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Advances in sequencing technologies have aided the discovery of millions of genome-wide
DNA
polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) which are an invaluable resource for marker-assisted breeding. Presently available bioinformatics tools assist the discovery of polymorphisms between target genotypes and the reference genome for a range of species. The discovery of polymorphisms between two genotypes within a breeding program is complicated by several factors such as bias in the number of reads from each genotype and residual heterozygosity within each genotype. In this chapter, we describe a novel approach where polymorphisms between a pair of genotypes are discovered from whole-genome re-sequencing data.