DETERMINATION OF CANDIDATE SEQUENCES CHALCONE ISOMERASE AND DIHYDROFLAVONOL REDUCTASE IN WHEAT

The aim of this work is identification of genes from the anthocyanins biosynthetic pathway that are responsible for grain color and comparison of their sequences. We used ripened caryopsis of spring wheat with different coloration of the aleurone and pericarp layers as an experimental material. Genotypes UC66049 and Tschermaks Blaukorniger Sommerweizen have blue aleurone. Genotypes Abissinskaja an-asajta and ANK-28B have purple pericarp. Genotype Novosibirskaja 67 was used as standard. It has a white caryopsis as well as a genotype Heroldo (a form of winter wheat). RNA from caryopsis was isolated using phenol-chloroform extraction and revese transcribed into cDNA. House keeping gene GAPDH (the gene for glyceraldehyde-3-phosphate dehydrogenase) was chosen as a control for PCR to verify the successful transcription of cDNA. For designing primers we used the cDNA sequences for CHI and DFR obtained from the database NCBI were used for primers designing. Via PCR reactions were amplified segments of sequences for CHI and DFR, which were used for direct sequencing of PCR product. The obtained candidate sequences were compared with sequences in the NCBI database and their polymorphisms were evaluated. We aquired a partial sequence for genes chalcone isomerase and dihydroflavanol reductase. By comparing sequences for each gene was found homology in the range from 95 to 100%. The variability caused single nucleotide polymorphism and indels. Sequences were sent to the Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc. There were compared our cDNA sequences with BAC library of wheat chromosomes. A sequence for CHI was found on chromosome 5B. In the case of DFR was found a sequence on 3B and on long arm 3A chromosome. Downstream step in this work is to get the whole sequence of the transcribed genes by 3'RACE and 5'RACE PCR reactions. Furthermore, the complete cDNA sequences will be compared with genomic DNA sequences to determine the presence of introns in genes. Finally, the data will be used for the design of qPCR for the study of genes expresion during caryopsis maturation.