Paralog-aware assembly and filtering strategies reveal minimal nucleotide variation on the macro germline-restricted chromosome of the zebra finch.

The germline-restricted chromosome (GRC) of passerines is a remarkable tissue-specific chromosome that accumulated paralogs of genes from the regular "A chromosomes" over millions of years, often amplified into dozens of gene copies. In addition to its repetitive content, typically uniparental inheritance, and lack of recombination, the GRC resembles non-recombining sex chromosomes and some B chromosomes, for all of which assembly and single-nucleotide polymorphisms (SNPs) calling are difficult. Here, we first show that much of the Australian zebra finch macro-GRC can be assembled using accurate long reads. We then describe a paralog-aware Snakemake pipeline, ParaVar, to map short reads from the GRC to retrieve GRC regions suitable for haplotype-based analysis. ParaVar reliably calls hundreds of SNPs across the GRC, thereby providing an estimate of nucleotide diversity on the highly repetitive zebra finch macro-GRC. Our results show significantly lower nucleotide diversity (20- to 50-fold lower) on the GRC compared to the mitogenome and autosomes, and a strong phylogenetic discordance between the GRC and the mitochondrial genome. Beyond the contribution of background selection, our results suggest that a single GRC haplotype recently spread through the populations while jumping across matrilines via occasional paternal inheritance. We anticipate that our paralog-aware pipeline will be useful for SNP calling and population genetics analyses of repetitive GRCs, sex chromosomes, and B chromosomes.