Peculiarity of the analysis of heterochromatic regions in small chromosomes of plants.

Abstract

Visual analysis of small chromosomes of plants is difficult in most cases, because the chromosomes are similar in morphology and banding patterns. The computer software applied to chromosome analysis extended the capabilities of karyotypic analysis of small-chromosome species, because precise mathematical parameters of chromosomes and chromosome bands became available [1‐4]. This approach made it possible to completely identify chromosomes of some small-chromosome species and compare their karyotypes with the use of C-banding, which is commonly accepted in plant chromosome analysis [3‐7]. However, standard C-banding of chromosome preparations is often ineffective, and the banding pattern does not enable chromosome identification and measurements. To elongate small chromosomes, DNA intercalators are usually used [5, 6, 8], which enhances the resolution of C-banding patterns, and, hence, the possibility of chromosome identification. However, variation in C-banding patterns increases as the chromosome lengths increase after treatment with DNA intercalators. At the same time, there are no exact principles determining the range of chromosome length enabling stable C-banding and accurate morphometry to ensure not only chromosome identification and construction of idiograms, but also the possibility of comparative cytogenetic analysis. In this study, we aimed to develop these criteria. We demonstrated that, in principle, genomic polymorphism of the small-chromosome plant species can be studied by means of morphometric chromosome analysis, and the basic rules of the latter were formulated. We studied changes in the sizes of heterochromatic regions in both large and small mitotic chromosomes during their shortening. Chromosome selection for further measurements was based on the following criteria: the average size in the karyotype, the presence of intercalary heterochromatic bands, the absence of the secondary constriction, and the simplest identification. We finally selected chromosomes of the fourth groups in the karyotypes of the Djau-Kabutac cultivar of barley ( Hordeum vulgare L.) [9] and large-flower flax ( Linum grandiflorum Desf.) [6]. We examined all chromosomes identifiable from C-banding patterns that did not overlap in the same preparation meristem from a single plant to exclude the possibility of individual C-banding polymorphism. Barley chromosome preparations were obtained using the standard technique described previously [9]. To obtain flax preparations, the intercalator 9-aminoacridine was used [6]. On every barley or flax metaphase plate, one of a pair of homologous chromosomes was analyzed morphometrically. Numerous preliminary measurements (at least 30) of the same barley or flax chromosome were made to determine the limits of error for the morphometry. Similar results were obtained with both types of chromosomes (0.056 μ m).