Draft Genome Sequence of Bradyrhizobium japonicum Is-34, Which Is Incompatible with Rj4 Genotype Soybeans.

We report here the draft genome sequence of Bradyrhizobium japonicum Is-34, which is incompatible with Rj4 genotype soybeans. A candidate gene involved in this incompatibility was found to be present in this genome.

GENOME ANNOUNCEMENT

Bradyrhizobium bacteria establish a symbiosis with soybeans (Glycine max) by forming root nodules. In the root nodules, Bradyrhizobium bacteria provide fixed nitrogen to the soybean, and in return acquire carbohydrates from the soybean. However, some soybean cultivars restrict nodulation of specific strains such as B. japonicum Is-34 and B. elkanii USDA61 (1). This restriction is due to the Rj4 gene in soybeans, and recent studies have revealed that the Rj4 gene encodes a thaumatin-like protein (1,2). In contrast, a causal gene in the incompatible Bradyrhizobium strains has not been identified.

Genomic DNA of B. japonicum Is-34 was extracted with the ISOPLANT kit (Nippon Gene, Tokyo, Japan), and fragmented to 400 bp using the Covaris S2-A system (Covaris, Woburn, MA, USA). A library for sequencing was prepared by a NEBNext DNA library prep master mix set for Illumina (New England Biolabs, Ipswich, MA, USA), and paired-end sequenced (2 × 300 bp) on a MiSeq sequencer using a MiSeq version 3 reagent kit (Illumina KK, Tokyo, Japan). Raw reads were trimmed and de novo assembled using the CLC Genomics Workbench version 7.5 (Qiagen, Valencia, CA, USA). Parameters for the trimming were as follows: ambiguous limit, 2; quality limit, 0.001; number of 5′ terminal nucleotides, 10; and number of 3′ terminal nucleotides, 40. Parameters for the de novo assembly were as follows: update contigs, yes; bubble size, 600; minimum contig length, 1,000; word size, 51; perform scaffolding, yes; auto-detect paired distances, yes; mismatch cost, 2; insertion cost, 3; deletion cost, 3; length fraction, 0.5; and similarity fraction, 0.8.

The draft genome of B. japonicum Is-34 was assembled into 248 contigs, with an accumulated length of 10,326,597 bp (N50 = 472,902 bp) and an average GC content of 63.1%. The genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP version 2.8), and a total of 9,145 coding sequences (CDSs), 6 rRNAs, and 69 tRNAs were predicted.

As a candidate gene for the incompatibility with Rj4 genotype soybeans, an effector protein of type III secretion system (T3SS) was postulated because disruptants of the T3SS structural genes in the incompatible Bradyrhizobium strain overcame the nodulation restriction of Rj4 genotype soybeans (3). In the present report, a candidate effector gene (MA20_12780) was found in the genome of B. japonicum Is-34. A tts box motif (3) was present in the upstream of this gene, indicating that this gene was a type III-secreted protein. An NCBI conserved domain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed that this gene belongs to the C48 peptidase (the ubiquitin-like protease 1; Ulp1) family. In addition, the candidate gene (MA20_12780)-disrupted mutant of B. japonicum Is-34 overcame the nodulation restriction of Rj4 genotype soybeans. Detailed characterization of this disruptant will be reported in the near future.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. JRPN00000000. The version described in this paper is version JRPN01000000.