Bacterial and fungal microbiome dataset from holobionts of CP4-EPSPS–containing hybrids and wild-type Brassica juncea in South Korea

Abstract

This dataset provides comprehensive profiles of bacterial and fungal communities associated with the holobionts of CP4-EPSPS–containing hybrids and wild-type B. juncea in a natural roadside habitat. The hybrids were genetically consistent with B. napus × B. juncea origin and possible backcrossing with B. juncea, though the site and mechanism of hybridization are unclear. A total of 120 holobiont samples, including flowers, leaves, dead leaves, roots, and surrounding soil, were collected from twelve wild-type and twelve hybrid individuals (60 samples per group), in a natural roadside environment. Bacterial 16S rRNA and fungal ITS genes obtained through the Illumina Miseq approach were employed to characterize the holobiont-associated microbiomes. Metadata and raw sequences collected in this study are available from the National Center for Biotechnology Information (BioProject ID: PRJNA1237916 and 1237917). Amplicon Sequence Variants (ASVs) of bacteria and fungi were processed using the DADA2 pipeline. After quality filtering, trimming, and eliminating the chimeric sequences, 15,131 bacterial and 5,353 fungal ASVs were identified in the holobiont. Proteobacteria in bacteria, and Ascomycota in fungi were the predominant groups in the holobiont. Given the reported unintended releases and hybridization of transgenic B. napus in South Korea, this dataset provides a comprehensive baseline of the microbial communities associated with wild-type and hybrids, offering novel insights into their holobiont structures.

Specifications Table

Subject	Biology
Specific subject area	16S rRNA and ITS metagenomics of CP4-EPSPS–containing backcrossed hybrids and wild-type B. juncea holobionts and bulk soil
Type of data	Amplicon sequencing data of 16S rRNA and Internal Transcribed Spacer (ITS) region Raw, filtered, and analyzed
Data collection	Collected and used for DNA library preparation based on amplicon sequencing of the 16s rRNA and ITS regions. DNA sequences: Illumina Miseq platform Data processing: DADA2 v. 1.16.0. Data analysis: R v. 4.4.2.
Data source location	Plant: Brassica juncea, Brassica juncea hybrid, Latitude and longitude: 36.95°N, 126.61°E Sampling site: Gunsan, Jeonbuk State Country: Republic of Korea Sampling date: April 2, 2024 Institution: National Institute of Ecology (Republic of Korea)
Data accessibility	Repository name: NCBI SRA Biosample Data identification number: PRJNA1237916 and PRJNA1237917 Direct URL to data: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1237916 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1237917 Accessions: SAMN47444638-SAMN47444657
Related research article	none

1. Value of the Data

• •These bacterial and fungal microbiome datasets can be used to analyze microbial dynamics within the holobionts of CP4-EPSPS–containing backcrossed hybrids and wild-type Brassica juncea in natural ecosystems.
• •This dataset provides a valuable basis for comparing microbial community structures between hybrids and wild-type in a shared natural habitat.
• •Environmental and agricultural scientists may use these datasets to evaluate the potential ecological risks associated with the unintended release of genetically modified crops.

2. Background

The cultivation and production of genetically modified (GM) crops have been steadily increasing worldwide [1]. The unintentional release of GM crops into natural ecosystems poses increasing ecological risks, as they may hybridize with wild relatives and lead to unintended gene flow and genetic contamination [[2], [3]–4]. In South Korea, CP4-5‐enolpyruvylshikimate‐3‐phosphate synthase (CP4-EPSPS)–containing GM canola (Brassica napus) has been unintentionally released into natural ecosystems [5]. Since 2019, plants morphologically resembling wild B. juncea but containing the CP4-EPSPS transgene have been observed in the Gunsan area, a coastal region in western South Korea where such releases have been previously reported [5,6]. However, whether these hybrids originated from local natural hybridization events in the field or were introduced from abroad after hybridization occurred elsewhere remains unclear. Therefore, it is crucial to understand the microbial community associated with these hybrids and wild-type plants to assess the potential impacts on natural ecosystems. We previously applied a similar sampling design in our study of feral (non-GM) B. napus holobionts [7]. In the present study, we adopted the same framework to investigate both wild-type and GM-hybrid B. juncea collected from natural habitats in South Korea. In this study, we investigated bacterial and fungal communities associated with the flower, leaf, dead leaf, root, and surrounding soil of twelve individuals from each group. Our goal was to generate and share a detailed dataset on the plant-associated microbiome, which can serve as a valuable resource for understanding potential shifts related to gene flow from GM crops into wild populations.

3. Data Description

Flower, leaf, dead leaf, root, and surrounding soil samples were collected from Gunsan (36.95°N, 126.61°E), South Korea in April 2024. Both bacterial and fungal microbiomes were analyzed using the Illumina Miseq platform. After filtering, trimming, and removing chimeric sequences, 15,131 bacterial ASVs (2,339,686 reads) and 5,353 fungal ASVs (5,397,162 reads) were identified and used for further analysis. Rarefaction curves indicated that the sequencing depth was sufficient for robust community analysis (Fig. 1). In bacteria (Fig. 2a), Pseudomonadales (13.78 ± 24.24%) was the most abundant order, followed by Burkholderiales (12.68 ± 8.45%). The relative abundance of Pseudomonadales was approximately two-fold higher in wild-type samples than in hybrid samples in the flower samples. In contrast, Burkholderiales showed approximately a nine-fold higher relative abundance in hybrid flower samples than in wild-type flower samples. For fungal samples (Fig. 2b), Pleosporales and Olpidiales were the most abundant phyla (26.64 ± 21.40% and 20.40 ± 30.77%, respectively). In the root samples, Olpidiales was the most abundant at 73.98 ± 20.52%, with no significant difference in its dominance between wild-type and hybrid plants.

Fig. 1.

Rarefaction curves. (A) bacterial samples and (B) fungal samples.

Fig. 2.

The relative abundance of microbial communities in holobiont at the order level. (A) bacterial and (B) fungal orders.

4. Experimental Design, Materials and Methods

4.1. Study site and sampling design

On April 2, 2024, twelve wild-type B. juncea and twelve hybrid plants morphologically resembling B. juncea were immediately confirmed on-site at a roadside using a CP4-EPSPS strip test (EnviroLogics). These hybrids containing the CP4-EPSPS transgene were located within approximately 300 meters of the previously detected GM canola individuals. Samples were collected from plants at similar growth stages and heights to ensure homogeneity, and all were obtained within 10 m. A total of 120 samples were obtained from 12 individual hybrid plants and 12 individual wild-type B. juncea plants (24 plants in total), with samples collected from five compartments of each plant: flowers, leaves, dead leaves, roots, and surrounding soil. To reduce cross-contamination among samples, all tools were sterilized with 80% ethanol prior to the collection of samples. Every sample was immediately stored at -80°C for further analysis.

4.2. DNA extraction and sequencing

DNA was isolated using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, CA, USA) following the manufacturer's instructions and measured using a Nanodrop 2000 UV spectrophotometer (Thermo Scientific, DE, USA). Bacterial 16S rRNA gene was amplified using a universal primer set of 341F (5′-CCTACGGGNGGCWGCAG-3’) and 805R (5′-GACTACHVGGGTATCTAATCC-3’), targeting the V3-V4 region of bacterial 16S rRNA gene [8]. The fungal ITS1 region was amplified using the fungal universal primer sets ITS1F_KYO1 (5′- CTHGGTCATTTAGAGGAASTAA-3’) and ITS2_KYO2 (5′- TTYRCTRCGTTCTTCATC-3’) [9]. Dual-PCR amplification, clean-up, and measurement were performed in accordance with the Illumina 16S metagenomic sequencing library protocol outlined previously [10]. The final products were prepared for paired-end read sequencing reactions and sequenced using MiSeq (2 × 300 bp reads) obtained from Macrogen Corporation (Seoul, South Korea).

4.3. Bioinformatic analysis

To explore the ASVs profiles, the ASVs of bacterial 16S rRNA and fungal ITS genes were analyzed using DADA2 (version 1.16.0) following the pipeline tutorial (1.16) (https://benjjneb.github.io/dada2/tutorial.html) and DADA2 ITS Pipeline Workflow (1.8) (https://benjjneb.github.io/dada2/ITS_workflow.html), respectively [11]. Singletons, doubletons, and tripletons were excluded from the 16S rRNA and fungal ITS gene datasets prior to analysis. The most recent Silva database (release 138.1) [12] and UNITE general FASTA release for fungi (version 10.0) [13] were utilized to identify bacterial and fungal sequences, respectively. Any reads recognized as chloroplast, mitochondrial, or archaeal sequences were eliminated from the 16S rRNA database.

Limitations

None

Ethics Statement

The authors have read and followed the ethical requirements for publication in Data in Brief and confirm that the current work does not involve human subjects, animals experiments or any data collected from social media platforms.

Credit author statement

Jihoon Kim: Investigation, Methodology, Visualization, Writing- original draft draft.

Kyong-Hee Nam: Investigation, Methodology.

Jun-Woo Lee: Investigation, Methodology.

Seong-Jun Chun: Conceptualization, Investigation, Writing- review & editing, Supervision.

Data Availability

• NCBI SRAFungal holobiont of Brassica juncea in South Korea (Original data)

• NCBI SRABacterial holobiont of Brassica juncea in South Korea (Original data)