Marker-based knockout genotyping in diploid model organisms

Analysis of gene function in diploid organisms typically relies on molecular methods to distinguish wild-type, mono-allelic knockout, and bi-allelic knockout individuals, creating a major practical bottleneck for large-scale developmental studies. Here, we establish a modular visual genotyping approach that enables organism-level discrimination of knockout zygosity by tagging alternative disrupted alleles with spectrally distinct fluorescent markers. Implemented in the red flour beetle Tribolium castaneum , a two-marker strategy permits reliable identification of bi-allelic knockouts within mixed cohorts in a semi-random insertional mutagenesis framework. Further, a four-marker strategy using a targeted CRISPR/Cas9-based gene editing approach enables systematic generation and unambiguous recognition of individuals homozygous for both a disrupted gene of choice and a fluorescent reporter transgene. This design substantially reduces the workload associated with routine molecular genotyping while enabling genotype-resolved long-term live imaging of embryonic morphogenesis. Application to the extra-embryonic specification factor zerknüllt 1 reveals haplosufficiency and a semi-lethal phenotype associated with variable morphogenetic outcomes. Together, our approach provides a scalable framework for functional analysis of essential developmental genes. Summary Statement Visual marker-based genotyping enables organism-level discrimination of knockout zygosity, reducing reliance on molecular assays and facilitating scalable, genotype-resolved live imaging of developmental gene function in diploid model organisms.