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The button mushroom (Agaricus bisporus) is one of the world's most cultivated mushroom species, but in spite of its economic importance generation of new cultivars by outbreeding is exceptional. Previous genetic analyses of the white bisporus variety, including all cultivars and most wild isolates revealed that crossing over frequencies are low, which might explain the lack of introducing novel traits into existing cultivars. By generating two high quality whole genome sequence assemblies (one de novo and the other by improving the existing reference genome) of the first commercial white hybrid Horst U1, a detailed study of the crossover (CO) landscape was initiated. Using a set of 626 SNPs in a haploid offspring of 139 single spore isolates and whole genome sequencing on a limited number of homo- and heterokaryotic single spore isolates, we precisely mapped all COs showing that they are almost exclusively restricted to regions of about 100kb at the chromosome ends. Most basidia of A. bisporus var. bisporus produce two spores and pair preferentially via non-sister nuclei. Combined with the COs restricted to the chromosome ends, these spores retain most of the heterozygosity of the parent thus explaining how present-day white cultivars are genetically so close to the first hybrid marketed in 1980. To our knowledge this is the first example of an organism which displays such specific CO landscape.

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