Oligonucleotide Probes for ND-FISH Analysis to Identify Rye and Wheat Chromosomes

Abstract

Genomic in situ hybridization (GISH) has been widely used to detect rye (Secale cereale L.) chromosomes in wheat (Triticum aestivum L.) introgression lines. The routine procedure of GISH using genomic DNA of rye as a probe is time-consuming and labor-intensive because of the preparation and labeling of genomic DNA of rye and denaturing of chromosomes and probes. In this study, new oligonucleotide probes Oligo-1162, Oligo-pSc200 and Oligo-pSc250 were developed. The three new probes can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) assays and replace genomic DNA of rye as a probe to discriminate rye chromosomes in wheat backgrounds. In addition, previously developed oligonucleotide probes Oligo-pSc119.2-1, Oligo-pSc119.2-2, Oligo-pTa535-1, Oligo-pTa535-2, Oligo-pTa71-2, Oligo-pAWRC.1 and Oligo-CCS1 can also be used for ND-FISH of wheat and rye. These probes have provided an easier, faster and more cost-effective method for the FISH analysis of wheat and hybrids derived from wheat × rye.

Figure 1

ND-FISH experiments on triticale chromosomes performing with Oligo-1162 as probe (red) showing the 14 rye-origin chromosomes of octoploid triticale MA (a) and octoploid triticale MK (b). Sequential ND-FISH and GISH experiments on octoploid triticale chromosomes performing with Oligo-1162 (red), Oligo-pSc200 (green), Oligo-pSc250 (green) and genomic DNA of rye (red) as probes showing the 14 rye-origin chromosomes of MA (c,d) and MK (e,f). Arrows indicate the rye-origin chromosomes. Chromosomes were counterstained with DAPI (blue). Scale bar 10 μm.

Figure 2

ND-FISH experiments on chromosomes of octoploid triticales and wheat-rye translocation lines performing with Oligo-1162 (red), Oligo-pSc119.2-1 (green), Oligo-pSc119.2-2 (green), Oligo-pTa535-1 (white) and Oligo-pTa535-2 (white) as probes. a Signals of Oligo-1162, Oligo-pSc119.2-1 and Oligo-pTa535-1 on chromosomes of octoploid triticale MA. b Signals of Oligo-1162, Oligo-pSc119.2-2 and Oligo-pTa535-2 on chromosomes of octoploid triticale MK. c Signals of Oligo-1162, Oligo-pSc119.2-1 and Oligo-pTa535-1 on chromosomes of wheat-rye 1BL·1RS translocation line 14T-78. d Signals of Oligo-1162, Oligo-pSc119.2-2 and Oligo-pTa535-2 on chromosomes of wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation line 14T-267. Chromosomes were counterstained with DAPI (blue). Scale bar 10 μm.

Figure 3

Sequential ND-FISH experiments on chromosomes of wheat-rye 1R disomic addition line 14T-71 performing with Oligo-1162 (green), Oligo-pTa71-2 (green), Oligo-pSc119.2-1 (green) and Oligo-pTa535-1 (red) as probes. a Oligo-1162 signals on chromosomes of line 14T-71. b Oligo-pTa71-2 signals on the same cell in (a). c Oligo-pSc119.2-1 and Oligo-pTa535-1 signals on the same cell in (a) and (b). Chromosomes were counterstained with DAPI (blue). Scale bar 10 μm.

Figure 4

ND-FISH experiments on chromosomes of octoploid triticale MA performing with Oligo-1162 (green), Oligo-pAWRC.1 (red) and Oligo-CCS1 (green) as probes. a Oligo-1162 and Oligo-pAWRC.1 signals on chromosomes of octoploid triticale MA. b Oligo-1162 and Oligo-CCS1 signals on chromosomes of octoploid triticale MA. Arrows indicate the rye-origin chromosomes. Chromosomes were counterstained with DAPI (blue). Scale bar 10 μm.