The complete reference genome of Tartary buckwheat and its mutation library provide important resources for genetic studies and breeding.

Tartary buckwheat (TB), Fagopyrum tataricum, is an important medicinal and edible crop with a worldwide distribution. However, reference genomes with gaps and mutant population scarcity have hindered functional genomics and genetic improvement of TB. Here, we present a telomere-to-telomere (T2T) gap-free genome assembly of the elite TB inbred line Guiku1 and its ethyl-methyl-sulfonate (EMS)-induced phenotypically rich mutation library. The Guiku1 gap-free genome spans 453.83 Mb, containing 43,441 predicted protein-coding genes. The mutation library includes 751 mutants with stably heritable phenotypes. Whole-genome resequencing of 320 mutants identified 105,682 single-nucleotide polymorphisms (SNPs) and 21,461 insertions/deletions (indels), affecting the protein-coding sequences of 25,986 genes. Genes responsible for the pink stem and petiole mutant trait and flavonoid content variation were identified using forward- and reverse-genetics approaches, respectively. Collectively, the T2T gap-free genome of Guiku1 and its EMS mutation library provide important resources for functional genomics studies and the genetic improvement of TB.