Direct detection of meiotic recombination events in the highly heterozygous amphioxus genome

Amphioxus, a basal chordate with highly heterozygous genomes (3.2∼4.2% in sequenced species), represents a key model for understanding vertebrate origins. However, the extreme heterozygosity poses challenges for many genomic analyses, including studying meiotic recombination. Here, we present a novel bioinformatic pipeline that enables direct detection of crossover (CO) and non-crossover (NCO) recombination events using short-read whole-genome sequencing of a two-generation pedigree (two parents and 104 F1 offspring) of the amphioxus Branchiostoma floridae . Using parental assemblies generated by Platanus-allee as a custom reference for read alignment, we tracked inheritance patterns in offspring and phased contig-level haplotypes in parents, allowing us to detect recombination events. We identified 2,329 paternal and 2,288 maternal COs, yielding recombination rates of 4.57 cM/Mb and 4.49 cM/Mb, respectively. We found CO coldspots spanning >140 Mb in each parent and these are likely associated with large-scale heterozygous inversions. CO rates were positively correlated with transposable element and gene density in both sexes, but showed weak or no correlation with GC content. We further identified ∼10,000 paternal and ∼5,800 maternal NCO events, predominantly shorter than 200 bp in tract length, and found evidence of GC-biased gene conversion. This work provides the first direct and genome-wide measurement of recombination in amphioxus and demonstrates how high heterozygosity, often considered a barrier, can be leveraged for fine-scale recombination mapping. Our findings illuminate conserved and divergent features of recombination in chordates and establish a framework for studying recombination in other highly heterozygous organisms.