A number of individuals with aberrant Y chromosomes have been tested for the presence of Y-chromosome-specific reiterated DNA. These studies locate Y-chromosome-specific reiterated sequences on the long arm of the Y chromosome. Correlation with phenotype and other known Y chromosome markers establish that the Y-chromosome-specific reiterated DNA discussed here has no evident role in male determination.

Flow cytometry can be used to obtain high-resolution estimates of nuclear DNA content (8,12,27). Much of the work in this area has been confined to clinical studies in the human, but the technology has been extended to organisms such as pocket gophers (241, triploid trout ( 2 3 , side-necked turtles (61, sex-reversed horses (16), and oysters (1).In these and similar studies, the DNA content of target cells is quantified relative to a standard DNA content in cells from a reference species (13,14,28). Standards may be used as internal references, when target cells and reference cells are mixed together and assayed simultaneously, or as external references, when target cells and cells from the reference species are analyzed independently. When external references are used, the flow cytometer must be checked for equilibration between analyses.The standard can be used for several calculations. The standard can be assigned a known DNA mass, against which picogram quantities of nuclear DNA from target cells can be estimated directly (15). Alternatively, a standard could be used as an internal reference in the analysis of individual samples, following which the reference is cancelled during estimation of a DNA Index. An internal reference would also be cancelled during the calculation of picogram quantities of nuclear DNA relative to a separate standard that has a known DNA mass (and a known relationship to the internal reference). These last two calculations do not require knowledge of the precise DNA mass of the internal reference.Because the use of reference standards provides a relative measure of DNA content, it is crucial that the reference cells and the target cells have a similar DNA content to minimize possible zero shift error (28) and, in the case of a n internal reference, that the values for the reference and target cells do not overlap.In order to gather information relating to the selection of reference standards for use in flow cytometry, we have surveyed a wide spectrum of taxa representing each of the major vertebrate classes. Here we present the results of our survey in 45 species. These species were selected because of their broad distribution and general accessibility, and because they provide an array of fluorescence peaks spanning the range of DNA masses that might be encountered in comparative studies. ~~~

We measured genome size (nuclear DNA content) by fluorescence flow cytometry in 55 species of birds representing 12 different orders. Similar studies were performed in approximately 100 species by laboratories using absorption cytophotometry of Feulgen-stained nuclei. Although there have been apparent discrepancies in the assigned values for the species used as a reference, the values obtained in the different laboratories are generally in agreement. When the data are standardized in relation to a diploid (2C) value of 2.5 picograms (pg) of DNA for the domestic chicken (Gallus gallus domesticus), the mean for DNA content in 135 species representing 17 orders is 2.82 +/- 0.33 (SD) pg with a range of 2.0-3.8 pg. Thus the genome size of birds is the most conservative of any vertebrate class and, all values considered, is smaller and more uniform in size than previous estimates would indicate. This could be explained by a previously unexplored hypothesis: that the genome of birds has evolved from a small ancestral genome that was reduced before emergence of the protoavian.

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