Haplotype-specific chromosome painting provides insights into the chromosomal characteristics in self-duplicating autotetraploid sugarcane

Whole genome duplication (WGD) has long been recognized as an important driving force in plant evolution and diversification. Cytogenetic studies on allopolyploids formed by synthetic and natural WGD have reported small and large-scale changes, including structural and numerical chromosome rearrangements. However, the high similarity of homologous copies makes it difficult to find robust DNA probes for investigating whether each homologous copy undergoes chromosomal aberrations after whole-genome doubling in autopolyploid. In this study, we discovered and characterized a spontaneously doubling autotetraploid sugarcane AP85–441, which provided an opportunity for studying the evolutionary fate of chromosomes in autopolyploids after WGD. Then, a set of haplotype-specific oligo-fluorescence in situ hybridization (FISH) probes was developed based on the autotetraploid sugarcane AP85–441 genome. These probes can be used to unambiguously identify chromosome 8 homologous copies 8A, 8B, 8C, and 8D in a cell. Interestingly, a haplotype genome assembly error (approximately 12.26 Mb) was found between 8A and 8C homologous copies by haplotype-specific oligo-FISH. The haplotype-resolved karyotype was also established for the first time. By comparative FISH assay, no chromosomal aberrations were observed between AP85–441 and spontaneously doubled AP85–441 at the cytogenetic level, which suggested that the duplication of each homologous chromosome copy was strictly regulated in autotetraploid sugarcane following WGD. This study successfully applied the haplotype-specific FISH technology to autotetraploid sugarcane for the first time, and further application may bring us deep insights into the genetic mechanism of 2 n + n chromosome transmission associated with accelerated the restoration of a high sugar content in sugarcane breeding.