Identification of Homologous Chromosomes Revealed Evolution at Single-Copy Gene Loci in Cucumis ×hytivus Chen and Kirkbride, a Nascent Allotetraploid Cucumis Species

Allopolyploidization lays foundation for the speciation of most plant species. However, the rapid genomic changes caused by initial hybridization and genome doubling are not easy to be traced in ancient allopolyploids due to millions of years of evolution. Newly synthesized allopolyploids provide good models to shed light on the underlying mechanism of plant speciation. Cucumis ×hytivus Chen and Kirkbride (2n=4x=38) is an allotetraploid Cucumis species derived from an inter-specific hybridization between cultivated cucumber C. sativus (2n=2x=14) and wild C. hystrix (2n=2x=24), followed by chromosome doubling. We performed sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using various types of probes to investigate distribution patterns and chromosomal changes in the first and latest generation (S₁₁) of allotetraploid Cucumis. All 19 homologous chromosome pairs were unambiguously distinguished by GISH using gDNA from two parents, combining with a BAC clone (457-F11) selected from BAC libraries of C. hystrix. FISH signals of 457-F11 BAC probe could be observed at centromeric regions of all 24 chromosomes of C. hystrix. Distribution of four major cucumber repeats, 45S and 5S rDNA was also investigated. The results showed that the positions of above probes were the same in both generations, indicating a stable subgenomic structure of C. ×hytivus. An in-depth FISH analysis was conducted using a set of 11 single-copy gene probes. At least three of them showed changes in signal position as compared to their parents, which may due to evolution of the allotetraploid genome. The karyotype constructed herein and identified chromosome-specific molecular probes would likely facilitate research in chromosome pairing, chromosome rearrangements, and gene dosage balance in the allotetraploid genome.