A telomere-to-telomere genome of Mojiang purple rice resolves a rare three-copy structural allele upstream of Kala4/OsB2

Background: Black and purple pericarp in rice requires activation of the basic helix-loop-helix (bHLH) regulator Kala4/OsB2, and the known black-rice allele is a complex promoter rearrangement. Such repeat-embedded alleles can be difficult to resolve in fragmented assemblies, and for many pigmented landraces it remains unclear whether they carry that allele or structurally different ones. We asked three questions about the purple-pericarp landrace Mojiang ZN65: what is the structure of its Kala4/OsB2 upstream region; how frequent is gain of its upstream block in a rice diversity panel, and do the accessions with that gain share its junction; and how is it distributed relative to a second regulatory variant, a duplicated repeat in the OsKala3 promoter. Results: We assembled a gap-free telomere-to-telomere genome of ZN65 (395.1 Mb; all 24 telomeres; consensus quality value 53.6; 99.6% complete Benchmarking Universal Single-Copy Orthologues; indica), in which the flavonoid pathway showed no pathway-wide copy-number expansion. Upstream of Kala4/OsB2, a ~5.87-kb segment (block A) is present in three non-tandem copies, spanned as single molecules by three ultra-long Nanopore reads (115,450 bp); each of 33 other assemblies yielded a single homologous hit (10 of them coverage- or divergence-limited). This arrangement differs from the canonical black-rice Kala4 allele. Among 533 accessions of the 3,000 Rice Genomes panel, read depth detected block A gain in four accessions (three at three or more copies, 0.56%; one at two). All four carry the ZN65 junction at the same base, and three carry a pigment term in their variety names from a lexicon fixed in advance (Fisher's exact test, P = 0.0023). All four also have OsKala3 promoter-repeat read depth consistent with the two-unit configuration reported for black-pericarp cultivars, a state found in 1.9% of the panel. Conclusions: ZN65 carries a rare Kala4/OsB2 structural variant whose junction is shared with other accessions named as pigmented rice and which, in this panel, occurs only in accessions whose OsKala3 promoter-repeat read depth indicates duplication. Its effect on pigmentation remains to be tested with measured pericarp phenotypes and functional assays.