Wheat (Triticum aestivum L.) is rich in tandem repeats, and this is helpful in studying its karyotypic evolution. Some tandem repeats have not been assembled into the wheat genome sequence. Alignment using the blastn tool in the B2DSC web server indicated that the genomic sequence of 5B chromosome (IWGSC RefSeq v2.1) does not contain the tandem repeat pTa-275, and the tandem repeat (GA)₂₆ distributed throughout the whole 5B chromosome. The nondenaturing fluorescence in situ hybridization (ND-FISH) using the oligonucleotide (oligo) probes derived from pTa-275 and (GA)₂₆ indicated that one signal band of pTa-275 and two signal bands of (GA)₂₆ appeared on the 5B chromosome of Chinese Spring wheat, indicating the aggregative distribution patterns of the two kinds of tandem repeats. Single-copy FISH indicated that the clustering region of pTa-275 and the two clustering regions of (GA)₂₆ were located in ~160–201 Mb, ~153–157 Mb, and ~201–234 Mb intervals, respectively. Using ND-FISH and single-copy FISH technologies, the translocation breakpoint on the 5BS portion of the translocation T7BS.7BL-5BS, which exists widely in north-western European wheat cultivars, was located in the region from 157,749,421 bp to 158,555,080 bp (~0.8 Mb), and this region mainly contains retrotransposons, and no gene was found. The clustering regions of two kinds of tandem repeats on wheat chromosome 5B were determined and this will be helpful to improve the future sequence assembly of this chromosome. The sequence characteristics of the translocation breakpoint on the translocation T7BS.7BL-5BS obtained in this study are helpful to understand the mechanism of wheat chromosome translocation.

Single-copy FISH analysis is a useful tool to physically locate a given sequence on chromosome. Centromeric single-copy sequences can be used to locate the position of centromere and disclose the subtle differences among different centromeres. Nine centromeric single-copy sequences 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R2, 6R3, and 7R1 were cloned from Kustro (Secale cereale L.). FISH analysis using these sequences as probes indicated that the signals of 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R1, 6R2, and 7R1 were located in the centromeric regions of rye 1R, 3R, 4R, 4R, 5R, 5R, 6R, 6R, and 7R chromosomes, respectively. In addition, for each of the centromeric single-copy sequences, high sequence similarity was observed among different Secale species. Combined with rye genomic sequence, single-copy FISH analysis indicated that the 1BL.1RS translocations in wheat cultivar CN17 and wheat line 20T363-4 contained the centromeric segment of 1R chromosome from 349,498,361 to 349,501,266 bp, and the 1BL.1RS translocations in the other two wheat cultivars did not contain this segment. The nine sequences are useful in determining the centromere location on rye chromosomes, and they have the potential to disclose the accurate structural differences of centromeres among the wheat-rye centric fusion translocation chromosomes; therefore, more centromeric single-copy sequences are needed.