17种金线鲃核DNA含量及倍性的研究

采用血涂片、Feulgen染色和显微分光光度技术 ,测定了金线属 (Sinocyclocheilus) 17个种的核DNA含量。结果显示 ,除侧条金线 (S .lateristritus)中采自云南沾益炎方山那边的样本之 2C值为 7 79pg外 ,其余的核DNA 2C值都集中分布在 4 19～ 4 86pg范围 ,大体与其近缘四倍体种 2C值相同或相近 ,是其近缘二倍体种 2C值的大约 2倍。据此我们推断 ,这 17种金线很可能都是四倍体 ,个别种还含有八倍体的类型 ,金线属可能是整属的四倍体起源。     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions

 Genome size (C-values) and pollen viability staining were applied as new criteria to investigate the species of the genus Helleborus Linnaeus (Ranunculaceae). All species have the same chromosome number (2n=32). However, the nuclear DNA content, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 19 pg to 35.7 pg. The different genome sizes of the species coincided to a large extent with earlier determined section boundaries based on morphology. Flow cytometry can be a convenient method to discriminate between some species. Received April 17, 2001 Accepted May 7, 2001     

Nuclear DNA contents, rDNAs, and karyotype evolution in Vicia subgenus Vicia: II. Section Peregrinae

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of rDNA spacers have been determined for the species of Vicia belonging to sect. Peregrinae, as well as for V. mollis. The phylogenetic data generated from the comparison of rDNA sequences and karyomorphological results would both indicate that Vicia mollis is a sister group to sect. Peregrinae. The relationships among the species belonging to the Peregrinae section and species enclosed in sections Faba, Narbonensis, and Bithynicae have been also investigated: a clade including V. mollis and sect. Peregrinae is a sister group to a clade including V. bithynica and sect. Narbonensis. With our choice of outgroup, Vicia faba (including subsp. paucijuga) is external to the above mentioned inclusive group. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Nuclear DNA contents, rDNAs, chromatin organization, and karyotype evolution inVicia sect,faba

Summary Automated karyotype analyses, nuclear DNA contents, and sequences of rDNA internal transcribed spacers of the nine species inVicia sect.faba are reported. As karyomorphological parameters are used to evaluate the karyotype evolution, so the determination of the heterochromatin by Feulgen absorption at different thresholds of optical density provided further evidence on the chromatin organization withinVicia sect.faba. The comparison of sequences of rDNA spacers has enabled the definition of the phylogenetic relationships between the analyzed species.     

Nuclear DNA contents, rDNAs, and karyotype evolution in subgenus Vicia: III. The heterogeneous section Hypechusa

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of internal transcribed spacers from ribosomal genes have been determined in the species belonging to section Hypechusa of the subgenus Vicia. Karyomorphological results and phylogenetic data generated from the comparison of rDNA (genes coding for rRNA) sequences showed that sect. Hypechusa is not monophyletic; however, some monophyletic units are apparent (one including Vicia galeata, V. hyrcanica, V. noeana, and V. tigridis, another including V. assyriaca, V. hybrida, V. melanops, V. mollis, and V. sericocarpa), which partly correspond to morphology-based infrasectional groups. The relationships among these species and the species in sections Faba, Narbonensis, Bithynicae, and Peregrinae have been also investigated. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Unexpected DNA content in the endosperm of Cupressus dupreziana A. Camus seeds and its implications in the reproductive process

 Nuclear DNA content of embryo and endosperm from mature and immature Cupressus dupreziana A. Camus seeds was estimated using laser flow cytometry. Relative DNA content of endosperm nuclei corresponded to four ploidy levels: 2C, 4C, 6C and 8C. The embryo nuclei invariably exhibited a diploid pattern. In all endosperm tissue analyzed no haploid nucleus was found. This is problematic since, in gymnosperms, endosperm and female gametes originate from one functional haploid megaspore produced by meiosis. The possible origin and derivation of C. dupreziana endosperm are discussed in light of previous results concerning the two other Mediterranean cypresses, C. sempervirens and C. atlantica. Received: 15 January 1998 / Revision accepted: 27 March 1998     

A Gaussian disorientation model for interpreting linear dichroism measurements of DNA-drug fibres and films

The optical linear dichroism of DNA-drug fibres and films can provide valuable information on the geometry of the binding and its extent, especially when used in conjunction with X-ray diffraction data from the same specimens. We have considered the macroscopic orientation of the helices within a fibre or film to be characterized by a Gaussian distribution of the helix axes about the fibre (or film) axis. Using this model we have obtained analytical expressions for the dichroic ratio without resorting to computer simulation techniques or numerical integration methods, and used them to interpret the results of experiments using DNA-phenanthridine fibres. As the humidity is increased, ethidium and dimidium bromide show an increased fraction of binding perpendicular to the helix axis, consistent with intercalation. Prothidium shows little preferred orientation in its binding, and the occurrence of a significant proportion of intercalation can be excluded.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Ploidy variation of progenies from intra- and inter-ploidy crosses with regard to trisomic production in asparagus (<Emphasis Type=Italic>Asparagus officinalis</Emphasis> L.)

Ploidy variation within progenies from intra- and inter-ploidy crosses of asparagus were determined in this study. The DNA content of most reduced microspores was half that of somatic cells in diploid, triploid and tetraploid plants as determined by flow cytometry. Many viable seeds were obtained from various 2x × 2x and 2x × 3x intra- and inter-ploidy crosses. Nuclear DNA contents of these progenies from reciprocal crosses between diploids and triploids were similar to those expected of diploids, but with a wider range: hyper- and hypo-diploids including trisomics seem to be involved in the progenies. Indeed, the number of chromosomes in the progenies of 2x × 3x crosses ranged from 18 to 23. Based on frequencies of viable seeds and trisomic phenotypic appearance, it was concluded that the 2x × 3x cross was most efficient for obtaining trisomic plants in Asparagus officinalis.     

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.     

Genome Size of Three <Emphasis Type="Italic">Miscanthus</Emphasis> Species

Environmental and economic factors have stimulated research in the area of bioenergy crops. While many plants have been identified as potential energy crops, one species in particular, Miscanthus x giganteus, appears to have the most promise. As researchers attempt to exploit and improve M. x giganteus, genome information is critical. In this study, the genome size of M. x giganteus and its two progenitor species were examined by flow cytometry and stomatal cell analyses. M. x giganteus was found to have genome size of 7.0 pg while Miscanthus sinensis and Miscanthus sacchariflorus were observed to have genome sizes of 5.5 and 4.5 pg respectively with stomatal size correlating with genome size. Upon computing the two tetraploid × diploid hybrids theoretical genome sizes, the data presented in this paper supports the hypothesis of the union of a 2x M. sacchariflorus and a 1x M. sinensis gamete for the formation of the allotriploid, M. x giganteus. Such genomic information provides basic knowledge that is important in M. x giganteus plant improvement.     

Intra- and interspecific variation in DNA content in Cistus (Cistaceae)

Flow cytometry, using propidium iodide and 4',6-diamidano-2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A-T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1.5-fold differences in absolute DNA amounts were observed, ranging from 3.92 pg in C. crispus to 5.88 pg in C. monspeliensis. The (AT) : (GC) ratio was close to 1, and was similar for all species examined, ranging from 47.87% A-T content in C clusii, to 50.67% in C. populifolius. Pink-flowered species (subgenus Cistus) had lower DNA amounts than white-flowered species (subgenera Leucocistus and Halimioides). However, the distribution of DNA amounts in Cistus appeared to be continuous and did not permit a clear separation of infra-generic ranks in the genus.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Utilization of intergeneric somatic hybrids as an index discriminating taxa in the genus Citrus and its related species

Embryogenic protoplasts of Shogun mandarin (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from Citropsis gabunensis Swing. & M. Kell, and two green embryoids were regenerated after 3 months of culture. Two months after transfer to the regeneration medium, numerous plantlets were obtained from the embryoids. These plants grew vigorously, had well-developed root systems, and exhibited leaf characteristics intermediate to those of the parents. The absolute nuclear genome size of the regenerated plant SH2 (1.75 pg/4C) was the sum of those of the Shogun mandarin (0.75 pg/2C) and C. gabunensis (0.97 pg/2C). The chromosome counts of the young leaves revealed that they were tetraploids (2n=4x=36). Random amplified polymorphic DNA (RAPD) analysis of the two lines (SH1 and SH2) verified their hybridity. Cytoplasmic genome analysis using universal primers reveal that their chloroplast (cp) DNA banding pattern is identical to that of C. gabunensis, while the banding pattern of the mitochondrial (mt) DNA is identical to that of the Shogun mandarin. These somatic hybrids are important materials for investigating the phylogenetic relationships between these two genera in the subfamily Aurantioideae.     

Temperature and developmental responses of body and cell size in Drosophila; effects of polyploidy and genome configuration

Increased adult body size in Drosophila raised at lower temperatures could be attributed both to an increase in the cell volume and cell number. It is not clear, however, whether increased cell size is related to (or even caused by) increased nuclear volume and genome size (or configuration). Experiments with Drosophila melanogaster stocks (Oregon-R and w1118) raised at 16, 22, 24, and 28 °C resulted in larger adult body and wing size with lower temperature, while eye size was less affected. The increase in wing size reflected an increase in cell size in both males and females of both stocks. The nucleus size, genome size, and DNA condensation of adult flies, embryos, and Schneider 2 cells (S2 cells, of larval origin) were estimated by flow cytometry. In both adult flies and S2 cells, both nucleus size and DNA condensation varied with temperature, while DNA content appears to be constant. From 12% to 18% of the somatic cells were tetraploid (4C) and 2–5% were octoploid (8C), and for the Oregon strain we observed an increase in the fraction of polyploid cells with decreasing temperature. The observed increase in body size (and wing size) at low temperatures could partly be linked with the cell size and DNA condensation, while corresponding changes in the haploid genome size were not observed.     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.     

Developmental events in ovules of the ornamental plant Rudbeckia bicolor Nutt

Microscopic observations of R. bicolor ovules showed that tetrasporic embryo sacs of Fritillaria type are formed. In the mature female gametophytes modifications in antipodal cell formation and egg apparatus organization were observed, e.g. morphological resemblance was evident of antipode or synergid to the egg cell. In the central cell cytoplasm of the mature gametophytes the presence of small bodies was a characteristic feature. Development of both embryo and endosperm was observed in ∼73% of ovules at the embryo stage, while retarded or arrested development of the endosperm was found in ∼26% of them. Occasionally, two embryos occurred in the embryo sac. This is the first record of polyembryony in this species. Although hemigamy has been previously described in Rudbeckia bicolor Nutt., in the present investigations mosaic structure of embryos was not detected. Measurements of the C-DNA amount (flow cytometry) revealed embryo nuclei with 2C DNA content only, and endosperm nuclei with 5C DNA content in the mature seeds. No peak corresponding to 1C nuclei was detected in the histograms obtained from the nuclear preparation of seeds or seedling parts. These results suggest that hemigamy is not an obligatory phenomenon in R. bicolor. The mean 2C DNA value was determined as 14.51 pg (the first estimation for this species).     

Chromosomal DNA content of sweet pepper determined by association of cytogenetic and cytometric tools

The nuclear DNA content of sweet pepper (Capsicum annuum L. var. annuum, 2n = 24) has been measured by flow and image cytometries but the DNA content of each chromosome of this species has not yet been regarded. DNA content of individual chromosomes has been quantified by the flow karyotyping technique, which requires a great quantity of intact metaphasic chromosomes and methods that allow the characterization of individual chromosomes; however, the obtainment of adequate number of metaphases can be difficult in some species like C. annuum. In order to estimate the DNA content of each C. annuum var. annuum cv. "New Mexican" chromosome, flow and image cytometries were associated with the cytogenetic methodology. First, the DNA amount (2C = 6.90 pg) was established by flow cytometry. Integrated optical density (IOD) values were calculated by image cytometry for each Feulgen stained metaphasic chromosome. Then, by distributing the correspondent metaphasic value (4C = 13.80 pg) proportionally to average IOD values, the following chromosomal DNA contents were obtained in pg: 0.74 (chromosome 1), 0.67 (2), 0.61 (3, 4), 0.60 (5), 0.59 (6, 7), 0.58 (8), 0.57 (9), 0.56 (10) and 0.39 (11, 12). This study reports an alternative and reproducible technique that makes quantifying the chromosomal DNA content possible.     

Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

Summary A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G_0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G_0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.