Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron...

Fluorescence in situ hybridization (FISH) is a basic tool that is widely used in cytogenetic research. The detection efficiency of conventional FISH is limited due to its time-consuming nature. Oligonucleotide (oligo) probes with fluorescent labels have been applied in non-denaturing FISH (ND-FISH) assays, which greatly streamline experimental processes and save costs and time. Agropyron cristatum, which contains one basic genome, "P," is a vital wild relative for wheat improvement. However, oligo probes for detecting P-genome chromosomes based on ND-FISH assays have not been reported. In this study, according to the distribution of transposable elements (TEs) in Triticeae genomes, 94 oligo probes were designed based on three types of A. cristatum sequences. ND-FISH validation showed that 12 single oligo probes generated a stable and obvious hybridization signal on whole P chromosomes in the wheat background. To improve signal intensity, mixed probes (Oligo-pAc) were prepared by using the 12 successful probes and validated in the diploid accession A. cristatum Z1842, a small segmental translocation line and six allopolyploid wild relatives containing the P genome. The signals of Oligo-pAc covered the entire chromosomes of A. cristatum and were more intense than those of single probes. The results indicate that Oligo-pAc can replace conventional genomic in situ hybridization (GISH) probes to identify P chromosomes or segments in non-P-genome backgrounds. Finally, we provide a rapid and efficient method specifically for detecting P chromosomes in wheat backgrounds by combining the Oligo-pAc probe with the Oligo-pSc119.2-1 and Oligo-pTa535-1 probes, which can replace conventional sequential GISH/FISH assays. Altogether, we developed a set of oligo probes based on the ND-FISH assays to identify P-genome chromosomes, which can promote utilization of A. cristatum in wheat improvement programs.