We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.

Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues

The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication of a more comprehensive list is overdue. This paper lists absolute nuclear DNA amounts for 753 angiosperm species. The data were assembled primarily for reference purposes, and so the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists whom, it is anticipated, will be among its major users. The paper also reviews aspects of the history, nomenclature, methods, accuracy and problems of nuclear DNA estimation in angiosperms. No attempt is made to reconsider those aspects of nuclear DNA estimation which have been fully revised previously, although the bibliography of such aspects is given. Instead, the paper is intended as a source of basic information regarding the terminology, practice and limitations of nuclear DNA estimation, especially by Feulgen microdensitometry, as currently practiced.

Nuclear DNA Amounts in Angiosperms

Highly differentiated heteromorphic sex chromosomes were found in the karyotypes of Pyxicephalus adspersus (= Rana adspersa) and analysed with the various banding methods. The W chromosome is considerably smaller than the Z chromosome and consists to a great proportion of constitutive heterochromatin. The chromosome lengths and the DNA content of this species have the lowest values determined in the Ranidae to date. The nucleolus organizer regions in the chromosomes reveal a high frequency of inter-individual variations (duplications, triplications) and aberrations. In one female animal, a translocation between the W chromosome and a nucleolus organizer was identified. Preliminary results indicate, that the same chromosome pair which in P. adspersus constitutes the heteromorphic sex chromosomes is still in an initial stage of morphological differentiation in P. delalandii. Although the ZZ/ZW sex chromosomes in Pyxicephalus have developed independently of the ZZ/ZW sex chromosomes in birds and reptiles, they seemed to have passed through identical stages of morphological differentiation. In this process, the heterochromatinization of the W chromosome had a decisive function.

Chromosome banding in Amphibia

Ribosomal RNA genes in plants are highly variable both in copy number and in intergenic spacer (IGS) length. This variability exists not only between distantly related species, but among members of the same genus and also among members of the same population of a single species. Analysis of inheritance indicates that copy number change is rapid, occurring even among somatic cells of individual plants, and that up to 90% or more of the gene copies are superfluous. Subrepetitive sequences within the IGS appear to be changing rapidly as well. They are not only variable in sequence from one species to the next, but can vary in number between neighboring gene repeats on the chromosome. In all species examined in detail they are located in the same region of the IGS and contain sequences that can be folded into stem-loop structures flanked by a pyrimidine-rich region. It has been suggested that these subrepeats function in transcriptional enhancement, termination or processing, or in recombination events generating the high multiplicity of ribosomal genes.

Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer.

The somatic and meiotic chromosomes of the South American leptodactylid toads Odontophrynus americanus, Ceratophyrys ornata, and C. cranwelli were analysed both with conventional staining and differential banding techniques. The karyotypes of O. americanus were tetraploid; those of C. ornata octaploid. Ceratophrys cranwelli is a diploid species whose karyotype displays great similarities with that of C. ornata. The high frequency of multivalent pairing configurations in the meioses of O. americanus and C. ornata indicate that these animals were of autopolyploid origin. The conventionally stained somatic chromosomes of O. americanus can be arranged into sets of four similar chromosomes (quartets); those of C. ornata, into sets of eight similar chromosomes (octets). The banding patterns revealed heterogeneity within some quartets of O. americanus, dividing each of them into two pairs of homologous chromosomes. In analogy, some octets of C. ornata can be subdivided into two quartets of chromosomes with homologous bands. These structural heterogeneities within the quartets and octets are interpreted as a "diploidization" of the polyploid karyotypes. Diploidization leads to genomes that are polyploid with respect to the amount of genetic material and diploid with respect to chromosomal characteristics and the level of gene expression. In tetraploid O. americanus, the number of nucleolus organizer regions (NORs) and their DNA content is proportional to the degree of ploidy. In contrast, up to eight NORs have been deleted in the octoploid C. ornata. These NOR losses are discussed as a possible reason for the reduction of genetic activity in polyploid genomes.

Chromosome banding in Amphibia. IX. The polyploid karyotypes of Odontophrynus americanus and Ceratophrys ornata (Anura, Leptodactylidae).

THERE is widespread variation between species of higher plants in amounts of nuclear DNA. In ten species a fifty-seven-fold variation in amounts of DNA per nucleus for equivalent cells was recorded1 and later reports showed wide ranges in the DNA contents of diploid species within single families (forty-fold in the Ranunculaceae2 and sixty to eighty-fold in the Droseraceae3). Most strikingly, large differences exist within a single genus between species with the same diploid chromosome number, for example, a three-fold range in the genus Lathyrus4 and between five and seven times as much nuclear DNA in Vicia faba as in V. sativa5–7. The structural basis of these variations is unclear although models of either a single stranded chromosome, in which DNA content increases by longitudinal multiplication of DNA sequences, or a multi-stranded chromosome, where increase is by lateral increase of strands, have been suggested (see reviews, refs 8, 9).

E. P. Maher

Papers

Multiplicity of ribosomal RNA genes in Vicia species with different nuclear DNA contents.