An introduction is given to the literature concerning methods and objectives for cytometric DNA analysis of plant nuclei. This area has gained relevance with applications in plant breeding and seed production industries, where laboratories unfamiliar with cytometry are adopting the method. An extensive graphical guide to interpreting DNA histograms and their problems is given. Conversely, cytometry laboratories unfamiliar with plant sciences will find herein a guide, and references, to adapt their methods to plant material. A table of 2C values reassessed by flow cytometry for 70 plant species, plus the genome composition (GC%) in many instances, is also included.

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A novel procedure for calculating basepair frequencies in whole genomes is reported. This has been developed during a study of the role of heterochromatin in microevolution. Closely related species of the Crepis praemorsa complex have similar karyotypes but for their heterochromatin. The changes in relative AT frequency between species have been attributed to heterochromatin sequences by in situ banding of chromosomes with two base-specific fluorochromes. The absolute genome size of species, measured by cytofluorometry, correlated positively with increased karyotypic heterochromatin, as did the proportion of AT bases in the DNA. However, the determination of base content has called for a curvilinear interpretation of data obtained with two base-specific fluorochromes (bisbenzimide Hoechst 33342 and mithramycin), in contrast to the commonly assumed but erroneous direct relationship between fluorescence intensity and base content. Essentially, the fluorochromes' requirements for a sequence of certain basepairs lead to the notion of Coefficients of Overspecificity: the result is a simple formula for calculating the AT proportion in a genome relative to a reference species from cytometric data, taking account of ligand binding statistics. These statistics and probabilities of oligonucleotide binding are essentially the same. Key terms: DNA amount, AT%, mithramycin, bisbenzimide Hoechst, ethidium bromide, fluorochrome banding, Crepis praemorsa complex Constitutive heterochromatin is a particular structure of eukaryote chromatin, differing from the euchromatin by its replication and condensation, frequently containing highly repeated sequences (1). It is revealed by several staining procedures (13) and may arise by transformation of euchromatin andlor by multiplication of repeated sequences (23). Addition of supplementary heterochromatic sequences has nucleotypic effects such as increase of cell cycle length or other phenomena linked to nuclear organization (20). The degree of multiplication of, for instance, a GC-rich region may be sufficient to produce a significant change in the measure of overall genomic GC. Results notably from (30) suggest that modification of heterochromatin is a rapid process relative to biological evolution: in closely related species one would therefore expect to find a close link between quantity and type of heterochromatin and quantity and composition of DNA.We have used fluorometric procedures to assess genome size and GC content in a complex of closely related plant species of Crepis, modifying the classical and often erroneous interpretation of fluorescence ratios to a n interpretation based upon binding statistics for base-specific dyes. GC-or AT-rich sequences were also sought at the chromosome level by f luorochromebanding.

The genome size (1C value) and base composition of 14 ecotypes of two species of tetraploid and diploid Medicago have been assessed by flow cytometry. These parameters vary both between and within species. The diploid annual Medicago truncatula Gaertn. had the smallest genome of the group studied (which also covered M. sativa L. subsp. sativa, M. sativa L. subsp. caerulea (Less. ex Ledeb.) Schmalh., M. sativa L. subsp. quasifalcata Sinsk., M. sativa L. subsp. x varia (Martyn) Arcangeli; however, its ecotypes revealed substantial intraspecific variation. The smallest M. truncatula genome observed was ecotype 108-1 with 1C = 0.49 pg and 38.1% GC and the largest was Jemalong with 1C = 0.57 pg and 38.6% GC. The degree of polysomaty in these Medicago was low, although in some tissues the frequency of cells with 4C nuclei reached 50%.