Isoform‐resolved genome annotation enables mapping of tissue‐specific betalain regulation in amaranth

Summary Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue‐specific regulation of the pathway remains mostly unknown. We enhance the high‐quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue‐specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock‐out of red betacyanins in natural accessions of amaranth. We reveal the tissue‐specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2 . Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co‐option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.