CMA/DAPI Banding of Plant Chromosomes.

Chromosome banding based on base-specific fluorochromes, mainly double staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI), has been widely used since the 1970s. This technique allows the differential staining of distinct types of heterochromatin. Afterward, the fluorochromes can be easily removed and leave the preparation ready for sequential procedures such as FISH or immunodetection. Interpretations of similar bands obtained with different techniques, however, merit certain caution. Here we present a detailed protocol for CMA/DAPI staining optimized for plant cytogenetics and call attention to the most common sources of misinterpretation of DAPI bands.

Cytogenetic analysis of two Coprophanaeus species (Scarabaeidae) revealing wide constitutive heterochromatin variability and the largest number of 45S rDNA sites among Coleoptera.

The Coleopterans of Scarabaeinae clade presents Coprophanaeus (Megaphanaeus) ensifer and C. (Coprophanaeus) cyanescens (Scarabaeidae) when they are studied cytogenetically by different techniques. The species present symmetric karyotypes, diploid number of 2n=20, and meta-submetacentric chromosomes. C. (M.) ensifer present an XY sex-determining mechanism and C. (C.) cyanescens an XY(p) parachute mechanism. Analysis of constitutive heterochromatin (CH) in the two species revealed the presence of diphasic autosomes, with log arm heterochromatics. Moreover, an additional heterochromatic block in four autosomal bivalents were observed in C. (M.) ensifer. CMA(3)/DA/DAPI fluorochrome staining detected CMA(3) positive heterochromatic blocks restricted to the sex chromosomes in C. (C.) cyanescens, whereas in C. (M.) ensifer CMA(3) positive pericentromeric blocks were present in all autosomes, in the Y chromosome and in the four additional heterochromatic blocks. DAPI staining was neutral in both species. Silver nitrate (AgNO(3)) staining was inefficient for the detection of the nucleolar organizer region (NORs), but showed affinity for the heterochromatic regions. Fluorescence in situ hybridization (FISH) revealed the presence of 45S rDNA sites in the terminal region of the three autosomal bivalents of C. (C.) cyanescens and in seven bivalents and the Y chromosome of C. (M.) ensifer. These results contribute to a better understanding of chromosome evolution in the genus Coprophanaeus, and demonstrate a wide CH variability and the largest number of ribosomal sites among Coleoptera.

Cytogenetic characterization of Eurysternus caribaeus (Coleoptera: Scarabaeidae): evidence of sex-autosome fusion and diploid number reduction prior to species dispersion.

Mitotic and meiotic chromosomes of several populations of Eurysternus caribaeus (Coleoptera: Scarabaeidae) were analysed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining and fluorescent in situ hybridization (FISH). All specimens showed 2n = 8 in their karyotypes, with a neo-XY sex system (Y is a submetacentric and X a metacentric) and three pairs of submetacentric autosomes. The analysis of constitutive heterochromatin (CH) revealed small blocks located in the centromeric region of all chromosomes which do not present positive staining under the fluorochromes CMA3 and DAPI. Silver nitrate staining revealed that the nucleolar organizer region (NORs) is associated with the sex chromosomes. The FISH technique revealed that rDNA sites in the X and Y are different in size. Data from different populations indicate that the diploid number reduction (2n = 8) observed in E. caribaeus is established and presumably has preceded the dispersion of this species. Moreover, this reduction occasioned the translocation of rDNA sites to the sex chromosomes, X and Y, an uncommon pattern in Scarabaeidae that was observed for the first time by the FISH in this work.