Genotyping-By-Sequencing libraries were prepared following the protocol by Poland et al. (2012). Libraries were sequenced by Illumina NextSeq 550 mid-output using 150 base-pairs single-end kits. Reads were mapped to a “combined” genome containing the Zavitan WEW_v2.0 genome (Zhu et al., 2019) and the Svevo.v1 genome (Maccaferri et al., 2019) using bwa-mem (Li, 2013). Mapped reads were converted to binary alignment map (BAM) format and filtered for high quality (>30), uniquely mapped and perfect matched using SAMtools package (Li et al., 2009). Zavitan and Svevo-specific reads served to build each “combined” genome. We found an average of 32,000 to 64,000 markers per chromosome, namely parent-specific reads. Each pair of chromosomes was divided into identical number of ∼1 Mb bins and the number of filtered reads was calculated for each bin using BEDtools (Quinlan and Hall, 2010). For each pair of matching bins (from Zavitan and/or Svevo) the number of mapped reads was summed together. For each bin, the ratio between Zavitan reads and Svevo reads was calculated. Each bin was then re-calculated as the mean ratio of the surrounding 15 bins. A bin was genotyped as homozygous if the calculated ratio was higher than 0.9 (Zavitan) or lower than 0.1 (Svevo), otherwise it was considered as heterozygous. Bins with less than 10 reads were ignored. COs were assigned to regions where bins changed from one genotype to another. Double COs were ignored if the distance between them was less than 8 Mb for subtelomeric regions or less than 70 Mb for pericentric regions. We applied this analysis on libraries of Zavitan and Svevo as well. Between 2 and 3% of the Bins were not consistent with parental genotypes and were removed from the progeny analysis.

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