Chromosomal detection of simple sequence repeats (SSRs) using nondenaturing FISH (ND-FISH)

Abstract

Simple Sequence Repeats (SSRs) are known to be scattered and present in high number in eukaryotic genomes. We demonstrate that dye-labeled oligodeoxyribonucleotides with repeated mono-, di-, tri, or tetranucleotide motifs (15-20 nucleotides in length) have an unexpected ability to recognize SSR target sequences in non-denatured chromosomes. The results show that all these probes are able to invade chromosomes, independent of the size of the repeat motif, their nucleotide sequence, or their ability to form alternative B-DNA structures such as triplex DNA. This novel and remarkable property of binding SSR oligonucleotides to duplex DNA targets permitted the development of a non-denaturing fluorescence in situ hybridization method that quickly and efficiently detects SSR-enriched chromosome regions in mitotic, meiotic, and polytene chromosome spreads of different model organisms. These results have implications for genome analysis and for investigating the roles of SSRs in chromosome structure and function.

Structural rearrangements detected in newly-formed hexaploid tritordeum after three sequential FISH experiments with repetitive DNA sequences

“…Therefore, the use of SSR probes in FISH experiments for the study of cereals could provide new insights about genomic restructuring among allopolyploids (Carvalho et al 2013) but also among newly-formed allopolyploids and respective parental species, contributing for the study of genomes evolution. According to Cuadrado and Jouve (2010), ND-FISH technique is suitable for SSR probes, and once it does not involves denaturing. The chromosome morphology is well maintained, being important for successive reprobing on the same slide.…”

“…The SSR (AG) 10 probe was used alone following the ND-FISH protocol described by Cuadrado and Jouve (2010). The genomic DNA from H. chilense and 45S rDNA sequence -pTa71 (Gerlach and Bedbrook 1979) were labelled with biotin and digoxigenin, respectively, using the Nick Translation kit (Roche Applied Science).…”

“…ND-FISH technique constitutes one variant of the conventional FISH which does not involve the denaturation of chromosomal DNA, is more cost-effective, less laborious and faster than conventional FISH, once the hybridization and the posthybridization washes could be performed in one single day (Cuadrado and Jouve 2010). These authors considered that ND-FISH is more suitable for SSR probes and when the chromosome spreads have to be reprobed several times.…”

Molecular Markers to Access Genetic Diversity of Castor Bean: Current Status and Prospects for Breeding Purposes

“…In this case, cytogenetic tools as fluorescent in situ hybridization (FISH) with SSR-like probes in mitotic chromosomes may be helpful in characterizing distribution and polymorphisms of large microsatellite repeats along the chromosome set (Cuadrado & Jouve, 2010). Cuadrado & Jouve (2007), for instance, analyzing the distribution pattern of trinucleotide repeats in barley (Hordeum vulgare) chromosomes by means of FISH, observed a preference of these large microsatellite clusters for heterochromatic regions, except for the ACT-based probe.…”

Chromosomal Evolution in the <b><i>Amolops mantzorum</i></b> Species Group (Ranidae; Anura) Narrated by Repetitive DNAs

“…FISH was performed using the procedure of Cuadrado and Jouve [2010] but with chromosome denaturation. After removal from the freezer, the slides were air-dried at room temperature for 1 h and then placed in an oven at 55 ° C for 1.5 h. Subsequently, the slides were denatured in a solution of 70% formamide, 10% 20× SSC at 72 ° C for 3 min.…”