MSc

Characterisation and evaluation of Njavara types of rice (Oryza sativa L.) was under taken in the Department of Plant Breeding and Genetics and Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2005 - 2007 with the objective of molecular characterization and gene sequencing in Njavara for developing suitable molecular markers for cultivar identification.

Njavara genotypes exhibited high variability with respect to lemma and palea colour with two major classes viz., yellow (straw colour) and black types. Detailed characterization revealed that yellow type could be further grouped into gold furrows on straw (N3 & N5) and brown furrows on straw (N4 & N7). Three genotypes viz., N1, N2 and N6 exhibited a lemma and palea colour dominated by black. N1 and N2 exhibited variations in black colour for lemma and palea as pure black, black furrows/black patches on straw background whereas N6 exhibited light shade of black. Variations in seed coat colour as red, light brown and brown were also observed among the genotypes.

The method suggested by Dellaporta et al. (1983) with slight modifications was found to be effective in isolating good quality genomic DNA from Njavara. Good amplifications were observed when RAPD analysis was performed with sequences OPA 1, OPA 4, OPA 6, OPA7 , OPA 9, OPN 6, OPN 18, OPP 6, OPP 11 and OPE 6. OPA 1 and OPP 11 were found to be promising in the amplification of rice genomic DNA with maximum amplification. Amplification of Njavara DNA with primer OPE 6 exhibited unique bands (1.375 kb , 1.29 kb and 0.44 kb ) for Njavara genotypes and hence are valuable as DNA markers for the identification of this unique cultivar. The dendrogram with RAPD markers showed distinct clusters for Njavara.

Cloning and sequencing of the unique molecular band with M 13 primer gave the sequence data of a gene segment of size 762 bp.The homology search of this sequence with BLASTN showed that it has maximum identity with genes from Oryza sativa (japonica cultivar-group) mitochondrial gene for tRNA-Asn, complete sequence, O. sativa mRNA for chilling-inducible protein, O.sativa rbbi2-5 gene for putative Bowman Birk tryspin inhibitor, O.sativa rbbi2-3 gene for putative Bowman Birk trypsin inhibitor, O.sativa rbbi2-4 gene for putative Bowman Birk trypsin inhibitor and O. sativa (japonica cultivar-group) mRNA for chilling tolerance related protein.The homology of cloned DNA fragments of Njavara (N5) with BBI genes (rbbi 2-3, rbbi 2-4 and rbbi 2-5 ) is a preliminary indication of the medicinal property (anticarcinogenic) of this unique medicinal cultivar of Kerala and also its thermostable nature.

Sequence analysis revealed the presence of five ORF’s . The longest open reading frame had 180 bases encoding 59 amino acids in the predicted coding region. Among the amino acids, serine was occurring more frequently than other amino acids. ORF in +3 reading frame was found to be of a residue length of 102 bases, encoding 33 amino acids. Genscan tool determined two internal exons from the clone with a length of 91 and 129 residues.

Sequence analysis of the data with VecScreen showed strong match to vector sequences in the database eventhough the sequences were not matching with pSCA (vector used in the present study) vector. Alignment of sequences through CLUSTAL W programme revealed poor homology with query sequence and vector sequence used for cloning. Homology was shown between the sequences when BLAST 2 SEQUENCES programme was used. These results are to be confirmed through further studies.