Barley grain at maturity can have yellow, purple, blue, and black pigmentations which are suggested to play a protective role under stress conditions. The first three types of the colors are caused by phenolic compounds flavonoids; the last one is caused by phytomelanins, oxidized and polymerized phenolic compounds. Although the genetic basis of the flavonoid biosynthesis pathway in barley has been thoroughly studied, there is no data yet on its regulation in purple and black barley grains. In the current study, genetic model of Hordeum vulgare ‘Bowman’ near-isogenic lines (NILs) was used to investigate the regulation of the flavonoid biosynthesis in white, purple, and black barley grains. Microsatellite genotyping revealed donor segments in the purple- and black-grained lines on chromosomes 2H (in region of the Ant2 gene determining purple color of grains) and 1H (in region of the Blp gene determining black lemma and pericarp), respectively. The isolated dominant Ant2 allele of the purple-grained line has high level of sequence similarity with the recessive Bowman’s ant2 in coding region, whereas an insertion of 179 bp was detected in promoter region of ant2. This structural divergence between Ant2 and ant2 alleles may underlie their different expression in grain pericarp: Bowman’s Ant2 is not transcribed, whereas it was up-regulated in the purple-grained line with coordinately co-expressed flavonoid biosynthesis structural genes (Chs, Chi, F3h, F3’h, Dfr, Ans). This led to total anthocyain content increase in purple-grained line identified by ultra-performance liquid chromatography (HPLC). Collectively, these results proved the regulatory function of the Ant2 gene in anthocyanin biosynthesis in barley grain pericarp. In the black-grained line, the specific transcriptional regulation of the flavonoid biosynthesis pathway genes was not detected, suggesting that flavonoid pigments are not involved in development of black lemma and pericarp trait.

Background Anthocyanins are plants secondary metabolites important for plant adaptation to severe environments and potentially beneficial to human health. Purple colour of barley grain is caused by the pigments synthesized in pericarp. One or two genes determine the trait. One of them is Ant2 mapped on chromosome 2HL and is known to encode transcription factor (TF) with a bHLH domain. In plants, bHLH regulates anthocyanin biosynthesis together with TF harboring an R2R3-MYB domain. In wheat, the R2R3-MYBs responsible for purple colour of grain pericarp are encoded by the homoallelic series of the Pp-1 genes that were mapped on the short arms of chromosomes 7. In barley, in orthologous positions to wheat’s Pp-1 , the Ant1 gene determining red colour of leaf sheath has been mapped. In the current study, we tested whether Ant1 has pleiotropic effect not only on leaf sheath colour but also on pericarp pigmentation. Results А set of near isogenic lines (NILs) carrying different combinations of alleles at the Ant1 and Ant2 loci was created using markers-assisted backcrossing approach. The dominant alleles of both the Ant1 and Ant2 genes are required for anthocyanin accumulation in pericarp. A qRT-PCR analysis of the Ant genes in lemma and pericarp of the NILs revealed that some reciprocal interaction occurs between the genes. Expression of each of the two genes was up-regulated in purple-grained line with dominant alleles at the both loci. The lines carrying dominant allele either in the Ant1 or in the Ant2 locus were characterized by the decreased level of expression of the dominant gene and scant activity of the recessive one. The Ant1 and Ant2 expression was barely detected in uncolored line with recessive alleles at both loci. The anthocyanin biosynthesis structural genes were differently regulated: Chs , Chi , F3h , Dfr were transcribed in all lines independently on allelic state of the Ant1 and Ant2 genes, whereas F3’h and Ans were activated in presence on dominant alleles of the both regulatory genes. Conclusions The R2R3-MYB-encoding counterpart ( Ant1 ) of the regulatory Ant2 gene was determined for the first time. The dominant alleles of both of them are required for activation of anthocyanin synthesis in barley lemma and pericarp. The R2R3-MYB + bHLH complex activates the synthesis via affecting ex...

Anthocyanins contribute to plants' defence against a number of abiotic and biotic stress agents. The anthocyanin pigmentation of the barley leaf sheath is genetically determined by Ant1, a gene which maps to a region of chromosome 7HS delimited by the microsatellite loci Xgbms0226 and Xgbms0240. The sequence of the maize gene C1 (encoding an R2R3 MYB factor regulating anthocyanin synthesis) was used for the PCRbased cloning of Ant1. In ant1 genotypes, no transcript is generated in the leaf sheath, whereas the gene is active in the presence of the dominant allele. A comparison of the coding and promoter sequences of Ant1 (which induces purple pigmentation in the leaf sheath) and ant1 (which does not) showed that the key polymorphisms lay in the promoter sequence. The transcription of four anthocyanin synthesis structural genes (Chi, F3h, Dfr, Ans) was dependent on the allelic status of Ant1.

In wheat, anthocyanin pigments can be accumulated in pericarp tissues (under control of the Pp genes) resulting in purple-colored grain. In the current study, a strategy, based on the use of molecular and morphological markers, was applied to create purple-grained bread wheat cultivars adapted to the West Siberian region. The breeding scheme started from crossing of recipients (elite cultivars and lines) with donor lines carrying dominant alleles of the complementary genes Pp3 and Pp-D1. The F2 hybrids passed three-step marker-assisted selection, and those having dominant Pp-D1Pp-D1Pp3Pp3 genotypes were backcrossed with the recurrent parents. The desired BC1F2-3 progenies were selected using morphological marker, while BC1F3 also passed through field evaluation. At this stage, 120 lines were selected and planted in individual 1 m2 “breeding nursery (BN) plots” for assessment of heading dates, duration of vegetation period, resistance to powdery mildew, stem and leaf rusts, protein and gluten content, as well as productivity. After these investigations, a total of 17 promising anthocyanin-rich purple-grained lines characterized by multiple resistance and having best yield/quality characteristics were finally candidates for selection of commercial cultivars adapted to the West Siberian climate and suitable for functional food production.