Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species

Complete and draft genome sequences of 12 Rathayibacter strains were generated using Oxford Nanopore and Illumina technologies. The genome sizes of these strains are 3.21 to 4.61 Mb, with high G+C content (67.2% to 72.7%) genomic DNA. Genomic data will provide useful baseline information for natural taxonomy and comparative genomics of members of the genus Rathayibacter.

For DNA extraction, biomass was grown in liquid peptone-yeast medium (13) inoculated with cells from a single colony, followed by cultivation at 28°C for 18 to 20 h on a rotary shaker. Genomic DNA was extracted using a QIAamp DNA minikit (Qiagen, Germany).
DNA libraries were prepared for long-read sequencing using the Nanopore rapid barcoding genomic DNA (gDNA) sequencing kit (catalog number SQK-RBK004; Oxford Nanopore Technologies) according to the manufacturer's protocol and were sequenced in-house using a MinION device. DNA libraries of strains VKM Ac-2754, VKM Ac-2759, VKM Ac-2760, VKM Ac-2762, VKM Ac-2804, and VKM Ac-2805 were prepared for short-read sequencing using the Nextera DNA flex library prep kit (Illumina) and Nextera DNA CD indexes (Illumina) according to the manufacturer's instructions. DNA libraries of strains VKM Ac-1601 T , VKM Ac-1602 T , VKM Ac-2761, VKM Ac-2801, VKM Ac-2802, and VKM Ac-2803 were prepared using NEBNext Ultra II FS DNA library prep kit for Illumina (New England BioLabs) following the protocol for use with inputs of Ն100 ng with the following modifications: TruSeq DNA CD indexes (Illumina) were used in place of NEBNext adaptors to eliminate the need for PCR steps. The USER enzyme addition was skipped for this reason, and the volume was adjusted with water to reach the necessary sample volume for size selection steps. No PCR amplification was performed on these libraries. Pooled DNA libraries were sequenced by Novogene Co., Ltd.
Default parameters were used for all software unless otherwise specified. Nanopore basecalling was performed by Guppy basecalling software 2.3.5, available from the Oxford Nanopore Technology (ONT) community website (with the following parameters: --flowcell, FLO-MIN106; --kit, SQK-RBK004), and demultiplexed by Deepbinner 0.2.0 (14) (with parameter --rapid). Adapter sequences from long reads were removed using Porechop 0.2.4 (https://github.com/rrwick/Porechop) with parameter --discard_middle. Adapter sequences and low-quality regions in short reads were cut using Trimmomatic 0.39 (15) with the following parameters: ILLUMINACLIP:adapters.fa:2:30:10; SLIDING-WINDOW:4:15; MINLEN:30, where adapters.fa is NexteraPE-PE.fa or TruSeq3-PE-2.fa depending on the kit used to prepare the library. Hybrid assembly was performed by Unicycler 0.4.8 (16). There was insufficient DNA quantity of VKM Ac-1601 T to make a library for Nanopore sequencing; thus, the genome assembly of this organism was performed on short reads only. The quality of assemblies was assessed with QUAST 5.0.2 (17). Assemblies were annotated with the NCBI PGAP (18) and the RAST Web server (19,20). A phylogenomic tree was inferred by the balanced minimum evolution method using JolyTree (21). Statistical information for the complete and draft genome sequences is given in Table 1. It is worth noting that plasmids were identified in the genome assemblies of Rathayibacter strains for the first time.
The tree (Fig. 1) shows that 9 of the 10 novel strains cluster separately from the Rathayibacter species with validly published names. The calculated average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values (well below the borderlines for species differentiation [22]; not shown) indicated the presence of seven putative new species among the strains studied. Further comparative phenotypic study and genome-wide analyses of these strains and other members of the genus Rathayibacter will result in valid descriptions of the revealed new species and facilitate insight into the molecular mechanisms involved in interactions between plants and bacteria.
Data availability. These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in Table 1