Abstract
Genomic In Situ Hybridization (GISH) is a powerful tool to identify and to quantify genomic constituents in allopolyploids, and is mainly based on hybridization of highly and moderate repetitive sequences. In animals, as opposed to plants, GISH has not been widely used in part because there are technical problems in obtaining informative results. Using the allopolyploid Squalius alburnoides Steindachner, 1866 fish complex as a model system, we succeeded in overcoming methodological constraints when dealing with parental species with a small genome size. This hybridogenetic complex has biotypes with different genome compositions and ploidy levels, but parental chromosomes are small, morphologically very similar and therefore cannot be distinguished by conventional cytogenetic approaches. Specimens have a small genome (C-value1.2 pg) with a low level of highly and moderate repetitive sequences, mainly located at pericentromeric chromosome regions. Since it is well known that probe annealing depends on probe concentration and hybridization time to obtain uniform hybridization signals along the chromosome arms, we progressively increased the amount of labeled probes from 100ng up to 1µg and the incubation time from overnight up to 5 days. We also made other smaller improvements. Results showed a clear enhancement of signals with respect to previous data, allowing an accurate and reproducible assignment of the parental genomes in both diploid and triploid fish.It was thus evidenced that high probes’ concentrations and long incubation time are the key to obtain, without extra image editing, uniform and reliable hybridization signals in metaphase chromosomes of animal hybrids from species with small genome size.
Highlights
Genomic In Situ Hybridization (GISH) was developed by Schwarzacher et al (1989) to identify parental chromosomes in Hordeum chilense Roemer et Schultes x Secale africanum Stapf hybrid plants where classical karyotyping and/or chromosome banding were unable to detect genomic differences
It is possible to distinguish parental chromosomes in interspecific hybrids using GISH if the parental genomes are divergent and the hybridization is relatively recent (Markova and Vyskot 2009)
The results showed non uniform, faint and spotted hybridization signals, mainly at pericentromeric chromosome regions and rDNA clusters as well as in some heterochromatic regions (Fig. 1a–d)
Summary
Genomic In Situ Hybridization (GISH) was developed by Schwarzacher et al (1989) to identify parental chromosomes in Hordeum chilense Roemer et Schultes x Secale africanum Stapf hybrid plants where classical karyotyping and/or chromosome banding were unable to detect genomic (chromosomal) differences. This technique uses labeled total genomic DNA as probes to recognize a genome in chromosome preparations of hybrid individuals. GISH uses labeled total genomic DNA (gDNA) as the probe in in situ hybridization experiments together with sheared unlabeled whole genomic DNA, usually from the other parental species (but see Markova and Vyskot 2009) as blocking DNA. It is possible to distinguish parental chromosomes in interspecific hybrids using GISH if the parental genomes are divergent and the hybridization is relatively recent (Markova and Vyskot 2009)
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