Fluorescent labeling of plant chromosomes in suspension by FISH

Abstract

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

“…As pointed out by several authors [6], [23], [24], [25], [39], [40], [41], [42], [43], all previous attempts to directly translate FISH protocols into chromosomes in suspension have failed, due to many problems, such as chromosome clumping, paucity in suspension, poor hybridization pattern reproducibility, and loss of chromosome morphology. Here we present a straightforward wash-less method for fluorescence in situ hybridization of plant chromosomes in suspension (FISHIS), using synthetic, fluorescence-labeled DNA probes ( Figure 1 ) that quantitatively assess specific hybridization patterns, thus allowing for precise flow sorting of individual chromosomes to high purity ( Figure 2 ).…”

“…Many efforts have been made to apply the classic FISH labeling procedure to nuclei and chromosomes in suspension both in human [23] and plant samples [24], [25] with generally unsatisfactory results. FISH involves denaturation of the probe and target DNAs and annealing under stringent enough conditions to provide specific and reproducible hybridization.…”

Individual chromosome identification, chromosomal collinearity and genetic-physical integrated map in <i>Gossypium darwinii</i> and four D genome cotton species revealed by BAC-FISH

“…Therefore, a simplified and reproducible karyotype method for identifying individual chromosomes in plants is necessarily to be developed. Recently, fluorescence in situ hybridization (FISH), which is used to localize DNA probes and identify chromosomes or chromosomal segments, have been adapted successfully to identify the chromosome for many plant species, including rice, potato, cotton and so on (Cheng et al, 2001;Dong et al, 2000;Gan et al, 2011;Kim et al, 2002;Ma et al, 2005;Peng et al, 2012;Wang et al, 2007Wang et al, , 2008Zhang et al, 2004). This technique has been developed from highly repeated copies sequences probe to singlecopy probe (Desel et al, 2001;Zhu et al, 1999) and from single-color to multiple-color (Tang et al, 2009).…”

Recent advances in rice genome and chromosome structure research by fluorescence <i>in situ</i> hybridization (FISH)

“…19) , 20) Recently, flow cytometry and sorting studies of plant chromosomes have been carried out using suspensions of intact chromosomes, and individual chromosomes have been discriminated in cereals. 21) , 22) 5S rDNA, 17S rDNA, and centromeric DNA have been used as probes in FISH for rye and barley chromosomes in suspension to label the specific chromosomes. Bright signals were detected at the specific regions of interest on the chromosomes.…”