mpH-1 is conferred by a mutation in TTHERM_00564170. (A) NGS-based mapping of variants co-segregating with mpH-1 in the micronuclear genome using the ACCA method. mpH-1 is linked to a region on chr1 around 7 Mb. (B) The domain organization of TTHERM_00564170 protein. (C and D) An AlphaFold2 model of the TPR domain of TTHERM_00564170 viewed from the protein’s C terminus (C) and from the side (D). The TPR segments are shown in blue. (E–H) Replacement of the A5354 variant base by the reference G base in the mpH-1 homozygotes rescues the mutant phenotype. (E–G) SR-SIM images of cells, grown at 30°C and stained with 20H5 anti-centrin (magenta) and DAPI (blue). GFP-mNeonGreen fluorescence was imaged directly (green). (E) WT; F, mpH-1 homozygotes; G, mpH-1 homozygotes with an introduced MpH-GFP-mNeonGreen fragment that replaced the A5354 variant base with the WT G and added MpH-GFP-mNeonGreen sequence to the 3′ end of the coding region of TTHERM_00564170 (see Fig. S1 C). (H) Quantification of the percentage of OA with 2 M rows in strains with the following macronuclear genotypes: WT (strain CU428), mpH-1 (strain IA483), and mpH-1 with A5354 variant base replaced by the reference G and GFP-mNeonGreen at the end of the coding region of TTHERM_00564170. Mean ± SD. N = 3 experiments indicated by symbol colors (15–124 cells scored per experiment, a total of 112–226 cells scored per genotype). P = 0.0223 in an unpaired t test with Welch’s correction.