First de novo draft genome sequence of Oryza coarctata, the only halophytic species in the genus Oryza

Introduction

Soil salinity is a major abiotic stress of rice cultivation globally (Molla et al., 2015), and rice cultivation areas under soil salinity stress are increasing gradually. Genetic potential for salt tolerance of rice that exists among the natural population has been largely exploited, and alternative useful alleles may further enhance salinity tolerance. Wild species are a potential source of many useful genes and QTLs that may not be present in the gene pool of the domesticated species.

Oryza coarctata, known as Asian wild rice, grows naturally in the coastal region of South-East Asian countries. It flowers and set seeds under as high as 40 E.Ce dS m^-1 saline soil (Bal & Dutt, 1986). It is the only species in the genus Oryza that is halophyte in nature. However, with the exception of one transcriptomic (Garg et al., 2014) and one miRNA (Mondal et al., 2014) experiment, no large scale generation of any other genomic resource is available for this important species, although several pinitol biosynthesis pathway genes have been cloned to study the functional genomics (Sengupta & Majumder, 2009).

Methods

The plants were collected from its native place, Sundarban delta of West Bengal, India (21º.36'N and 88º.15' E) and established to our institute NET house. To determine the genome size, 20 mg of young leaf tissue from Net house grown plants was chopped into small pieces and stained with RNase containing propidium iodide (50 μg/ml) (BD Science, India) as per the protocol of Dolezel et al. (2007). The samples were filtered through a 40-μM mesh sieve (Corning, USA), before analysis in (CFM) BD FACS Calibur (BD Biosciences, San Jose, CA, USA). Pisium sativum leaf was used as standard for calculating the genome size. Further, high-quality genomic DNA from 100 mg young leaf was extracted using CTAB method (Ganie et al., 2016) for the preparation of various genomic DNA libraries. We used Illumina 4000 GA IIx sequencer (San Diego, CA, USA), with 150-bp paired-end libraries, four mate-pair library (with 150-bp paired-end libraries) of four different sizes (average of 2, 4, 6 and 10 kb size). In addition, we also used third generation sequencing (Oxford Nanopore) technology for better assembly. Sequencing was performed on MinION Mk1b (Oxford Nanopore Technologies, Oxford, UK) using SpotON flow cell (R9.4) in a 48h sequencing protocol on MinKNOW 1.4.32. Base calling was performed using Albacore. Base called reads were processed using poRe version 0.24 (Watson et al., 2015) and poretools version 0.6.0 (Loman & Quinlan, 2014). The simple sequence repeats (SSRs) of each scaffold were identified by MISA perl script (Thiel et al., 2003). Gene model prediction was done by AUGUSTUS 3.1 (Stanke & Waak, 2003) and genes were functionally analysed using InterProScan version 5.16.55 (Jones et al., 2014). The InterProScan results were further parsed for additional functional evidence (GO terms and KEGG pathway) using interproscanParser script available at iPlant (Brozynska et al., 2016). Noncoding RNAs, such as miRNA, tRNA, rRNA, snoRNA, snRNA, were identified by adopting Infernal v1.1.2 (Nawrocki & Eddy, 2013) using Rfam (Nawrocki et al., 2015). Transfer RNA was predicted using tRNAscane-SE v 1.23 (Schattner et al., 2005)

Discussion

The genome (KKLL) of O. coarctata is tetraploid (2n=4X=48) with a genome size estimated by flow cytometer is found to be approximately 665Mb. The Illumina 4000 GA IIx sequencer pair-end generated 137 Gb data. Further four mate-pair libraries together generated 104.35 Gb and Nanopore generated 6.35 Gb sequence data. Hence, we achieved 372.48 X depth of the genome of O. coarctata. The final assembly generated 58362 number of contigs with a minimum length of 200 bp to maximum length of 7,855,609 bp and 1,858,627 bp N50 value, making a total contig length of 569994164 (around 570 Mb) assembled genome, resulting 85.71 % genome coverage. It has been calculated that data contain very small amount of non-ATGC character. Further, we also found that the repeat contain 19.89% of the genome. We also identified approximately 1605 different non-coding RNAs and around 105673 SSRs. Gene ontology analysis identified several salt responsive genes.

Data availability

Raw sequence data are available at NCBI SRA under the BioProject ID: PRJNA396417.