Analysis of Anthocyanin-Less 2 Diversity in Barley Reveals a Specific Allele to Cause Purple-Colored Grains

The purple color of the barley (Hordeum vulgare L.) grain is attributed to anthocyanins which are beneficial for human health. Synthesis of these pigments in both grain and vegetative tissues is controlled by ANT1 and ANT2 that belong to the families R2R3-MYB and bHLH of transcription factors, respectively. Here, we investigated the role of the Ant2 gene in pigmentation of grains compared to vegetative tissue. After screening of 504 barley accessions, six relevant alleles of Ant2 were identified. These involve distinct insertions in the promoter and/or in intron 6. Allele Ant2.l was found in all purple-grained barley accessions and was more strongly expressed compared to the alleles present in the other lines. The crucial role of Ant2.l in grain pigmentation was validated by targeted mutagenesis using RNA-guided endonuclease Cas9. It is further shown that the other Ant2 alleles do not affect grain pigmentation while being essential for pigmentation of vegetative tissues.