Simple sequence repeat identification for RBOHB gene in multiple varieties of Oryza sativa
ABSTRACT: Studies have shown that Respiratory Burst Oxidase Homolog B (RBOHB) are involved in stress response in rice plants. Primers were developed for amplification via Polymerase Chain Reaction (PCR) of a region that contained a simple sequence repeat (SSR) in RBOHB. PCR was performed on 6 different varieties of Oryza sativa. PCR product was sequenced on an ABI 3730 capillary sequence machine. Sequence data was aligned to observe differences in SSR length between each rice variety.
Project description:Studies have shown that Rice Salt Sensitive 1 (RSS1) is involved in stress response in rice plants. Primers were developed for amplification via Polymerase Chain Reaction (PCR) of a region that contained a simple sequence repeat (SSR) in RSS1. PCR was performed on 6 different varieties of Oryza sativa. PCR product was sequenced on an ABI 3730 capillary sequence machine. Sequence data was aligned to observe differences in SSR length between each rice variety.
Project description:Information about protein expression in rice grain across both pigmented and non-pigmented rice varieties is still relatively scarce. The data provided here represent proteomic data obtained from selected 6 Malaysian local rice varieties with varying pigmentations (black, red and white). The selected pigmented rice varieties such as black (BALI and Pulut hitam 9) and red rice (MRQ100 and MRM16) have shown high antioxidant activities and non-pigmented rice (MRQ76 and MR297) contain amino acid and micronutrient contents. This project aimed to obtain global protein expression profile as well as differential protein expression between the selected pigmented and non-pigmented rice varieties particularly proteins with their functions responsible for nutritional (i.e. antioxidant, folate and low glycaemic index) and quality (i.e. aromatic) traits. Integration of this proteomics dataset with other available in-house omics data could facilitate the identification of significant functional markers related to nutritional and quality traits. Total proteins were prepared from dehusked matured seeds harvested from three different rice plants of each variety (3 protein samples per variety). The proteins were trypsin digested before subjected to SWATH-MS proteomics analysis. Proteins were identified by matching tandem mass (MS/MS) spectra from both 1D and 2D IDA to Oryza sativa japonica and indica rice databases available at UniProt by using ProteinPilot software (v4.2) (AB Sciex). Quantification of proteins was carried out by determining protein peak areas extracted from SWATH analysis data sets using PeakView (v2.1) (AB Sciex) software. Differentially expressed protein between varieties were identified using T-test analysis with a set threshold for fold change ± 1.5 and p‐value < 0.05.
Project description:In order to evaluate the genome differences and find the more tolerant cultivar, first eleven Malaysian rice cultivars namely, MR219, MR276, MR220, MR211, MR219-4, MR253, Q50, Q74, MR159, Masuri and MR263 were subjected under water deficit. Then, based on the morphological and physiological traits, the more drought-tolerant and -susceptible cultivars were screened and time-course gene expression profiling established by a comprehensive transcriptome database sequencing of the leaf RNA of tolerant rice. The current investigation provides pivotal data for understanding the rice drought tolerance mechanisms.
Project description:Detailed analysis of genome-wide transcriptome profiling in rice root is reported here, following Cr-plant interaction. Such studies are important for the identification of genes responsible for tolerance, accumulation and defense response in plants with respect to Cr stress. Rice root metabolome analysis was also carried out to relate differential transcriptome data to biological processes affected by Cr (VI) stress in rice. The rice variety IR-64 was germinated and allowed to grow for 5 d at 37 C and then transferred to Hewitt solution for growth. After 10 d of growth, seedlings of uniform size and growth were treated with 100 µM of Cr (VI), As (V), Cd, and Pb under standard physiological conditions of 16 h light (115 μmol m−2 s−1) and 8 h dark photoperiod at 25 ± 2 C for 24 h. Total RNA was extracted from the treated rice roots and microarray was performed using one-cycle target labeling and control reagents (Affymetrix platform).
Project description:MicroRNAs (miRNAs) are endogenous, noncoding, short RNAs directly involved in regulating gene expression at the post-transcriptional level. In spite of immense importance, limited information of P. vulgaris miRNAs and their expression patterns prompted us to identify new miRNAs in P. vulgaris by computational methods. Besides conventional approaches, we have used the simple sequence repeat (SSR) signatures as one of the prediction parameter. Moreover, for all other parameters including normalized Shannon entropy, normalized base pairing index and normalized base-pair distance, instead of taking a fixed cut-off value, we have used 99% probability range derived from the available data. We have identified 208 mature miRNAs in P. vulgaris belonging to 118 families, of which 201 are novel. 97 of the predicted miRNAs in P. vulgaris were validated with the sequencing data obtained from the small RNA sequencing of P. vulgaris. Randomly selected predicted miRNAs were also validated using qRT-PCR. A total of 1305 target sequences were identified for 130 predicted miRNAs. Using 80% sequence identity cut-off, proteins coded by 563 targets were identified. The computational method developed in this study was also validated by predicting 229 miRNAs of A. thaliana and 462 miRNAs of G. max, of which 213 for A. thaliana and 397 for G. max are existing in miRBase 20. There is no universal SSR that is conserved among all precursors of Viridiplantae, but conserved SSR exists within a miRNA family and is used as a signature in our prediction method. Prediction of known miRNAs of A. thaliana and G. max validates the accuracy of our method. Our findings will contribute to the present knowledge of miRNAs and their targets in P. vulgaris. This computational method can be applied to any species of Viridiplantae for the successful prediction of miRNAs and their targets. Small RNA sequencing was done for 10 days old seedlings of Phaseolus vulgaris Cv. Anupam
Project description:Traditional rice varieties found in India have many desirable characteristics. Amongst them, their differential responses to abiotic and biotic stresses are of great agricultural importance. Drought or osmotic stress is one of the major abiotic stresses afflicting crop plants in India. Indigenous varieties like Dagad deshi have been found to be drought resistant and, thereby, are being studied in great detail by plant breeders and biotechnologists alike. In this study, we have analyzed the transcriptomes of two contrasting cultivars, i.e. Dagad deshi (tolerant) and IR20 (susceptible), under control and stress conditions to elucidate the differences in their responses to drought stress using Affymetrix microarray platform. Hydroponically grown seven-day-old seedlings of Dagad deshi and IR20 were subjected to drought stress by placing them on 3 mm Whatmann sheets under light for 3 h and 6 h at 28±1oC. For control samples, seedlings were kept in RGM (root growth medium) for 6 h at 28±1oC. RNA extracted from each sample was hybridized on rice Affymetrix microarrays. Three biological replicates of each sample were used for microarray analysis. Overall, eighteen samples were analyzed representing control, 3 h and 6 h of dehydration stress for each cultivar (Dagad deshi and IR20).
Project description:Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR. Examination of 2 different small RNA expression profilings in 2 developmental stages of meristems.
Project description:The aim of this study was to analyze potential brown planthopper (BPH) resistant genes in Rathu Heenati (RHT) by Affymetrix whole rice genome array,BPH susceptible and resistant rice varieties of TN1（Taichung Native 1）as control. All the resistant related genes derived from RHT will be analyzed according to the SSR markers interval flanked on the chromosome 3, 4, 6 and 10. It will be benefit to the gene clone and marker assistant breeding for Bph3 gene in the near future. We used microarrays to detail the global differential gene expression before and after brown planthopper attack in two different varieties, and identified distinct classes of high enriched genes induced by BPH or constituent in RHT The 2nd to 3rd instar nymphs of BPH were transferred to tillering stage seedings (10 BPH nymphs per plant) in a box covered with nylon-mesh. Stems of the rice plant infected by BPH were collected at 0h (T0), 8h (T8), 24h (T24) after BPH attack, total RNA were extracted for the microarray hybirdlization.
Project description:Transcriptional profiling of developing rice endosperm at seven days after flowering comparing aleurone layers with central starchy endosperm. Cereal productivity is dependent on the accumulation of storage compounds in the endosperm, a nutritive tissue that is composed of aleurone cells in the outermost regions and starchy endosperm in the inner region. The transcriptional analyses provides clues to the molecular basis for different metabolic pathways in response to the spatial and nutritional differences between rice aleurone cells and starchy endosperm. Two-condition experiment, Aleurone layers vs. central starchy endosperm. 3 biological replicates with color swap for each biological replicate
Project description:We report the application of methylacytosine immunoprecipetation combined with Illumina sequencing (MeDIP-seq) for high-throughput profiling of DNA methylation in rice embryo 3, 6, 9 DAP and endosperm 2, 3, 6, 9 DAP. A total number of 12-14 million of 2×49 bp paired-end reads was generated for each sample, and BOWTIE2 was used for read mapping. We generated genome-wide DNA methylation profiles of rice embryo and endosperm. This study provides a framework to systemically characterize the effect of DNA methylation in developing seeds and will help to illustrate the epigenetic regulation of rice seed development. Rice embryo and endosperm were selected for DNA extraction and methylacytosine immunoprecipetation combined with Illumina sequencing. We sought to obtain the genome-wide DNA methylation profilings of embryo at 3,6,9 days after pollination(DAP) and endosperm at 2,3,6,9 DAP. To that end, we hand-selected embryo at 3,6,9 DAP and endosperm at 2,3,6,9 DAP according to morphological criteria.