We developed an improved protocol for identification of causative mutations in Tetrahymena using pooled linkage and comparative whole genome sequencing. The workflow involves an outcross of a mutant of interest, uniparental cytogamy of a single F1 clone, pooling of a number of wild-type and mutant F2 clones, whole genome sequencing of mutant and wild-type pools, variant subtractions from the mutant set, and filtering for nitrosoguanidine-induced transition mutations. Based on a few mutants we have analyzed thus far, this workflow reveals either a single or a small number of closely linked variants containing 100% of mutant reads, as candidates for the causative mutation. The procedure can be used for both recessive and dominant alleles. We successfully used this approach for identification of the causative mutation for cdaI-1, a temperature-sensitive “hammerhead” mutant, in which the oral primordium and the cleavage furrow shift toward the anterior end, resulting in an incomplete and unequal cell division (isolated by L.M. Jenkins and J. Frankel, University of Iowa; Frankel J., Eukaryotic Cell 2008,10:1617-1639). The cdaI-1 cause is a substitution in a STE20-related kinase encoded by TTHERM_00971920. A wild-type fragment of TTHERM_00971920 rescued the mutant phenotype of the cdaI-1 cells grown at the restrictive temperature. We applied the same strategy to a more challenging case, identification of a genetic suppressor of a cilia length regulation mutant. A loss of function of a Tetrahymena ortholog of the ”long flagella 4” (LF4) protein of Chlamydomonas reinhardtii leads to longer cilia. LF4 encodes a conserved protein kinase. We aim at identifying the mechanism by which LF4 regulates the length of cilia and its kinase substrate(s). An MTT1-driven cadmium-induced overexpression of LF4 shortens cilia, which leads to cell paralysis, an easily selectable phenotype. A suppressor locus of the LF4 overexpression could encode a phosphorylation substrate or a regulator of LF4. We isolated a number of suppressors of the LF4 overexpression, all of which but one are intragenic (due to a loss of the overproduced LF4 kinase activity). For the single extragenic suppressor, we identified a candidate causative mutation in a conserved ciliary protein gene. We are currently testing whether an introduction of the mutant candidate gene fragment suppresses the consequences of LF4 overexpression.