RAD-seq data of 5463 single-nucleotide polymorphism (SNP) markers from 275 full-sib individuals, without parents, were used as input into Lep-MAP2 (44). The RAD-seq data were generated from nextRAD technology by SNPsaurus (Oregon, USA) (note S10). To obtain genotype data, the RAD-seq data were mapped to the reference genome using BWA mem (45), and SAMtools (46) was used to produce sorted bam files of the read mappings. On the basis of read coverage (SAMtools depth), Z chromosomal regions were identified from the genome, and the sex of offspring was determined. Custom scripts (47) were used to produce genotype likelihoods (called posteriors in Lep-MAP) from the output of SAMtools mpileup.

The parental genotypes were inferred with the Lep-MAP2 ParentCall module using parameters “ZLimit=2 and ignoreParentOrder=1,” first calling Z markers and second calling the parental genotypes by ignoring which way the parents are informative (the parents were not genotyped, so we could not separate maternal and paternal markers at this stage). Scripts provided with Lep-MAP2 were used to produce a linkage file from the output of ParentCall, and all single-parent informative markers were converted to paternally informative markers by swapping parents, when necessary. Filtering by segregation distortion was performed using the Filtering module.

Following this, the SeparateChromosomes module was run on the linkage file, and 25 chromosomes were identified using a logarithm of the odds (LOD) score limit of 39. Then, the JoinSingles module was run twice to add more markers on the chromosomes with an LOD score limit of 20. Then, SepareteChromosomes was run again but only on markers informative on single parents with an LOD limit of 10 to separate paternally and maternally informative markers. Fifty-one linkage groups were found, and all were ordered using the OrderMarkers module. On the basis of likelihood improvement of marker ordering, paternal and maternal linkage groups were determined. This was possible as there is no recombination in females (achiasmatic meiosis), and thus the order of the markers does not improve likelihood on the female map. The markers on the corresponding maternal linkage groups were converted to maternally informative, and OrderMarkers was run on the resulting data twice for each of the 25 chromosomes (without allowing recombination in female). The final marker order was obtained as the order with the higher likelihood from the two runs.

A maternal marker map was constructed from a different family of P. napi in which a female from Abisko, Sweden, was crossed with a male from Catalonia, Spain. Genomic DNA libraries were constructed for the mother, father, and four offspring (two males and two females). RNA libraries were constructed for an additional six female and six male offspring. All sequencing was performed on an Illumina HiSeq 2500 platform using High Output mode, with PE 2 × 100-bp reads at SciLifeLab (Stockholm, Sweden). Both DNA and RNA reads were mapped to the genome assembly with bbmap using default settings. SAMtools was used to sort read mappings and merge them into an mpileup file (note S6). Variants were called with BCFtools (48) and filtered with VCFtools (49). Linkage between SNPs was assessed with PLINK (50). A custom script was used to assess marker density and determine sex-specific heterozygosity.

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