The longitudinal banding patterns obtained from each of the techniques described here differ from each other yet each provide reliable reference points across the chromosome in relation to the centromere and telomeres. The light and dark bands delineate regions providing a visual karyotypic map that can be used to describe the approximate location of mapped genes and their relative physical distance from other loci on the same chromosome. Breaks and reattachment points of chromosomal rearrangements can be described in relation to band location. Comparison of banding patterns between related species or their hybrids can suggest the possible types of rearrangements leading to chromosomal differences.

Objective: 

The objective of this paper is to outline recommendations concurred in by participants at the Guelph meeting. A standardized banding map is necessary for construction of adequate physical maps that integrate cytogenetic, molecular and classical mapping activity. It is recommended that general guidelines presented here be applied to other avian species. This will be particularly useful for comparative studies.

Materials and Methods

The primary fibroblast cell lines used to obtain chromosomes used in Figs. 1(a) and 2(a) were isolated from 9-day-old chicken embryos by trypsinization. Cultures were synchronized as described by Ladjali et al. (1995) with a double thymidine block during S phase in order to increase the yield of metaphase and early metaphase cells. To obtain the RBG-FPG bands [R-bands obtained incorporating bromodeoxyuridine (BrdU) followed by fluorochrome-photolysis and Geimsa stain], BrdU (Sigma; final concentration 10 mg/ml) was added to the cell culture as previously described (Romagnano and Richer 1984; Viegas-Péquignot et al., 1989; Schmid et al., 1989; Ladjali et al., 1995). The slides were incubated in Hoechst 33258 (1 mg/100 ml) for 15 to 20 min at room temperature. The slides were washed and placed in fresh 2X SSC, exposed to UV light for 90 min at a distance of 15 cm, then stained for 6 - 10 min in 6% Giemsa solution.

GTG-banding (G-bands obtained with trypsin and Giemsa) was done by a modification of Seabright (1971). Briefly, 3 to 10-day-old slides were incubated for 10 s in a fresh trypsin solution (final concentration 0.25%). The preparations were washed twice in phosphate buffer solution and stained for 10 min in 6% Giemsa solution (pH = 6.8).

Results and discussion

The emphasis at the Guelph meeting was to establish landmarks on the larger chicken chromosomes and to identify some major bands. The following points were agreed on:

1. There are 78 chromosomes in the chicken; 38 pair of autosomes and the Z and W sex chromosomes. ISCN (1978) will be the basis for chicken chromosome nomenclature. Chromosomes will be numbered consecutively based on size, with the largest being chromosome 1. The Z and W chromosomes will not be numbered.

3. G-band landmarks are the guiding landmarks and will be used as the standard. Those presented here have been modified from previous publications mainly due to improvements in procedures. All other bands are tentative pending verification. The banding map is a dynamic map, and as technologies change, refinements in the map are expected. The detailed G-banding map will be revised, but the landmarks as suggested in this paper should be retained for consistency.

The availability of a wide array of fluorescent reporter molecules suitable for fluorescence microscopy as well as the success of chromosomal gene localization using fluorescent in situ hybridization (FISH) technology has resulted in the development of karyotype standards based on BrdU incorporation during DNA replication with fluorescent staining. This allows simultaneous identification of fluorescent bands along a chromosome and the gene FISH signals, thereby allowing gene assignments to specific bands within a chromosome. When FISH is the preferred method for gene mapping, researchers are encouraged to use the RBG standard banding pattern. However, when using a fluorescent compound instead of Giemsa to stain BrdU treated chromosomes, bands will not be markedly differentiated as seen on Giemsa stained chromosomes. Due to this variation in resolution that was observed when comparing published karyotypes produced by different laboratories, some differences in banding patterns when comparing Giemsa and fluorescent stained karyotypes could not be resolved by the standardization committee. Readers are referred to Ponce de Leon et al. (1992) when RBP banding (Reverse bands by BrdU using propidium iodide) is used for gene mapping.

4. R-bands are presented, however these are tentative. R-bands are not exactly equivalent to G-bands, and different procedures and laboratory environments will produce slightly different maps. R-band maps will need further refinement and standardization.

5. In the near-metacentric Z chromosome, the large heterochromatic light staining G-band region marks the telomeric end of the long (q) arm. This is the large dark staining C-band region.

6. Q-bands and C-bands were considered. While Q-bands are useful for some studies, they are too variable for mapping purposes. C-bands are not consistent enough in the chicken genome to be useful. The exceptions are the heterochromatic region on the long (q) arm of the Z chromosome (5. above) and the W chromosome, which stains as a large dark object after C-band treatment.

7. It is difficult to establish with certainty which end of the chromosome is the centromeric end and which is the telomeric end on chromosomes smaller than number 8. The committee decided that techniques at that time would not allow an unequivocal assignment of centromeres, which is needed to assign band numbers. Newer technology will need to be used to develop maps of the smaller chromosomes. For instance, microchromosomes can be identified by specific molecular probes (Fillon et al., 1998).

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