S U M M A R Y Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus "Leghorn" nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229-235, 2010)

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showing DNA sequence fidelity for the tested SSR primers. In conclusion, the adopted procedure for in vitro CSD is reproducible for induction, regeneration and propagation of Coffea polyploids and potentially other shrubbery and woody species. In view of the novelty of this procedure to generate new germplasm, we show the key issues and the steps of the CSD procedure.

Karyotype and nuclear 2C-value data are considered important in taxonomic and evolutionary approaches in Coffea. Still, new methods are needed to further support such studies, especially to determine the progenitors of Coffea arabica. In this work, new cytogenetic and flow cytometry data were used to compare Coffea arabica, Coffea canephora and Coffea congensis. These data corroborate the hypothesis that C. canephora and C. congensis originated from a single ancestor, whose basic chromosome number was x = 11. In agreement with the observations of other authors, the karyotype and mean 2C-values confirm that C. arabica is a true allotetraploid originating from two diploid Coffea species with similar genomes. Although C. canephora and C. congensis have been considered potential progenitors of C. arabica, karyotype comparison revealed that only one of these species may be parental to C. arabica. These accurate cytogenetic and flow cytometry data contribute to expand our knowledge of the Coffea genome, as well as of possible progenitors of C. arabica.

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