Single-Gene Detection and Karyotyping Using Small-Target Fluorescence in Situ Hybridization on Maize Somatic Chromosomes

Abstract

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.

A Transgenomic Cytogenetic Sorghum (Sorghum propinquum) Bacterial Artificial Chromosome Fluorescencein SituHybridization Map of Maize (Zea maysL.) Pachytene Chromosome 9, Evidence for Regions of Genome Hyperexpansion

“…Even so, the linkage maps are based on recombination frequencies that vary widely in relation to physical distances (Anderson et al , 2004Wang et al 2006), and the FPC physical maps may not accurately predict the physical distance between the ends of adjacent contigs. Even as the maize genome sequence approaches completion, cytogenetic tools will remain useful for evaluating the variation between different species, subspecies, and cultivars within the genus Zea (White and Doebley 1998;Liu et al 2003;Kato et al 2004;Buckler et al 2006;Lamb and Birchler 2006;Lamb et al 2007).…”

“…The development of cytogenetic FISH maps of maize has progressed from mapping repeat sequences such as knobs, centromeres, and telomeres to mapping RFLP markers, single-copy genes, and individual transposons on mitotic and meiotic chromosomes (Shen et al 1987;Coe 1994;Chen et al 2000;Sadder et al 2000;Weber 2001, 2002;Koumbaris and Bass 2003;Kato et al 2004Kato et al , 2005Wang et al 2006;Lamb et al 2007). FISH mapping of pachytene chromosomes has proved to be useful for many other plant species such as tomato (Zhong et al 1996a,b;Peterson et al 1999), potato , Arabidopsis (Fransz et al 1996;Lysak et al 2001), Medicago (Kulikova et al 2001), rice (Cheng et al 2001a(Cheng et al ,b, 2002, sorghum (Islam-Faridi et al 2002;Kim et al 2005a,b), Brassica (Howell et al 2002), and soybean (Walling et al 2006).…”

Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat

“…For genomes enriched with repeats, a repeat-free probe for a particular genic region either can be produced from cDNA or developed from genomic DNA by sequence analysis and PCR amplification of the repeat-free region, and used in direct FISH as was shown in maize (Wang et al 2006;Lamb et al 2007;Danilova and Birchler 2008) and barley (Ma et al 2010). In direct FISH, fluorochromes are incorporated directly into DNA probes and the procedure does not need detection step.…”

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

“…Oligo probes have allowed the visualization of single-copy viral DNA as well as individual mRNA molecules using branched DNA signal amplification (27) or a handful to a few dozen short oligo probes (26,28), and, by targeting blocks of repetitive sequences as a strategy to amplify signal, enabled the first FISHbased genome-wide RNAi screen (29). Oligo probes have also been generated directly from genomic DNA using parallel PCR reactions (30,31). However, the high cost of synthesizing oligo probes has limited their use.…”