Metagenome analysis of the root endophytic microbial community of Indian rice (O. sativa L.).

This study reports the root endophytic microbial community profile in rice (Oryza sativa L.), the largest food crop of Asia, using 16S rRNA gene amplicon sequencing. Metagenome of OS01 and OS04 consisted of 11,17,900 sequences with 300 Mbp size and average 55.6% G + C content. Data of this study are available at NCBI Bioproject (PRJNA360379). The taxonomic analysis of 843 OTU's showed that the sequences belonged to four major phyla revealing dominance of Proteobacteria, Firmicutes, Cyanobacteria and Actinobacteria. Results reveal the dominance of Bacillus as major endophytic genera in rice roots, probably playing a key role in Nitrogen fixation.

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Resource details

As rice yield is enormously affected by large number of pathogenic organisms, nematodes, fungi, insects and virus hence, understanding and exploitation of the root endophytic community for this high demand Asian crop can result in the promotion of crop health. This could be an alternative approach towards eco-friendly potential natural source for biological control in disease management [1]. Advances in high-throughput environmental genomic DNA sequencing or metagenomic sequencing as well as various analytical tools and data resources has enabled us to understand the vast diversity of microorganisms, specially rare and uncultured microorganism and their phylogeny in community analysis. It also gives us insight into the enormous amount of functional gene diversity of a microenvironment [2]. The low cost of this technology and easy generation of draft genomes from complex dataset has made metagenomics study a much popular technique bypassing the need for isolation and lab cultivation of individual microorganism.

In this study, we thoroughly investigated the root endophytic microbial community present in the local cultivar of rice (Oryza sativa L.) at different field condition of West Bengal. The rice plants were selected at 60 days stage and were dug out from some selected wet land local rice fields which produces the bulk of the requirement of the population of Kolkata. Average temp of the area was 86 °F and soil pH ranges from pH 7.2 to pH 8.1.

Among 10 collected samples, two samples OS01 and OS04 were randomly selected for metagenomic sequencing. The total number of reads obtained for sample OS01 was 5,66,012 and that for sample OS04 was 5,51,888 (Fig. 1). In both the samples, the community study revealed an abundance of over 50% for the members of Firmicutes. In sample OS01 the percentage was found to be 58.4% while in sample OS04 it was 97%. At the genus level Bacillus was the most dominant microbial member with abundance of over 50% in both the samples. In sample OS01 gammaproteobacteria had a high abundance of 34.9% but in sample OS04 the percentage was as low as 0.2% as evidenced in the heat map prepared with OTU abundance of 200 or more (Fig. 2).

Fig. 1

Histogram representing Total number of reads for both samples (OS01 and OS04).

Fig. 2

Heatmap representing OTUs of 200 or more hits.

Materials and methods

Samples were collected directly from field in and around Kolkata in triplicates and were stored in sterile plastic bags. Root samples were thoroughly washed in tap water, several times. Surface sterilization of the root tips were then performed with 70% ethanol for 1 min followed by 1.2% (w/v) NaOCl solution for 15 min. Samples were then washed three times with sterile distilled water with shaking (10 min). Root samples were finally dried and stored at − 20 °C. The DNA of each sample was isolated according to the protocol reported by Bonet et al. 2012 [3].The DNA was quantified using QubitdsDNA HS Assay kit (Life Tech). 1 μl of each sample was used for determining concentration using Qubit®2.0 Fluorometer. The amplicon libraries were prepared using Nextera XT Index Kit (Illuminainc.) as per the 16S Metagenomic Sequencing Library preparation protocol (Part # 15044223 Rev. B). Primers for the amplification of the V3-V4 hyper-variable region [V3 Forward Oligo: CCTACGGGNBGCASCAG and V4 Reverse Oligo: GACTACNVGGGTATCTAATCC] of 16S rDNA gene of bacteria and Archaea were used. The amplicons with the Illumina adaptors were amplified by using i5 and i7 primers that add multiplexing index sequences as well as common adapters required for cluster generation (P5 and P7) as per the standard Illumina protocol. The amplicon libraries were purified by 1 × AMpureXP beads and checked on Agilent DNA 1000 chip on Bioanalyzer 2100 and quantified on fluorometer by QubitdsDNA HS Assay kit (Life Technologies). The library size of Sample OS01 and Sample OS04 were 634 bp and 622 bp respectively. The libraries were sequenced using the Illumina sequencing chemistry to generate ~ 150 Mb of data per sample. After obtaining the Qubit concentration for the library and the mean peak size from Bioanalyzer profile, library was loaded onto Illumina Platform at appropriate concentration (10–20 pM) for cluster generation and sequencing. The kit reagents were used in binding of samples to complementary adapter oligos on paired-end flow cell. The adapters were designed to allow selective cleavage of the forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand was then used to perform sequencing from the opposite end of the fragments.

Verification and authentication

Our study revealed that in both the samples, the dominant member of rice root microbiome is Bacillus and this data is well supported by other literatures [4], [5].QIIME analysis indicated that Shannon α-Diversity = 3.10 and no. of observed species = 420 and the Shannon α-Diversity = 2.40 and no. of observed species = 297 for Sample OS01and for sample OS04 respectively. At phylum level, both the samples are enriched with Firmicutes followed by Proteobacteria, Bacilli, whereas Gammaproteobacteria were the most abundant at class level in both the samples. At genus level, Bacillus and Acinetobacter were found to be the most abundant genus enriched in both the root samples. Moreover, our findings were also consistent with the reports of Ji et al. 2014 [6] where three major diazotrophic endophytic communities were identified as Actinobacteria, Gammaproteobacteria and Bacillus. Although, the functional annotations of the endophytic bacterial community of our samples are still pending, however, the dominant groups suggests their probable role of atmospheric N2 fixation, a primary requisite for plant growth particularly in rice.

Nucleotide sequence accession numbers

Metagenome sequence data from this study are available at the NCBI Sequence Read Archive (SRA) and Biosamples under accession numbers: SAMN06209694 and SAMN06209718.

Funding

The work was supported by the grants given to Subhadipa Sengupta (Grant No. PSW-039/14-15 ERO) by the University Grants Commission (UGC), New Delhi, India.

Name of resource	Metagenome of rice root endophytic community
Institution	aBidhannagar CollegebAIIST, PALTA - 743122cThe Biome, Kolkata – 700064
Person who created resource	Subhadipa Senguptaa, Sayak Gangulib and Pankaj K Singhc
Contact person and email	bansubha@gmail.com
Date archived/stock date	31/08/2016
Type of resource	Raw sequence reads of rice root endophytic metagenome
Link to directly related literature that employed/validated this resource	https://www.ncbi.nlm.nih.gov/sra/SRX2524586https://www.ncbi.nlm.nih.gov/sra/SRX2527833
Information in public databases	https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=360379