In order to design simple PCR markers we aligned the sequence of chromosome 1A of “Svevo” and “Zavitan” and we screened for InDels (20–200 bp) which are easy to distinguish on a simple agarose electrophoresis gel. The InDels were detected by an in house developed pipeline that utilized public tools. Specially, initially alignment was done between Zavitan chromosome and the Svevo genome (160802_Svevo_v2_pseudomolecules.1.fasta) using the program NUCmer from MUMmer (version 3.23; parameters : -maxmatch -l 100 -c 500) (Kurtz et al., 2004). The output was analyzed with the program Assemblytics (parameter: 200)2. The bed output variants_between_alignments was filtered (using awk) to contain InDels that are between 20 and 200 bases long that align to chromosome 1A of Svevo. We found more than 2000 such InDels. Annotation of the InDel region was added using Homer script annotatePeaks.pl3. The 150 base sequence surrounding the InDel was extracted using bedtools getfasta4. In addition, to ensure that the certain sequence of Svevo does not have an homologous region in Svevo or and additional homologous region in Zavitan genome, blastn was run (version 2.5.0, parameters: -outfmt 7 -max_target_seqs 1) against the relevant genomes in which the InDel regions were masked by running bedtools program maskfasta.

We choose 12 deletions (in “Svevo” compared to “Zavitan”) spreading all along the chromosome. Primers were carefully designed for chromosome-specific amplification, namely sequences of both “Zavitan” and “Svevo” chromosome 1A, but not of the homoeologous chromosome 1B nor from paralogous loci (Supplementary Table 3). DNA was purified from the first or second leaves of seedlings using Nucleospin DNA Plant© kit (MACHEREY-NAGEL). PCR reactions were done in 96 plates in total volume of 15 μl using Hy-Taq ready mix© (Hy-Labs, Israel) and products were analyzed by gel electrophoresis.

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