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.

Introduction

Soil salinity is a major abiotic stress of rice cultivation globally ( Molla ), 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 ) and one miRNA ( Mondal ) 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 . 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 ) 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 ) and poretools version 0.6.0 ( Loman & Quinlan, 2014). Assembly of the high quality reads was performed using PLATANUS v1.2.4 ( Kajitani ) and SSPACE v3.0 ( Boetzer ) with default parameter. The simple sequence repeats (SSRs) of each scaffold were identified by MISA perl script ( Thiel ). 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 ) 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 ) 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 ) is self-pollinated, ( Sarkar ) 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.

The authors have included the requested details about the source of plant materials, estimate of genome size, and genome assembly methods. Although still not sure how they arrived at the estimate of 19.86% repeat sequences, this is a minor point.

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 work describe the whole genome sequence of wild species of Oryza coarctata species that 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.

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 Oryza sativa and Oryza coarctata. How this 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 O. sativa. How many different genes were 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” should be  inserted in the first paragraph last line of Introduction. So the correct sentence will be "...in the primary gene pool...".

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 high-copy repeats, leading to an estimate of about 30% total repeat content.

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, however I have significant reservations, as outlined above.

The authors report a draft genome sequence of a halophyte Oryza species collected from Sunderbans, and provides a glimpse into the adaptive strategies employed by Oryza against salinity stress. Undoubtedly, it will be an useful resource for future functional characterization, comparative genomic studies, and developing salinity tolerance in rice. I understand that the present format is only for reporting, and look forward to reading the full manuscript with all the analysis. There are small language edits that that authors need to incorporate.

Comments:

Please add a reference or sufficient information for general readers as to how genome types of rice (for instance, KKLL in case of O. coarctata) was assigned.

Language corrections:

Change “have been used” to “has been used”

Change “We report for the first time that more than 85.71 % of the genome coverage and the data have been deposited in NCBI SRA, with BioProject ID PRJNA396417” to “deposited in NCBI SRA, with BioProject ID PRJNA396417”

Change “and established to our institute NET” to “and established at our institute NET”

Change “resulting 85.71 % genome coverage” to “resulting in 85.71 % genome coverage”

Change “we also found that the repeat contain 19.89% of the genome.” to “we also found that the 19.89% of the genome is repetitive in nature”.

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.