**1. Introduction**

In several plant groups, especially those with polyploid complexes as *Triticum* (the wheat genus, Poaceae), related species can be used as important sources of genes. In the tribe Triti‐ ceae as a whole, which comprises other important cereals as barley (*Hordeum vulgare*) and rye (*Secale cereale*), there are high rates of successful interspecific hybridization [1-2]. Due to the ease in obtaining these hybrids, plus the high amount of available information on the genomes of the species, the interspecific hybrids are potentially useful for the genetic im‐ provement of these crops [3-4]. Thus, the hybrids and their derivatives from breeding pro‐ grams can be analyzed by means of different approaches, aiming the full knowledge on the phenotypic constitution of the plant material for its subsequent utilization.

Many cytogenetic methods can be applied during the process of crop improvement, mainly regarding the characterization of chromosome types among accessions of a germplasm col‐ lection [5-6]. Since the discovery of the nucleic acid hybridization reaction by Hall & Spiegel‐ mann [7], and later by using fluorescent detection rather than radioactive isotopes [8], the fluorescent *in situ* hybridization (FISH) and its variations have been largely employed in karyotype characterization of plants [9]. The technique basically consists on the pairing of a given probe (a DNA or RNA fragment) with a specific sequence on the target genome, aim‐ ing to indicate its exact location in a chromosome.

When the objective is to distinguish parental chromosomes (or chromosome segments) in an interspecific hybridization or the distinct genomes of an allopolyploid, the entire genome of one parent should be labeled and used as probe [10]. In this case, the technique is called genom‐

© 2013 Patussi Brammer et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Patussi Brammer et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ic *in situ* hybridization (GISH). On the other hand, the genome of the second parent (unlabeled) is used as blocking DNA, aiming to avoid non-specific hybridizations due to the similarity of the two parental genomes. Thus, both parental genomes (the probe and the blocking DNA) must be used together in the same hybridization mixture. The proportion probe:blocking DNA should be adequate to avoid the detection of the second parent (see Fig. 1).

**Figure 1.** Main steps of the genomic *in situ* hybridization (GISH). (A) Direct and indirect probe labeling. (B) Fragmenta‐ tion of the blocking DNA. (C) Slide preparation. (D) Probe and blocking DNA denaturation in a hybridization mixture. (E) Addition of the hybridization mixture with the probe and the blocking DNA. (F) Denaturation of the chromosome DNA. (G) *In situ* hybridization of probe and blocking DNA in the target sequence of the chromosome. (H) Detection of the probe in the chromosome DNA of one parent, in an indirect labeling. (I) Chromosome DNA molecule of the sec‐ ond parent associated to the unlabeled blocking DNA. (J) Visualization of hybridization signals associated to a probe (green) in a fluorescence microscope. Unmarked chromosomes are visualized with a counter-staining (blue). When the probe labeling is direct, the detection step of the GISH can be excluded. The fluorochromes are the signaling mol‐ ecules and can be directly visualized in a fluorescence microscope with the appropriate filter. Santelmo Vasconcelos & Ana C. Brasileiro-Vidal.

The probe labeling can be either direct or indirect (Fig. 1A). In the direct labeling, the marked nucleotides are associated to fluorochromes, which can be directly visualized in a fluorescence microscope with the proper filter, after *in situ* hybridization procedures. On the other hand, in the indirect labeling, the marked nucleotides are associated to marker molecules (Fig. 1A), which cannot be visualized in microscopes. Thus, after *in situ* hybridization procedures, the la‐ beled probes are recognized by antibodies conjugated to fluorochromes, allowing for the probe detection and visualization (Fig. 1J).

The GISH has direct applications on the understanding of the genome evolution of poly‐ ploid hybrids, partial allopolyploids and recombinant inbred lines, as well as in detecting the amount of introgressed chromatin during the production of new lineages [11-13]. There‐ fore, the GISH has efficiently contributed for the analysis on the karyotypic stability of plant materials, indicating the best genotypes, and helping the assisted selection in different phas‐ es of crop improvement [9, 14].

Here we describe and discuss the main methodological steps of the GISH process as well as the importance of such an approach for the establishment of successful inbred lines, using as example hybrids between common wheat (*T. aestivum*, 2*n* = 6*x* = 42, AABBDD genome) and rye (*S. cereale*, 2*n* = 2*x* = 14, RR genome), hexaploid (2*n* = 6*x* = 42, AABBRR genome) and octo‐ ploid (2*n* = 8*x* = 56, AABBDDRR genomes) triticale lines and their derivatives. For this analy‐ sis, genomic DNA from rye was used as probe and wheat genomic DNA was used as blocking agent in a proportion of 1:10 (probe:blocking agent). Variations of the technique for other Triticeae species will also be discussed.
