ABSTRACT
We report draft genome sequences of 12 Agrobacterium strains isolated from coastal plants in Taiwan. Assemblies range from 4.95 to 5.42 Mb with >97.5% completeness. Genomic analyses reveal species identities and secretion system genes, providing resources for future research.
KEYWORDS: genome, Agrobacterium, endophyte, Taiwan
ANNOUNCEMENT
The genus Agrobacterium comprises diverse species associated with soil and plants. Some strains harbor oncogenic plasmids and can be used for Agrobacterium-mediated transformation (AMT) (1). A major limitation of AMT is that many plants are recalcitrant to common laboratory strains. Early work demonstrated that wild-type Agrobacterium strains differ substantially in host range, suggesting that strain diversity could broaden AMT application to different crops (2). Guided by this rationale, we screened draft genome assemblies from our collection of endophytic bacteria isolated from coastal plants in Taiwan, identified 12 strains as members of the genus Agrobacterium, and report their genome sequences in this work (Table 1).
TABLE 1.
Metadata and genome assembly statistics of the strains included in this studya
| Strain | Species | Similarity to type strain (AF/ANI %) | Collection date | Location | GPS coordinates | Host | SRA accession | Assembly accession | Number of reads |
Coverage (fold) | Number of contigs | Contig N50 (bp) | Assembly size (bp) | GC content (%) | Number of annotated genes | Completeness (%) | Contamination (%) | T6SS genes |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NCHU4840 | Agrobacterium cavarae | 85.5/96.2 | 02-03-2022 | Taiwan: Miaoli County, Tongxiao Township | 24.495968 N 120.671137 E | Bidens pilosa | SRR34682073 | GCA_051590745.1 | 5,511,444 | 159.6 | 27 | 422,750 | 5,048,636 | 58.2 | 4,644 | 97.72 | 0.68 | Complete |
| NCHU4860 | Agrobacterium cavarae | 84.7/96.2 | 02-03-2022 | Taiwan: Miaoli County, Tongxiao Township | 24.495968 N 120.671137 E | Urena lobata | SRR34681896 | GCA_052261035.1 | 6,259,710 | 183.5 | 15 | 884,046 | 4,951,969 | 58.2 | 4,579 | 97.76 | 0.95 | Complete |
| NCHU4975 | Agrobacterium cavarae | 85.7/96.2 | 02-03-2022 | Taiwan: Miaoli County, Tongxiao Township | 24.495968 N 120.671137 E | Ipomoea pes-caprae subsp. brasiliensis | SRR34682131 | GCA_052261055.1 | 5,653,282 | 162.8 | 29 | 403,161 | 5,048,170 | 58.2 | 4,647 | 97.72 | 0.68 | Complete |
| NCHU4981 | Agrobacterium pusense | 89.3/98.2 | 02-03-2022 | Taiwan: Miaoli County, Tongxiao Township | 24.495968 N 120.671137 E | Ipomoea pes-caprae subsp. brasiliensis | SRR34692460 | GCA_052261075.1 | 6,124,078 | 166.2 | 37 | 654,217 | 5,355,332 | 59.1 | 5,079 | 99.60 | 0.77 | Absent |
| NCHU5031 | Agrobacterium pusense | 88.6/98.2 | 09-05-2022 | Taiwan: Miaoli County, Zhunan Township | 24.675569 N 120.830987 E | Oenothera laciniata | SRR34685741 | GCA_052261095.1 | 7,600,106 | 206.6 | 23 | 1,004,322 | 5,409,650 | 59.1 | 5,115 | 99.33 | 0.95 | Absent |
| NCHU5032 | Agrobacterium pusense | 87.7/97.6 | 09-05-2022 | Taiwan: Miaoli County, Zhunan Township | 24.675569 N 120.830987 E | Oenothera laciniata | SRR34687319 | GCA_052261115.1 | 6,567,210 | 178.4 | 30 | 440,007 | 5,416,145 | 59.3 | 5,172 | 99.55 | 0.62 | Partial |
| NCHU5334 | Agrobacterium salinitolerans | 89.2/99.2 | 04-06-2022 | Taiwan: Tainan City, Qigu District | 23.157924 N 120.077311 E | Bidens pilosa | SRR34682062 | GCA_052261135.1 | 6,247,712 | 175.8 | 29 | 443,794 | 5,207,595 | 59.3 | 4,955 | 99.71 | 0.29 | Complete |
| NCHU5640 | Agrobacterium cavarae | 85.7/96.2 | 22-08-2022 | Taiwan: Yilan County, Toucheng Township | 24.913217 N 121.877840 E | Murdannia keisak | SRR34682063 | GCA_052261155.1 | 5,375,518 | 155.8 | 43 | 405,051 | 5,006,747 | 58.2 | 4,677 | 97.52 | 1.30 | Complete |
| NCHU5641 | Agrobacterium cavarae | 85.4/96.2 | 22-08-2022 | Taiwan: Yilan County, Toucheng Township | 24.913217 N 121.877840 E | Murdannia keisak | SRR34685756 | GCA_052261175.1 | 6,621,420 | 193.0 | 43 | 405,051 | 5,006,273 | 58.2 | 4,680 | 97.52 | 1.30 | Complete |
| NCHU5644 | Agrobacterium cavarae | 85.6/96.2 | 22-08-2022 | Taiwan: Yilan County, Toucheng Township | 24.913217 N 121.877840 E | Murdannia keisak | SRR34692412 | GCA_052261195.1 | 5,096,434 | 147.9 | 41 | 421,673 | 5,006,542 | 58.2 | 4,676 | 97.79 | 1.30 | Complete |
| NCHU5645 | Agrobacterium cavarae | 85.2/96.2 | 22-08-2022 | Taiwan: Yilan County, Toucheng Township | 24.913217 N 121.877840 E | Ipomoea pes-caprae subsp. brasiliensis | SRR34685760 | GCA_052261215.1 | 5,920,386 | 170.7 | 44 | 405,051 | 5,006,869 | 58.2 | 4,671 | 97.52 | 1.30 | Complete |
| NCHU5738 | Agrobacterium deltaense | 92.0/97.9 | 14-10-2022 | Taiwan: Changhua County, Fangyuan Township | 23.928038 N 120.314438 E | Pluchea indica | SRR34685761 | GCA_052261235.1 | 5,704,246 | 161.2 | 41 | 350,740 | 5,158,671 | 59.9 | 4,914 | 99.47 | 1.70 | Complete |
AF, alignment fraction; ANI, average nucleotide identity; T6SS, type VI secretion system.
Unless otherwise noted, methods followed the cited references, kits were used according to the manufacturers’ instructions, and bioinformatics tools were used with default settings. For strain isolation, root samples were surface-sterilized, homogenized in distilled water, serially diluted, spread onto Luria Broth (LB) agar, and incubated at 30°C for 48 h to obtain single colonies, as described (3). Colonies were re-streaked at least three times to ensure purity. For DNA extraction, a single colony was cultured in 1 mL LB broth at 30°C for 24 h, then processed sequentially using the Wizard Genomic DNA Purification Kit (Promega) and DNeasy Blood and Tissue Kit (Qiagen).
For shotgun sequencing, libraries were prepared using the SATLite DNA Prep Kit with the SATLite 2.0 Library Prep Automation Device (GeneReach Biotechnology) and sequenced on a NovaSeq X Plus using the 10B Reagent Kit (Illumina) to obtain paired-end 150 bp reads. Raw reads were trimmed using fastp v1.0.1 (4) and assembled using SPAdes v3.15.0 (5) with the “--isolate” option (k = 21, 33, 55, and 77). Contigs >500 bp were retained. Trimmed reads were mapped to contigs using BWA v0.7.17-r1188 (6), and coverage was assessed using SAMtools v1.9 (7). For species identification, assemblies were compared against NCBI RefSeq genomes (8) with FastANI v1.33 (9). Assemblies were submitted to GenBank and annotated using the NCBI Prokaryotic Genome Annotation Pipeline v6.10 (10). Completeness and contamination were estimated using CheckM v1.2.4 (11). MacSyFinder v2.1.4 (12) was used to identify genes encoding type IV and type VI secretion systems, which are relevant to Agrobacterium virulence and ecology, respectively (13). To identify contigs corresponding to oncogenic plasmids, coding sequences from reference plasmids (14, 15) were queried using BLASTP v2.11.0 (16) against annotated coding sequences.
Results are summarized in Table 1. All strains showed >84% alignment fraction and >96% average nucleotide identity to their respective type strains, allowing confident species assignment (17). Contig counts ranged from 15 to 44, N50 from 351 to 1,004 kb, assembly sizes from 4.95 to 5.42 Mb, GC content from 58.2 to 59.9%, and annotated genes from 4,579 to 5,172. All assemblies showed >97.5% completeness and <2% contamination. No oncogenic plasmid contigs nor type IV secretion system genes were detected. The feasibility of introducing oncogenic plasmids into these strains by conjugation remains to be tested. These genome assemblies provide useful references for future studies of these strains and comparative genomics of Agrobacterium.
ACKNOWLEDGMENTS
We thank members of GeneReach Biotechnology Corp., HHH lab, and CHK lab for discussions and technical assistance.
This work was supported by Academia Sinica and the National Science and Technology Council of Taiwan (107-2321-B-005-009, 108-2321-B-005-004, 109-2321-B-005-025, 110-2321-B-005-008, 110-2313-B-005-008, 111-2313-B-005-009-MY3, 114-2313-B-005-004). Additional support was provided by the Innovation and Development Center of Sustainable Agriculture and the Advanced Plant and Food Crop Biotechnology Center through the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education of Taiwan. Illumina library preparation and sequencing were performed by GeneReach Biotechnology Corp (Taichung, Taiwan). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Contributor Information
Hau-Hsuan Hwang, Email: hauhsuan@dragon.nchu.edu.tw.
Chih-Horng Kuo, Email: chk@gate.sinica.edu.tw.
Silvia T. Cardona, University of Manitoba, Winnipeg, Canada
DATA AVAILABILITY
The raw sequencing reads and draft genome assemblies have been deposited in NCBI GenBank and are publicly available; accession numbers are listed in Table 1.
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Associated Data
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Data Availability Statement
The raw sequencing reads and draft genome assemblies have been deposited in NCBI GenBank and are publicly available; accession numbers are listed in Table 1.