Duplicated and genes in barley genome.

BACKGROUND

Anthocyanin compounds playing multiple biological functions can be synthesized in different parts of barley ( L.) plant. The diversity of anthocyanin molecules is related with branching the pathway to alternative ways in which dihydroflavonols may be modified either with the help of flavonoid 3'-hydroxylase (3') or flavonoid 3',5'-hydroxylase (3'5')-the cytochrome P450-dependent monooxygenases. The ' and '5' gene families are among the least studied anthocyanin biosynthesis structural genes in barley. The aim of this study was to identify and characterise duplicated copies of the ' and 3'5' genes in the barley genome.

RESULTS

Four copies of the '5' gene (on chromosomes 4HL, 6HL, 6HS and 7HS) and two copies of the ' gene (on chromosomes 1HL and 6HS) were identified in barley genome. These copies have either one or two introns. Amino acid sequences analysis demonstrated the presence of the flavonoid hydroxylase-featured conserved motifs in all copies of the 3' and 3'5' genes with the exception of 3'5'-3 carrying a loss-of-function mutation in a conservative cytochrome P450 domain. It was shown that the divergence between ' and '5' occurred 129 million years ago (MYA) before the emergence of monocot and dicot plant species. The ' copy approximately occurred 80 MYA; the appearance of '5' copies occurred 8, 36 and 91 MYA. qRT-PCR analysis revealed the tissue-specific activity for some copies of the studied genes. The '-1 gene was transcribed in aleurone layer, lemma and pericarp (with an increased level in the coloured pericarp), whereas the ' gene was expressed in stems only. The '5' gene was expressed only in the aleurone layer, and in a coloured aleurone its expression was 30-fold higher. The transcriptional activity of '5' was detected in different tissues with significantly higher level in uncoloured genotype in contrast to coloured ones. The '5' gene expressed neither in stems nor in aleurone layer, lemma and pericarp. The '5' gene copy was weakly expressed in all tissues analysed.

CONCLUSION

' and '5'-coding genes involved in anthocyanin synthesis in were identified and characterised, from which the copies designated ', ', '5' and '5' demonstrated tissue-specific expression patterns. Information on these modulators of the anthocyanin biosynthesis pathway can be used in future for manipulation with synthesis of diverse anthocyanin compounds in different parts of barley plant. Finding both the copies with tissue-specific expression and a copy undergoing pseudogenization demonstrated rapid evolutionary events tightly related with functional specialization of the duplicated members of the cytochrome P450-dependent monooxygenases gene families.