Primer efficiency determination for flavanone 3-hydroxylase gene in wheat (Triticum aestivum L.)

Anthocyanins are water-soluble pigments that may be red, purple, or blue. They belong to a parent class of molecules called flavonoids. Anthocyanins occur in all tissues of higher plants, including leaves, stems, roots, blossoms, and fruits. Flavanon 3-hydroxylase (F3H) is one of the key enzymes of the flavanoid biosynthesis pathway. F3H converts flavanones to dihydroflavonols. The main aim was to study the F3H gene in wheat (Triticum aestivum L.). In our experiment 5 genotypes with nonstandard coloured caryopses were used (2 genotypes with blue aleurone, 2 genotypes with purple pericarp and 1 genotype with white caryopses-without anthocyanins). Total RNA was isolated by phenol-chloroform method and complementary DNA was obtained by reverse transcription. Specific primers for each of the F3H genes (F3H_A, F3H_B and F3H_D) that were found in the National Center for Biotechnology Information (NCBI) database were designed. The first results of quantitative PCR (qPCR) contained two or more products. New primers for F3H-D gene were found and qPCR was repeated. The only one PCR product of F3H_D amplification from all five genotypes was sent for sequence analysis. Obtained sequences of F3H_D have 99-100% conformity with the database NCBI.