Genetic recombination is the major mechanism behind evolution and crop improvement. Recombination typically consists of the exchange of genetic material between homologous chromosomes through the formation of chiasmata and crossing over (CO). In wheat, proper CO and chiasmata formation is regulated by the Pairing homoeologous-1 (Ph-1) gene. However, homoeologous chromosome pairing can be promoted if the Ph1 locus is absent (i.e. ph1b deletion mutant). Recently, soft kernel durum wheat lines were developed through ph1b-mediated 5DS-5BS chromosomal translocation, resulting in several independent 5DS-5BS recombinant lines. However, the size of the translocation and the physical location of the translocation breakpoint were not determined for any of these lines. In the present study, the size of the 5DS-5BS translocation was first analyzed in a set of soft kernel durum genotypes derived from a single 5DS-5BS recombination line (Langdon 1-674) using Illumina’s 90k wheat single nucleotide polymorphism (SNP) array. Results of this analysis indicated a nearly reciprocal translocation, as the analyzed lines gained a 5DS segment of at least 24.4 Mbp and lost a 5BS segment of ~20.0 Mbp. A subset of the polymorphic SNPs was then selected and used to analyze the 5DS-5BS translocation in 12 other soft kernel durum wheat lines derived from four independent Langdon 5DS-5BS translocation lines, which included Langdon 1-674. The 5DS-5BS translocation breakpoint interval was subsequently delimited by chromosome walking and sequenced. Results of this analysis revealed that each of the analyzed lines lost a 5BS segment of ~ 20.7 bp and gained a 5DS segment of ~ 28.1 bp. Moreover, sequencing analysis identified the recombination breakpoint in a conserved 39 bp region within a predicted gene in each of the lines, suggesting the presence of a possible recombination hotspot. In order to accurately identify the 5DS-5BS translocation associated with the soft kernel trait, results of the sequencing analysis were further explored to develop a KASP marker specific for the translocation breakpoint. A subset of the soft kernel durum wheat lines was concurrently analyzed by genomic in situ hybridization (GISH). Results of the GISH analysis were in agreement with the molecular analysis and confirmed the presence of a unique

D-genome fragment in the soft kernel durum wheat lines. Results of this study provide a better understanding of the 5DS5BS chromosomal translocation associated with soft kernel texture in durum wheat and elucidates genetic recombination in wheat.