Target gene mutation discovery and amplicon sequencing for evaluating maduramicin resistance in Eimeria tenella.

Anticoccidial drug resistance in Eimeria spp. presents a growing threat to global poultry health and food security, necessitating robust molecular tools for resistance surveillance. In this study, we report the identification and functional validation of a single nucleotide mutation (c.1255 C > T) in the ETH2_0402100 gene of Eimeria tenella that confers resistance to maduramicin, a widely used ionophore anticoccidial drug. Using genetic crossing, backcrossing, and QTL-seq, we narrowed the resistance locus to a 76 kb region on chromosome HG994964.1. CRISPR/Cas9-mediated site-directed mutagenesis and gene overexpression confirmed that the leucine-to-phenylalanine substitution in ETH2_0402100 confers maduramicin resistance. To translate this discovery into a practical tool, we developed a PCR-based amplification sequencing assay that enables rapid, quantitative detection of maduramicin-resistant alleles from field samples. Together, these findings establish the molecular basis and monitoring strategy for maduramicin resistance in Eimeria spp., and provide a field-deployable molecular diagnostic tool for detecting ionophore resistance.