Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis

Citrus sinensis is the most cultivated and economically valuable Citrus species in the world, whose genome has been assembled by three generation sequencing. However, chromosome recognition remains a problem due to the small size of chromosomes, and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome. We employed fluorescence in situ hybridization (FISH) with nine chromosome painting probes, thirty oligo pools and eight repetitive sequences to visualize 18 chromosomes. Then we developed an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3 (CMA) staining. By this approach, we constructed a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions, which enable us to visualize significant differences between homologous chromosomes. Based on the map, we located several highly repetitive sequences on chromosomes, and estimated sizes and copy numbers of each site. In particular, we discovered the translocation regions of chromosomes 4 and 9 in C. sinensis 'Valencia'. The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.