Genome sequence of the Trifolium rueppellianum -nodulating Rhizobium leguminosarum bv. trifolii strain WSM2012.

Rhizobium leguminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an ineffective root nodule recovered from the roots of the annual clover Trifolium rueppellianum Fresen growing in Ethiopia. WSM2012 has a narrow, specialized host range for N2-fixation. Here we describe the features of R. leguminosarum bv. trifolii strain WSM2012, together with genome sequence information and annotation. The 7,180,565 bp high-quality-draft genome is arranged into 6 scaffolds of 68 contigs, contains 7,080 protein-coding genes and 86 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.


Introduction
Atmospheric dinitrogen (N2) is fixed by specialized soil bacteria (root nodule bacteria or rhizobia) that form non-obligatory symbiotic relationships with legumes. The complex, highly-evolved legume symbioses involve the formation of specialized root structures (nodules) as a consequence of a tightly controlled mutual gene regulated infection process that results in substantial morphological changes in both the legume host root and infecting rhizobia [1]. When housed within root nodules, fully effective N2-fixing bacteroids (the N2-fixing form of rhizobia) can provide 100% of the nitrogen (N) requirements of the legume host by symbiotic N2-fixation. Currently, N2-fixation provides ~40 million tonnes of nitrogen (N) annually to support global food production from ~300 million hectares of crop, forage and pasture legumes in symbioses with rhizobia [2]. The most widely cultivated of the pasture legumes is the legume genus Trifolium (clovers). This genus inhabits three distinct centers of biodiversity with approximately 28% of species in the Americas, 57% in Eurasia and 15% in Sub-Saharan Africa [3]. A smaller subset of about 30 species, almost all of Eurasian origin, are widely grown as annual and perennial species in pasture systems in Mediterranean and temperate regions [3]. Globally important commonly cultivated perennial species include T. repens (white clover), T. pratense (red clover), T. fragiferum (strawberry clover) and T. hybridum (alsike clover). Trifolium rueppellianum is an important annual self-pollinating species grown in the central African continent as a food and forage legume. Clovers usually form N2-fixing symbiosis with the common soil bacterium Rhizobium leguminosarum bv. trifolii, and different combinations of Trifolium spp. hosts and strains of R. leguminosarum bv. trifolii can vary markedly in symbiotic compatibility [4] resulting in a broad range of symbiotic development outcomes ranging from ineffective (non-nitrogen fixing) nodulation to fully effective N2-fixing partnerships [5]. Rhizobium leguminosarum bv. trifolii strain WSM2012 (syn. MAR1468) has a narrow, specialized host range for N2 fixation [6] and was isolated from a nodule recovered from the roots of the annual clover T. rueppellianum growing in Ethiopia in 1963. This strain is a good representative of one of the six centers of biodiversity, Africa, and can be used to investigate the evolution and biodiversity of R. leguminosarum bv. trifolii strains [6]. Here we present a preliminary description of the general features for R. leguminosarum bv. trifolii strain WSM2012 together with its genome sequence and annotation.

Classification and general features
R. leguminosarum bv. trifolii strain WSM2012 is a motile, Gram-negative rod ( Figure 1 Left and Center) in the order Rhizobiales of the class Alphaproteobacteria. It is fast growing, forming colonies within 3-4 days when grown on half Lupin Agar (½LA) [7] at 28°C. Colonies on ½LA are white-opaque, slightly domed, moderately mucoid with smooth margins (Figure 1 Right). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of R. leguminosarum bv. trifolii strain WSM2012 in a 16S rRNA sequence based tree. This strain clusters closest to Rhizobium leguminosarum bv. trifolii T24 and Rhizobium leguminosarum bv. phaseoli RRE6 with 99.9% and 99.8% sequence identity, respectively.

Genome sequencing and annotation information Genome project history
This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [25] and an improved-high-quality-draft genome sequence in IMG. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 2.  Phylum Proteobacteria TAS [10] Class Alphaproteobacteria TAS [11,12] Order Rhizobiales TAS [12,13] Family Rhizobiaceae TAS [14,15] Genus Rhizobium TAS [14,16- Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a g enerally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [22].  [24] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Strains with a g enome sequencing project reg istered in GOLD [25] are in bold print and the GOLD ID is me ntioned after the accession number. Published g enomes are indicated with an asterisk.

Growth conditions and DNA isolation
Rhizobium leguminosarum bv. trifolii strain WSM2012 was grown to mid logarithmic phase in TY rich medium [27] on a gyratory shaker at 28°C. DNA was isolated from 60 ml of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [28].

Genome sequencing and assembly
The genome of Rhizobium leguminosarum bv. trifolii strain WSM2012 was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [29] and 454 technologies [30]. An Illumina GAii shotgun library which produced 63,969,346 reads totaling 4,861.7 Mb, and a paired end 454 library with an average insert size of 8 Kb which produced 428,541 reads totaling 92.6 Mb of 454 data were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI user homepage [28]. The initial draft assembly contained 158 contigs in 6 scaffolds. The 454 paired end data was assembled with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data were assembled with Velvet, version 1.0.13 [31], and the consensus sequences were computationally shredded into 1.5 Kb overlapping fake reads (shreds). The 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library were integrated using parallel phrap, version SPS -4.24 (High Performance Software, LLC). The software Consed [32][33][34] was used in the following finishing process. Illumina data were used to cor-rect potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible misassemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [35], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 167 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 6.7 Mb and the final assembly is based on 49.8 Mb of 454 draft data which provides an average 7.4× coverage of the genome and 2,010 Mb of Illumina draft data which provides an average 300× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [36] as part of the DOE-JGI Annotation pipeline [37], followed by a round of manual curation using the JGI GenePRIMP pipeline [38]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Noncoding genes and miscellaneous features were predicted using tRNAscan-SE [39], RNAMMer [40], Rfam [41], TMHMM [42], and SignalP [43]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [44].

Genome properties
The genome is 7,180,565 nucleotides with 60.89% GC content (Table 3) and comprised of 6 scaffolds ( Figure 3) of 68 contigs. From a total of 7,166 genes, 7,080 were protein encoding and 86 RNA only encoding genes. The majority of genes (72.87%) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4.