Role of non-coding RNAs against salinity stress in Oryza species: Strategies and challenges in analyzing miRNAs, tRFs and circRNAs.

Salinity is an imbalanced concentration of mineral salts in the soil or water that causes yield loss in salt-sensitive crops. Rice plant is vulnerable to soil salinity stress at seedling and reproductive stages. Different non-coding RNAs (ncRNAs) post-transcriptionally regulate different sets of genes during different developmental stages under varying salinity tolerance levels. While microRNAs (miRNAs) are well known small endogenous ncRNAs, tRNA-derived RNA fragments (tRFs) are an emerging class of small ncRNAs derived from tRNA genes with a demonstrated regulatory role, like miRNAs, in humans but unexplored in plants. Circular RNA (circRNA), another ncRNA produced by back-splicing events, acts as target mimics by preventing miRNAs from binding with their target mRNAs, thereby reducing the miRNA's action upon its target. Same may hold true between circRNAs and tRFs. Hence, the work done on these ncRNAs was reviewed and no reports were found for circRNAs and tRFs under salinity stress in rice, either at seedling or reproductive stages. Even the reports on miRNAs are restricted to seedling stage only, in spite of severe effects on rice crop production due to salt stress during reproductive stage. Moreover, this review sheds light on strategies to predict and analyze these ncRNAs in an effective manner.

miRPreM and tiRPreM: Improved methodologies for the prediction of miRNAs and tRNA-induced small non-coding RNAs for model and non-model organisms.

In recent years, microRNAs (miRNAs) and tRNA-derived RNA fragments (tRFs) have been reported extensively following different approaches of identification and analysis. Comprehensively analyzing the present approaches to overcome the existing variations, we developed a benchmarking methodology each for the identification of miRNAs and tRFs, termed as miRNA Prediction Methodology (miRPreM) and tRNA-induced small non-coding RNA Prediction Methodology (tiRPreM), respectively. We emphasized the use of respective genome of organism under study for mapping reads, sample data with at least two biological replicates, normalized read count support and novel miRNA prediction by two standard tools with multiple runs. The performance of these methodologies was evaluated by using Oryza coarctata, a wild rice species as a case study for model and non-model organisms. With organism-specific reference genome approach, 98 miRNAs and 60 tRFs were exclusively found. We observed high accuracy (13 out of 15) when tested these genome-specific miRNAs in support of analyzing the data with respective organism. Such a strong impact of miRPreM, we have predicted more than double number of miRNAs (186) as compared with the traditional approaches (79) and with tiRPreM, we have predicted all known classes of tRFs within the same small RNA data. Moreover, the methodologies presented here are in standard form in order to extend its applicability to different organisms rather than restricting to plants. Hence, miRPreM and tiRPreM can fulfill the need of a comprehensive methodology for miRNA prediction and tRF identification, respectively, for model and non-model organisms.

Draft genome sequence of first monocot-halophytic species Oryza coarctata reveals stress-specific genes.

Oryza coarctata (KKLL; 2n = 4x = 48, 665 Mb) also known as Porteresia coarctata is an extreme halophyte species of genus Oryza. Using Illumina and Nanopore reads, we achieved the assembled genome size of 569.9 Mb, accounting 85.69% of the estimated genome size with N50 of 1.85 Mb and 19.89% repetitive region. We also found 230,968 simple sequence repeats (SSRs) and 5,512 non-coding RNAs (ncRNAs). The functional annotation of predicted 33,627 protein-coding genes and 4,916 transcription factors revealed that high salinity adaptation of this species is due to the exclusive or excessive presence of stress-specific genes as compared to rice. We have identified 8 homologs to salt-tolerant SOS1 genes, one of the three main components of salt overly sensitive (SOS) signal pathway. On the other hand, the phylogenetic analysis of the assembled chloroplast (134.75 kb) and mitochondrial genome (491.06 kb) favours the conservative nature of these organelle genomes within Oryza taxon.

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.