Identification and analysis of weak linear banding patterns of fish chromosomes with a computer-based densitometric method.

Identification of individual chromosomes by banding patterns is an essential step for physical genetic mapping of fish and other species where chromosomes are numerous and similar in size. Many techniques, such as Giemsa banding, reverse banding and restriction enzyme banding can generate well-defined patterns on mammalian chromosomes. However, these linear banding techniques are not useful with fish species, presumably due to structural homogeneity of fish chromosomes (4). Efforts have been made to produce banding patterns on fish chromosomes with techniques such as replication banding, in which DNA is labeled chemically during replication (10). However, the resultant patterns are difficult to analyze with the naked eye because distinct borders do not exist between dark and light bands, thus making these observations subjective and labor-intensive. Computer-assisted methods have been utilized to automate karyotyping and to examine subtle differences in chromosome size (2,8). Use of densitometry-based techniques has been reported in the field of molecular biology for quantitation of radiographic assays, electrophoretic bands (3,6) and DNA hybridization signals on chromosomes (7). Little information exists on application of densitometry to karyotyping or numerical analysis of chromosomal bands, especially for problematic groups such as fish. We report in this study, a computer-based densitometric method for analysis of weak banding patterns, thus providing a fast and objective solution to this problem. Pro-metaphase chromosomes were prepared from cultured leukocytes of channel catfish (Ictalurus punctatus) with standard fluorouracil-bromodeoxyuridine replication banding procedures and stained by fluorochrome plus Giemsa (5,10). Images of the chromosomes were captured directly with a 24bit video capture board (Imaging Technology, Bedford, MA, USA) at 1000× magnification from a Microphot-SA Light Microscope (Nikon, Melville, NY, USA) equipped with a high-resolution RGB color video camera (Model A206A; Microimage Video Systems, Boyertown, PA, USA). The Optimas computer software package (Bioscan, Edmonds, WA, USA), a Windows-based application, was used for processing of captured images. The linear measurement tool was used to draw a line along the long axis of individual chromosomes. By using this line as a transect, length and luminance of chromosomes were measured simultaneously. In this program, the line transect can be analyzed as 1–4096 sample units (segments), and a luminance value can be acquired from each. The number of units used varied with chromosome size and was standardized in preliminary trials. In channel catfish, chromosomes were divided into 32 sample units per 1% of relative length, which was calculated using the following formula: