The Determination of Nuclear DNA Content (2C value) on Some Representative Genus and Species of Magnoliaceae

The plant nuclear DNA content (2C value) is a principal characteristic parmeter to describe biodiversity of species,which has important significance on the study of plant resources. In this study, we choosed 23 species from 10 representative genera (Magnolia、Michelia、Manglietia、Liriodendron、Talauma、 Paramichelia、Tsoongiodendron、Manglietiastrum、Kmeria、Parakmeria) of Magnoliaceae in China. All samples were determined using a flow cytometry technique with a standard of Zea mays (545pg/2C). The amount of nuclear DNA among these species ranged from 325pg (317850Mbp) to 1361pg (1331058Mbp) for Pgrandiflora, Mofficinalis subsp. biloba respectively, and the coefficient of variation (CV) were less than 5%. The results of the study will not only provide references for determination of the nuclear DNA content of Magnoliaceae and other plants, but also lay the foundation for the utilization and conservation of Magnoliaceae plant resources.     

应用流式细胞术测定17种中国野生蔷薇核DNA含量

以17种中国野生蔷薇为试材,采用改良的LB 01裂解液,以4种不同的标准植物——大豆（Glycine max Merr.‘Polanka’）、绿豆（Vigna radiata（L.） Wilczek）、番茄（Lycopersicon esculentum Miller）和玉米（Zea mays L.）为外标,以二倍体材料丽江蔷薇（Rosa×lichiangensis Yü et Ku）为内部参照,利用流式细胞术对其核DNA含量及染色体倍性进行检测,并采用常规染色体压片法验证倍性准确性。本研究首次检测了3个二倍体种——商城蔷薇（Rosa shangchengensis T.C.Ku）、广东蔷薇（Rosa kwangtungensis Yü et Tsai）和无刺刺梨（Rosa roxburghii f.inermis S.D.Shi）,1个三倍体种——伞房蔷薇（Rosa corymbulosa Rolfe）和1个四倍体种——弯刺蔷薇（Rosa beggeriana Schrenk）的核DNA含量及基因组大小。结果表明,流式细胞术检测结果与常规染色体压片法结果一致,可对中国野生蔷薇的倍性研究进行补充。本研究结果可丰富中国蔷薇属植物的细胞遗传学背景资料并为繁育新品种提供理论依据。     

流式细胞仪检测高等植物细胞核DNA含量的方法

相对于动物和微生物而言,流式细胞术在植物科学上的应用会因植物组织与细胞(如细胞壁、中央液泡、特殊细胞器等)的特殊结构以及次生代谢产物等特殊成分,造成样品在前期处理、染色及测试等方面的困难,甚至导致检测失败或结果不准确。笔者在长期运用流式细胞仪测试工作中,积累了大量的植物样本检测经验,并参考国内外相关文献,总结出从植物取材、样品制备到植物细胞核DNA流式检测的方法和技巧,可为植物科学研究者及从事流式细胞检测的技术人员提供实验参考。     

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.     

保存方法和保存时间对竹子核DNA含量 （2C-值） 检测的影响

竹子核DNA含量（2C 值）的检测对竹资源的科学研究具有重要意义,而大部分野外采集的样本,通过不同方法保存后,均使用流式细胞仪技术进行核DNA含量检测。本文选取麻竹（Dendrocalamus latiflorus）、筇竹（Chimonobambusa tumidissinoda）和毛花酸竹（Acidosasa purpurea）三种竹子样本,使用硅胶保存法和Sample protector试剂保存法分别保存4d、8d、12d、16d后,采用流式细胞术检测样品2C 值。CV值可以用来反映数据检测结果质量,是对检测结果准确性以及精确性的评价标准,我们通过样品CV值的大小和2C 值的变异率来评价保存方法和保存时间对竹子2C 值检测的影响。方差分析显示,保存时间对CV值具有显著性影响（P < 0001）,随着保存时间增大,CV值增大;保存方法对2C 值变异率有显著性影响(P < 0001)。硅胶保存法保存后的样品,测量值比新鲜材料大;Sample protector试剂保存法保存后,测量值比新鲜材料小。因此,随着保存时间的增加,样品CV值增大,引起检测结果质量降低。研究发现,硅胶保存法和Sample protector试剂保存法会影响竹子样本2C 值大小,但2C 值大小的变化小于10%。     

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.     

3种淡水环境中低核酸含量细菌的滤过性

【目的】增加低核酸含量(LNA)细菌与过滤性细菌之间的认识。【方法】采用流式细胞技术(FCM)、变性梯度凝胶电泳(DGGE)及统计学分析研究3种典型淡水环境中细菌群落与滤过性。【结果】LNA细菌与过滤性细菌在数量上具有很好的相关性(y=0.646x+22.42,R2=0.984,P0.01),细菌的滤过性不仅与细菌大小有关,还与细胞整体形状及其伸缩性有关;细菌群落组织与LNA细菌比重呈负相关性,而与HNA细菌呈正相关性。【结论】0.45μm膜过滤对水样微生物群落中的LNA细菌具有极强的筛选效果;细菌群落组织与其基于流式细胞技术测定的基因含量具有密切联系。     

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.