Cross-reference read-template discordance constrains proxy-marker screening for paternal DNA in polyploid gibel carp (*Carassius gibelio*)

Short-read screens for paternal DNA in polyploid gynogenetic fish are difficult to interpret when the parents were sequenced too shallowly and the markers therefore come from public genomic resources, and when the two reference genomes need not recover the same reads. We sequenced six offspring of the spawning female YJ1, from one batch of allogynogenetic gibel carp (Carassius gibelio) activated by common carp (Cyprinus carpio) sperm, and applied a dual-reference rule at 4,389 proxy single-nucleotide variant (SNV) markers. None of the 26,334 fish-by-marker states passed the rule because the dual-reference ALT-count criterion was never met. Despite this zero-candidate outcome, strong cross-reference ALT discordance was evident. On the carp reference, 1,393 depth-evaluable states at 263 markers carried at least three alternate-allele (ALT) reads, and 183 of those markers showed the pattern in all six offspring, whereas the gibel-reference ALT count never reached three. A targeted audit of 20 sample–locus combinations with strong carp-reference ALT support found that the carp-reference ALT-supporting read templates did not cover the reciprocal gibel coordinate; they aligned elsewhere in the gibel assembly. Simulations showed reference-dependent recovery loss; matching input depth raised the simulated fraction reaching carp ALT ≥ 10 from 7.3% to 34.7%, leaving a residual gap to the observed 58.9%. Reciprocal marker coordinates therefore did not necessarily recover the same read-template sets, limiting how confidently a negative proxy-marker screen can be interpreted as paternal-DNA exclusion.