Comprehensive survey and evolutionary analysis of genome-wide miRNA genes from ten diploid Oryza species.

BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs that play versatile roles in post-transcriptional gene regulation. Although much is known about their biogenesis, and gene regulation very little is known about their evolutionary relation among the closely related species. RESULT: All the orthologous miRNA genes of Oryza sativa (japonica) from 10 different Oryza species were identified, and the evolutionary changes among these genes were analysed. Significant differences in the expansion of miRNA gene families were observed across the Oryza species. Analysis of the nucleotide substitution rates indicated that the mature sequences show the least substitution rates among the different regions of miRNA genes, and also show a very much less substitution rates as compared to that of all protein-coding genes across the Oryza species. Evolution of miRNA genes was also found to be contributed by transposons. A non-neutral selection was observed at 80 different miRNA loci across Oryza species which were estimated to have lost ~87% of the sequence diversity during the domestication. The phylogenetic analysis revealed that O. longistaminata diverged first among the AA-genomes, whereas O. brachyantha and O. punctata appeared as the eminent out-groups. The miR1861 family organised into nine distinct compact clusters in the studied Oryza species except O. brachyantha. Further, the expression analysis showed that 11 salt-responsive miRNAs were differentially regulated between O. coarctata and O. glaberrima. CONCLUSION: Our study provides the evolutionary dynamics in the miRNA genes of 10 different Oryza species which will support more investigations about the structural and functional organization of miRNA genes of Oryza species.

Identification of Novel and Conserved miRNAs from Extreme Halophyte, Oryza coarctata, a Wild Relative of Rice.

Oryza coarctata, a halophyte and wild relative of rice, is grown normally in saline water. MicroRNAs (miRNAs) are non-coding RNAs that play pivotal roles in every domain of life including stress response. There are very few reports on the discovery of salt-responsive miRNAs from halophytes. In this study, two small RNA libraries, one each from the control and salt-treated (450 mM NaCl for 24 h) leaves of O. coarctata were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of O. coarctata which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through in silico analysis. In total, 36 known and 7 novel miRNAs were up-regulated whereas, 12 known and 7 novel miRNAs were down-regulated under salinity stress. Further, 233 and 154 target genes were predicted for 48 known and 14 novel differentially regulated miRNAs respectively. These targets with the help of gene ontology analysis were found to be involved in several important biological processes that could be involved in salinity tolerance. Relative expression trends of majority of the miRNAs as detected by real time-PCR as well as predicted by Illumina sequencing were found to be coherent. Additionally, expression of most of the target genes was negatively correlated with their corresponding miRNAs. Thus, the present study provides an account of miRNA-target networking that is involved in salinity adaption of O. coarctata.

Identification and analysis of novel salt responsive candidate gene based SSRs (cgSSRs) from rice (Oryza sativa L.).

BACKGROUND: Majority of the Asian people depend on rice for nutritional energy. Rice cultivation and yield are severely affected by soil salinity stress worldwide. Marker assisted breeding is a rapid and efficient way to develop improved variety for salinity stress tolerance. Genomic microsatellite markers are an elite group of markers, but there is possible uncertainty of linkage with the important genes. In contrast, there are better possibilities of linkage detection with important genes if SSRs are developed from candidate genes. To the best of our knowledge, there is no such report on SSR markers development from candidate gene sequences in rice. So the present study was aimed to identify and analyse SSRs from salt responsive candidate genes of rice. RESULTS: In the present study, based on the comprehensive literature survey, we selected 220 different salt responsive genes of rice. Out of them, 106 genes were found to contain 180 microsatellite loci with, tri-nucleotide motifs (56%) being most abundant, followed by di-(41%) and tetra nucleotide (2.8%) motifs. Maximum loci were found in the coding sequences (37.2%), followed by in 5'UTR (26%), intron (21.6%) and 3'UTR (15%). For validation, 19 primer sets were evaluated to detect polymorphism in diversity analysis among the two panels consisting of 17 salt tolerant and 17 susceptible rice genotypes. Except one, all primer sets exhibited polymorphic nature with an average of 21.8 alleles/primer and with a mean PIC value of 0.28. Calculated genetic similarity among genotypes was ranged from 19%-89%. The generated dendrogram showed 3 clusters of which one contained entire 17 susceptible genotypes and another two clusters contained all tolerant genotypes. CONCLUSION: The present study represents the potential of salt responsive candidate gene based SSR (cgSSR) markers to be utilized as novel and remarkable candidate for diversity analysis among rice genotypes differing in salinity response.