CHARACTERIZATION, VALIDATION AND SEQUENCE ANALYSIS OF CHLOROPLAST GENOME SPECIFIC REGIONS AS CANDIDATE LOCI FOR DNA BARCODING OF RICE (Oryza spp.)
| dc.contributor.advisor | BANERJEE, SHUBHA | |
| dc.contributor.advisor | CHANDEL, GIRISH | |
| dc.contributor.advisor | KOTASTHANE, A. S. | |
| dc.contributor.advisor | SAXENA, R. R. | |
| dc.contributor.author | SINGH, JYOTI | |
| dc.date.accessioned | 2018-02-01T10:02:28Z | |
| dc.date.available | 2018-02-01T10:02:28Z | |
| dc.date.issued | 2018 | |
| dc.description.abstract | The Oryza sativa species have well established and ancient divergence between two major subspecies, indica and japonica which are further divided into five sub groups aromatic, aus, indica, temperate japonica, and tropical japonica of cultivated rice. Chhattisgarh is rich in rice biodiversity of cultivated rice (containing the wild progenitors also). The biodiversity has been the cradle of crop improvement thus to conserve and catalogue such natural treasure is of utmost importance. Molecular characterization of the rice biodiversity in Chhattisgarh is also important for establishing its uniqueness and to protect the IPR of the state. Characterization and validation of chloroplast specific regions as candidate loci is important for assessing the genetic variation in germplasm accession and study genetic relationship between genotypes. DNA barcoding is a technique that makes use of short sequences from a standardized region of a genome to provide quick and reliable identification of species among all forms of life. The presence of uniqueness and variability required for DNA barcoding is well reported in animal system based on mitochondrial gene Cytochrome c oxidase I (CO1). On the other hand, limited information is available on universal barcode for plants. Candidate loci belonging to chloroplast genome and nuclear genome have been analyzed in various plants to identify universal barcoding loci capable of inter and intra-species discrimination. In this study, genetic variation and phylogenetic relationship analysis was carried out in a set of 231 rice genotypes including diverse germplasm lines, elite lines, landraces and wild rice collection from undivided Madhya Pradesh and Chhattisgarh along with popular rice varieties and advanced breeding lines for various traits. A total of 31 physiological traits were recorded at different stages (as per SES, IRRI 2002). Thirty one potential candidate loci recommended for barcoding of plants were used for validation in 231 rice genotypes, and subsequent selection of suitable barcoding loci based on success in amplification and universality. Amplification results indicated that only one of the chloroplast genome specific primer pair “psbA-trnH” showed 100% amplification efficiency followed by “rbcL” 89.61%, “atpH-atpl” 68.39%, “matK” 66.2% and “petA-psbJ” 62.33% which were evaluated for the first time as candidate loci for DNA Barcode at intraspecies level in rice. While rest of the 9 primers showed lower amplification efficiency ranges from 5.19% to 52.81%. Based on amplification efficiency, reproducibility and amplicon size (as per Consortium for the Barcode of Life standard) seven loci ( atpH-atpI, petA-psbJ, trnK, rbcL ,matK, trnlc-trnld , psbA-trnH ) were amplified for sequencing. Sequencing of both strands was done by Sanger’s method with around 550bp read length. The sequence information was used for anenmat of phylogenetic and phylogeographical relationships among 24 genotypes. The multiple sequence alignment (Clustal W using MEGA 7.0.25) revealed divergence among the cultivars, nucleotide variation and distinctness for identify the candidate loci for barcoding. In our study we reporte the average frequency of nucleotide composition varied from G=22.5, C=22.8 % A= 36.5 and T/U= 36.2%. The highest Maximum likelihood values of transition rate were 62.06 and transversion rate were 17.12, also transition / transversion bais 2.04 were estimated in forward strand sequence of psbA-trnH loci substitution of nucleotides based on the seven markers for 24 genotypes of rice calculated by MEGA 7.0.25. The estimated evolutionary divergence between sequences ranged from 0.000 to 3.061 (average 0.153 to 1.236). The parsimony informative sites were recorded maximum 305 sites in both the strands sequence of matK loci, followed by 264 sites were reported in our study on both the strands sequence of rbcl loci and 226 sites in both the strands sequence of trnk loci and number of variable sites were 672 which is highest in both the strands sequence of rbcl loci, also 274 in both the strands sequence of trnk loci followed by 246 in forward strand sequence of matK loci. While nucleotide diversity (per site pi) reported maximum in rbcl 0.21613 and maximum numbers of haplotypes 17 were reported based on forward strand sequence of psbA-trnH loci sequence analysis. Selective neutrality tests were negative and insignificant maximum Tajima’s D =2.75355. The nucleotide diversity of each of the 7 loci among 24 rice genotypes were evaluated by FINGERPRINT to depict most variable locus. The sequences were further submitted to BOLD (Barcoding of Life Database) for generation of illustrative barcode. The barcode reported by these seven loci is the first scientific information that has been reported at the intra species level in rice. The overall analysis indicated matk, rbcl, psbA-trn ,trnk and trnl were as the most suitable candidate loci for DNA barcoding in rice. The efficacy of the selected barcoding loci must be tested as potentially useful loci for generation of DNA barcoding in rice at cultivar level. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810040231 | |
| dc.keywords | DNA BARCODING; CHLOROPLAST; LOCI; RICE | en_US |
| dc.language.iso | en_US | en_US |
| dc.pages | 281P. | en_US |
| dc.publisher | Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.) | en_US |
| dc.sub | Agricultural Biotechnology | en_US |
| dc.subject | null | en_US |
| dc.theme | PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY | en_US |
| dc.these.type | Ph.D | en_US |
| dc.title | CHARACTERIZATION, VALIDATION AND SEQUENCE ANALYSIS OF CHLOROPLAST GENOME SPECIFIC REGIONS AS CANDIDATE LOCI FOR DNA BARCODING OF RICE (Oryza spp.) | en_US |
| dc.type | Thesis | en_US |