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

Oryza coarctata plant, collected from Sundarban delta of West Bengal, India, has been used in the present study to generate draft genome sequences, employing the hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. We report for the first time the draft genome with the coverage of 85.71 % and deposited the raw data in NCBI SRA, with BioProject ID PRJNA396417.


Amendments from Version 1
In this revision, we have addressed all the issues raised by the referee. In additions, we have made few grammatical corrections. We revised the manuscript as a result of reanalysis of the data. We have also improved clarity in the Methods regarding assembly of the genome. Further, as per the suggestions of the referee as well as requirement, we incorporated 6 more references.

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 primary 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., 2015) 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 plant was collected from its native place, Sundarban delta of West Bengal, India (21°.36'N and 88°.15' E) and established at our institute Net house through clonal propagation. 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 of a single plant was extracted using CTAB method (Ganie et al., 2016) for the preparation of various genomic DNA libraries. We used standard Illumina HiSeq 4000 platform (San Diego, CA, USA) to construct 151-bp paired-end libraries and four mate-pair libraries of four different sizes (average of 2, 4, 6 and 8 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). Assembly of the high quality reads was performed using PLATANUS v1. 2.4 (Kajitani et al., 2014) andSSPACE v3.0 (Boetzer et al., 2011) with default parameter. The simple sequence repeats (SSRs) of each scaffold were identified by MISA perl script (Thiel et al., 2003). Gene model prediction was done by ab initio gene predictor AUGUSTUS 3.1 (Stanke & Waak, 2003) and sequence evidence based annotation pipeline, MAKER v2.31.8 (Campbell et al., 2014) with O. sativa ssp. japonica as reference gene model. The protein-coding genes were annotated by using BLAST based approach against a database containing functional plant genes downloaded from NCBI with Blast2GO (version 4.01) (Conesa & Gotz, 2008). Genes with significant hits were assigned with GO (Gene Ontology) terms and EC (Enzyme Commission) numbers. InterProScan search and pathway analyses with KEGG database were also performed by using Blast2GO. Non-coding RNAs, such as miRNA, tRNA, rRNA, snoRNA, snRNA, were identified by adopting Infernal v1.1. 2 (Nawrocki & Eddy, 2013) using Rfam database (release 9.1) (Nawrocki et al., 2015) and snoscan distribution. Transfer RNA was predicted using tRNAscan-SE v 1.23 (Lowe & Eddy, 1997)

Discussion
The O. coarctata genome (2n=4X=48; KKLL; Sanchez et al., 2013) is self-pollinated, (Sarkar et al., 1993) tetraploid plant with a genome size estimated by flow cytometry is found to be approximately 665Mb. The Illumina 4000 GA IIx sequencer pair-end generated 123.78 Gb data. Further four mate-pair libraries together generated 36.54 Gb and Nanopore generated 6.35 Gb sequence data. Hence, we achieved 250.66 X depth of the genome of O. coarctata. The final assembly generated 58362 numbers of scaffolds 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 scaffold length of 569994164 (around 570 Mb) assembled genome, resulting in 85.71% genome coverage. It has been calculated that data contain very small amount of non-ATGC character. Further, we also found that the 19.89% of the assembled genome is repetitive in nature. We also identified approximately 5512 different non-coding RNAs and around 230,968 SSRs. Gene ontology analysis identified several salt responsive genes.

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

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work. The work describe the whole genome sequence of wild species of species that Oryza coarctata exclusively grow under saline water and thus will be an important source of salinity tolerance genes. These genes can later be used to introduce salinity tolerance in commercial cultivars of rice. The authors used Illumina and Oxford nanopore sequencing platforms to generate 372.48X data.

Open Peer Review
The genome sequencing methods seems good enough but authors have discussed very little about the annotation of the genome data. I can understand that there is word limit under Data Note in F1000Research, but still by looking at the discussion, I think analysis portion is weak point in this paper. Authors should provide a comparative note on the genome of and How this Oryza sativa Oryza coarctata. species is tolerating such a high saline conditions, which kind of genes/osmoregulators are involved in this adaptation should be discussed along with comparison to . How many different genes were O. sativa predicted should be mentioned. Authors found approximately 1605 non-coding RNAs? I am not sure, what are trying to tell here, this number should be high as per my opinion.
There are some minor mistakes like; in the affiliation the word "Delhi" is not required. The word, "Primary" 1.
There are some minor mistakes like; in the affiliation the word "Delhi" is not required. The word, "Primary" should be inserted in the first paragraph last line of Introduction. So the correct sentence will be "...in the primary gene pool...".

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound? Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format? Yes No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors report a whole genome sequence dataset for a halophytic wild rice species. These data will be useful for discovery of novel alleles for rice improvement, and for comparative/evolutionary genomics within the Oryza genus.
The report would benefit from more details on the plant accession used as source of DNA for sequencing. It is stated O. coarctata is tetraploid. Was that determined by the authors, or is there a citation to include? Is it known whether O. coarctata is typically self or cross-pollinated, or other information about expected degree of heterozygosity? When grown in greenhouse to generate the plant tissue used for DNA extraction, were the plant(s) established from seeds, or via clonal propagation? Was the genomic DNA used to prepare sequencing libraries from a single plant, or a pool from multiple plants? This information is important to assess expected frequencies of variant types such as alleles or homeologs due to tetraploidy, which are likely collapsed to varying degrees in the subsequent assembly.
There is mention of an assembly and its quality, but not about the method(s) used to produce it or key parameters that guided the assembly. Can the authors provide that information, so that others have a benchmark upon which to compare future assemblies using the datasets?
The sentence "Further, we also found that the repeat contain 19.89% of the genome." Is not completely clear. I believe what the authors intend to say is that approximately 20% of the genome assembly is comprised of repeats. How was this sequence fraction defined as repeats, via tool for matching to known repeat sequences, or a de novo approach? By inference, it is also likely that the approximately 100-kb of the estimated genome size not covered by the assembly is comprised of

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes No competing interests were disclosed. Competing Interests: Referee Expertise: Comparative genomics, brassica, polyploidy, regulatory evolution I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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