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.

Towards a FISH-based karyotype of Rosa L. (Rosaceae)

“…FISH-based markers have shown their effectiveness and ease-to-use. The modern methods of probe labeling and the application of directly labeled oligonucleotides make FISH-based chromosome identification a robust and fast procedure (Kato et al 2004, Fu et al 2015, Tang et al 2014, Cuadrado et al 2009). Up-to-date FISH based karyotyping was established for many plant species including wheat, maize, rice, soybean, common bean and others (Cheng et al 2001, Kato et al 2004, Findley et al 2010, Iwata-Otsubo et al 2015).…”

New Oligonucleotide Probes for ND-FISH Analysis to Identify Barley Chromosomes and to Investigate Polymorphisms of Wheat Chromosomes

“…The chromosome spreads of materials were prepared through the methods described by Han et al [20]. ND-FISH analysis was operated according to the methods described by Fu et al [9]. In addition, (AAG) 6 , Oligo-pSc119.2-1 and Oligo-pTa535-1 [9] were also used to help identify wheat and barley chromosomes.…”

“…ND-FISH analysis was operated according to the methods described by Fu et al [9]. In addition, (AAG) 6 , Oligo-pSc119.2-1 and Oligo-pTa535-1 [9] were also used to help identify wheat and barley chromosomes. Probe Oligo-pSc119.2-1 was 5′-end-labeled with TAMRA.…”

“…These oligonucleotide probes can replace the role of the repetitive sequences for FISH analysis of wheat and rye [8]. In addition, these oligonucleotides were shown to be suitable for non-denaturing fluorescence in situ hybridization (ND-FISH) analysis and the hybridization time was reduced to one hour [9,10]. Therefore, this technique provides a convenient way to use the oligonucleotide probes to analyze chromosomes of wheat and its relatives.…”

New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents

“…A new oligo-probe, Oligo-pSt122 (5 0 GGCT CACATT AGGGAAGAAT CGGTGAACAA AGAAAA-GACA AATTCACCGT ATAGAGCT 3 0 ) was synthesized and labeled with 5 0 6-carboxyfluorescein (FAM) based on high copy number of SLAF reads sequences. The protocol of non-denaturing FISH by the synthesized probes was described by Fu et al (2015). After the oligo-based FISH, the sequential Giemsa C-banding was done according to Yang et al (2009).…”