). Genome-based analysis of average nucleotide identity and digital DNA-DNA hybridization of strain LB2T with A. gottschalkii LBS3T showed respective values of 70.8 and 13.4 %. Based on phylogenetic, genomic, chemotaxonomic and physiological properties, strain LB2T is proposed to represent the first species of a novel genus, for which the name Alkalicella caledoniensis gen. nov., sp. nov. is proposed (type strain LB2T=DSM 100588T=JCM 30958T).

Characterisation of hydrocarbon degradation, biosurfactant production, and biofilm formation in Serratia sp. Tan611: a new strain isolated from industrially contaminated environment in Algeria.

A novel bacterial strain was isolated from industrially contaminated waste water. In the presence of crude oil, this strain was shown to reduce the rate of total petroleum hydrocarbons (TPH) up to 97.10% in 24 h. This bacterium was subsequently identified by 16S rRNA gene sequence analysis and affiliated to the Serratia genus by the RDP classifier. Its genome was sequenced and annotated, and genes coding for catechol 1,2 dioxygenase and naphthalene 1,2-dioxygenase system involved in aromatic hydrocarbon catabolism, and LadA-type monooxygenases involved in alkane degradation, were identified. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of crude oil after biological treatment showed that Serratia sp. Tan611 strain was able to degrade n-alkanes (from C13 to C25). This bacterium was also shown to produce a biosurfactant, the emulsification index (E24) reaching 43.47% and 65.22%, against vegetable and crude oil, respectively. Finally, the formation of a biofilm was increased in the presence of crude oil. These observations make Serratia sp. Tan611 a good candidate for hydrocarbon bioremediation.

Complete Genome Sequences of Four Atrazine-Degrading Bacterial Strains, Pseudomonas sp. Strain ADPe, Arthrobacter sp. Strain TES, Variovorax sp. Strain 38R, and Chelatobacter sp. Strain SR38.

We report here the complete genome sequences of four atrazine-degrading bacteria. Their genomes will serve as references for determining the genetic changes that have occurred during an evolution experiment.

Convergent Rewiring of the Virulence Regulatory Network Promotes Adaptation of Ralstonia solanacearum on Resistant Tomato.

The evolutionary and adaptive potential of a pathogen is a key determinant for successful host colonization and proliferation but remains poorly known for most of the pathogens. Here, we used experimental evolution combined with phenotyping, genomics, and transcriptomics to estimate the adaptive potential of the bacterial plant pathogen Ralstonia solanacearum to overcome the quantitative resistance of the tomato cultivar Hawaii 7996. After serial passaging over 300 generations, we observed pathogen adaptation to within-plant environment of the resistant cultivar but no plant resistance breakdown. Genomic sequence analysis of the adapted clones revealed few genetic alterations, but we provide evidence that all but one were gain of function mutations. Transcriptomic analyses revealed that even if different adaptive events occurred in independently evolved clones, there is convergence toward a global rewiring of the virulence regulatory network as evidenced by largely overlapping gene expression profiles. A subset of four transcription regulators, including HrpB, the activator of the type 3 secretion system regulon and EfpR, a global regulator of virulence and metabolic functions, emerged as key nodes of this regulatory network that are frequently targeted to redirect the pathogen's physiology and improve its fitness in adverse conditions. Significant transcriptomic variations were also detected in evolved clones showing no genomic polymorphism, suggesting that epigenetic modifications regulate expression of some of the virulence network components and play a major role in adaptation as well.

Complete Genome Sequence of Microbacterium sp. Strain Nx66, Isolated from Waters Contaminated with Petrochemicals in El Saf-Saf Valley, Algeria.

Microbacterium sp. strain Nx66 was isolated from waters contaminated by petrochemical effluents collected in Algeria. Its genome was sequenced using Illumina MiSeq (2 × 150-bp read pairs) and Oxford Nanopore (long reads) technologies and was assembled using Unicycler. It is composed of one chromosome of 3.42 Mb and one plasmid of 34.22 kb.

Complete Genome Sequence of the Type Strain Pectobacterium punjabense SS95, Isolated from a Potato Plant with Blackleg Symptoms.

Pectobacterium punjabense is a newly described species causing blackleg disease in potato plants. Therefore, by the combination of long (Oxford Nanopore Technologies, MinION) and short (Illumina MiSeq) reads, we sequenced the complete genome of P. punjabense SS95T, which contains a circular chromosome of 4.793 Mb with a GC content of 50.7%.

Diversity of Pectobacteriaceae Species in Potato Growing Regions in Northern Morocco.

Dickeya and Pectobacterium pathogens are causative agents of several diseases that affect many crops worldwide. This work investigated the species diversity of these pathogens in Morocco, where Dickeya pathogens have only been isolated from potato fields recently. To this end, samplings were conducted in three major potato growing areas over a three-year period (2015-2017). Pathogens were characterized by sequence determination of both the gapA gene marker and genomes using Illumina and Oxford Nanopore technologies. We isolated 119 pathogens belonging to P. versatile (19%), P. carotovorum (3%), P. polaris (5%), P. brasiliense (56%) and D. dianthicola (17%). Their taxonomic assignation was confirmed by draft genome analyses of 10 representative strains of the collected species. D. dianthicola were isolated from a unique area where a wide species diversity of pectinolytic pathogens was observed. In tuber rotting assays, D. dianthicola isolates were more aggressive than Pectobacterium isolates. The complete genome sequence of D. dianthicola LAR.16.03.LID was obtained and compared with other D. dianthicola genomes from public databases. Overall, this study highlighted the ecological context from which some Dickeya and Pectobacterium species emerged in Morocco, and reported the first complete genome of a D. dianthicola strain isolated in Morocco that will be suitable for further epidemiological studies.

Hydrostatic Pressure Helps to Cultivate an Original Anaerobic Bacterium From the Atlantis Massif Subseafloor (IODP Expedition 357): Petrocella atlantisensis gen. nov. sp. nov.

Rock-hosted subseafloor habitats are very challenging for life, and current knowledge about microorganisms inhabiting such lithic environments is still limited. This study explored the cultivable microbial diversity in anaerobic enrichment cultures from cores recovered during the International Ocean Discovery Program (IODP) Expedition 357 from the Atlantis Massif (Mid-Atlantic Ridge, 30°N). 16S rRNA gene survey of enrichment cultures grown at 10-25°C and pH 8.5 showed that Firmicutes and Proteobacteria were generally dominant. However, cultivable microbial diversity significantly differed depending on incubation at atmospheric pressure (0.1 MPa), or hydrostatic pressures (HP) mimicking the in situ pressure conditions (8.2 or 14.0 MPa). An original, strictly anaerobic bacterium designated 70B-AT was isolated from core M0070C-3R1 (1150 meter below sea level; 3.5 m below seafloor) only from cultures performed at 14.0 MPa. This strain named Petrocella atlantisensis is a novel species of a new genus within the newly described family Vallitaleaceae (order Clostridiales, phylum Firmicutes). It is a mesophilic, moderately halotolerant and piezophilic chemoorganotroph, able to grow by fermentation of carbohydrates and proteinaceous compounds. Its 3.5 Mb genome contains numerous genes for ABC transporters of sugars and amino acids, and pathways for fermentation of mono- and di-saccharides and amino acids were identified. Genes encoding multimeric [FeFe] hydrogenases and a Rnf complex form the basis to explain hydrogen and energy production in strain 70B-AT. This study outlines the importance of using hydrostatic pressure in culture experiments for isolation and characterization of autochthonous piezophilic microorganisms from subseafloor rocks.

The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution.

The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives, including numerous extremophile species. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade and a sunflower-specific whole-genome duplication around 29 million years ago. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs.

Comparative Analysis of Ralstonia solanacearum Methylomes.

Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

ABI5 Is a Regulator of Seed Maturation and Longevity in Legumes.

The preservation of our genetic resources and production of high-quality seeds depends on their ability to remain viable and vigorous during storage. In a quantitative trait locus analysis on seed longevity in Medicago truncatula, we identified the bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5). Characterization of Mt-abi5 insertion mutant seeds revealed that both the acquisition of longevity and dormancy were severely impaired. Using transcriptomes of developing Mt-abi5 seeds, we created a gene coexpression network and revealed ABI5 as a regulator of gene modules with functions related to raffinose family oligosaccharide (RFO) metabolism, late embryogenesis abundant (LEA) proteins, and photosynthesis-associated nuclear genes (PhANGs). Lower RFO contents in Mt-abi5 seeds were linked to the regulation of SEED IMBIBITION PROTEIN1 Proteomic analysis confirmed that a set of LEA polypeptides was reduced in mature Mt-abi5 seeds, whereas the absence of repression of PhANG in mature Mt-abi5 seeds was accompanied by chlorophyll and carotenoid retention. This resulted in a stress response in Mt-abi5 seeds, evident from an increase in α-tocopherol and upregulation of genes related to programmed cell death and protein folding. Characterization of abi5 mutants in a second legume species, pea (Pisum sativum), confirmed a role for ABI5 in the regulation of longevity, seed degreening, and RFO accumulation, identifying ABI5 as a prominent regulator of late seed maturation in legumes.

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula.

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.