Haplotyping interspecific hybrids by dual alignment to both parental genomes.

Sequencing-based genotyping of heterozygous diploids requires sufficient depth to accurately call heterozygous genotypes. In interspecific hybrids, alignment of reads to both parental genomes simultaneously can generate haploid data, potentially eliminating the problem of heterozygosity. Two populations of interspecific hybrid rootstocks of walnut (Juglans) and pistachio (Pistacia) were genotyped using alignment to the maternal genome, paternal genome, and dual alignment to both genomes simultaneously. Downsampling was used to examine concordance of imputed genotype calls as a function of sequencing depth. Dual alignment resulted in datasets essentially free of heterozygous genotypes, simplifying the identification and removal of cross-contaminated samples. Concordance between full and downsampled genotype calls was always highest after dual alignment. Nearly all single nucleotide polymorphisms (SNPs) in dual alignment datasets were shared with the corresponding single-parent datasets, but 60%-90% of single-parent SNPs were private to that dataset. Private SNPs in single-parent datasets had higher rates of heterozygosity, lower levels of concordance, and were enriched in fixed differences between parental genomes ("homeo-SNPs") compared to shared SNPs in the same dataset. In multi-parental walnut hybrids, the paternal-aligned dataset was ineffective at resolving population structure in the maternal parent. Overall, the dual alignment strategy effectively produced phased, haploid data, increasing data quality and reducing cost.