= Solution
The first task is to map the <DNA> associated with the centromeric <histone> variant <CENP-A>; the second is to determine how that occupancy is maintained. These require different evidence. A shared <DNA> sequence is not sufficient evidence for an inherited centromeric state.
Use validated anti-<CENP-A> <chromatin immunoprecipitation sequencing> with input <DNA> and an appropriate negative immunoprecipitation control. Check antibody recognition separately in human, horse and chicken; an endogenous epitope-tagged allele can provide an orthogonal control if its chromosome-segregation function is retained. Native digestion can recover CENP-A-containing <nucleosomes>, whereas crosslinking can retain indirect associations. <CUT&RUN> provides a complementary occupancy assay with different recovery biases. Establish centromeric function by colocalization with <kinetochore> <proteins> and by normal <chromosome segregation>, not merely by calling a sequencing peak.
Mapping repetitive <DNA> is the main analytical obstacle. Use centromere-resolved assemblies where possible, retain information from multimapping reads, and analyse satellite-family enrichment as well as uniquely mappable sequence. Absence of a peak in an incomplete reference is not evidence of absent <CENP-A>. Long-read sequence assemblies, informative <single-nucleotide polymorphisms>, and <DNA fluorescence in situ hybridization> can connect a recovered sequence to a particular <chromosome> and distinguish homologues.
For precise positioning, compare enriched domains with input-normalized background at several resolutions. Map nucleosome-protected fragments and use fibre immunofluorescence or sequence probes to check domain extent and heterogeneity. A broad population peak may represent alternative positions in different cells or homologues, rather than one broad <CENP-A> domain on every <chromosome>.
The expected outcome is \b[a common centromeric function without a universal centromeric <DNA> sequence]. Human <CENP-A> commonly occupies selected portions of alpha-satellite arrays, but human <neocentromeres> show that such arrays are not obligatory. Horse <chromosome> 11 provides a normal satellite-free <centromere>. Chicken <chromosomes> include both repeat-rich <centromeres> and comparatively short non-tandem-repetitive centromeric regions.<Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences by Wei-Hao Shang and others>[Comparative chicken centromere mapping]{ref}<Birth, evolution, and transmission of satellite-free mammalian centromeric domains by Solomon G. Nergadze and others>[Satellite-free mammalian centromeres and inherited positional variation]{ref}
To test mitotic stability, start from single-cell clones and remap <CENP-A> on the same haplotype after successive divisions, while checking that the underlying <DNA> has not changed. Pulse labelling of old versus newly incorporated <CENP-A> can distinguish retention from replenishment. Follow both <chromosome> identity and domain position: stable centromeric function does not require an invariant nucleotide boundary. To test meiotic transmission, use informative pedigrees or gamete-and-offspring material and allele-resolved mapping, with matched cell types where possible. An inherited positional state, or bounded movement of that state on unchanged <DNA>, supports <epigenetic centromere inheritance>. Artificial <centromere> deletion and neocentromere recovery provide an additional perturbation test, but establishment after deletion is different from ordinary meiotic inheritance.<Chromosome Engineering Allows the Efficient Isolation of Vertebrate Neocentromeres by Wei-Hao Shang and others>[Chicken neocentromere formation and CENP-A domain mapping]{ref}
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