Kuster, R. D.; Sisler, P.; Sandhu, K.; Yin, L.; Niece, S.; Krueger, R.; Dardick, C.; Keremane, M.; Ramadugu, C.; Staton, M. E.

Background Pangenomes are a promising new approach to genomics that can reduce reference bias in genotyping, but the reliability of such a data model remains unclear in tracking variation across species. To test the utility of graph-based pangenomes for interspecific breeding, we developed a Minigraph-Cactus super-pangenome representing four Citrus species derived from the founder lines of a citrus breeding program. To benchmark SNP calling accuracy using graph and linear-based approaches, we performed whole genome short read sequencing for two sets of pedigreed progeny: 30 F1 hybrids and 244 advanced hybrids from an F1 crossed with a parent not included in the pangenome. Results The linear approach yielded more SNP calls than the graph-based approach, however, both methods exhibited similar Mendelian Inheritance Error Rates (MIER) in a tool-dependent manner. Reconstruction of parental haplotype blocks in the advanced hybrids revealed a striking improvement in performance in the pangenome graph-based calls, suggesting MIER is vulnerable to error when reference bias influences both parental and progeny genotype calls. Masking of regions diverged from the reference path improved MIER accuracy metrics and haplotype block reconstruction in both the linear and graph-based SNP calls. Conclusions In non-model systems, inheritance patterns observed from pedigreed hybrids provide a framework for benchmarking variant-calling accuracy using pangenomes. SNP miscalls originating from diverged regions can falsely satisfy MIER filters, thus we recommend haplotype blocks. The inherent structure of the pangenome graph has promising applications for removing regions of unreliable mapping quality, which cannot otherwise be reliably removed using traditional filtering metrics.