基于流式细胞术的北美冬青基因组大小测定

以10个北美冬青品种的嫩叶为材料,采用水稻‘日本晴’和二倍体大豆嫩叶为标样,检测北美冬青品种的核DNA含量及基因组大小,为北美冬青种质资源鉴定、基因组学研究以及新品种培育提供理论依据。结果表明：北美冬青品种间基因组大小存在显著差异。’Oosterwijk’、’Earlibright’、’Winter Red’、’Winter Gold’、’Apollo’、’Afterglow’和’Southern Gentleman’7个北美冬青品种基因组大小为728.46～852.99 Mb,推测这些品种为二倍体;’Cacapon’、’Red Sprite’和’Shaver’这3个品种北美冬青基因组大小为1954.99-2108.95 Mb,推测这3个品种为多倍体。’Apollo’和’Southern Gentleman’这两个雄性品种的基因组大小平均值明显小于其他雌性品种。本研究基于流式细胞术建立的北美冬青基因组大小测定方法可为该属其他植物的相关研究提供借鉴。     

基于流式细胞术的海菜花属植物基因组大小测定

利用改良的裂解液P1,以中国古代莲(Nelumbo nucifera Gaertn.Fruct.et Semin)为外标,采用流式细胞术(FCM)对海菜花属(Ottelia Pers.)6个代表性物种及3个存疑类群的基因组大小(C值)进行测定,并对海菜花属系统发育关系进行评估。结果显示:所测定的材料中,水菜花(Ottelia cordata(Wall.)Dandy)C值最小(6.759 pg),灌阳水车前(O.guanyangensis Z.Z.Li,S.Wu&Q.F.Wang)C值最大(12.929 pg);对基因组大小与该属系统发育树进行比较分析,结果发现该属植物基因组大小与系统发育关系具有一致性;对海菜花属3个存疑类群进行分子系统学研究,结果发现存疑类群与嵩明海菜花(Ottelia acuminata var.songmingensis Z.T.Jiang,H.Li&Z.L.Dao)及灌阳水车前的关系最近,而与水菜花的关系较远,这与基因组大小变异相一致。根据基因组大小进一步推测3个存疑类群很可能为二倍体。本研究结果可为海菜花属植物的系统学研究提供新资料,同时为该属植物基因组学研究提供基础数据。     

稀有鮈鲫与其他模式实验鱼类基因组大小的比较

稀有(鱼句)鲫 (Gobiocypris rarusYe et Fu)是我国特有的小型实验鱼类,具有性成熟期短、繁殖季节长、产卵频率高等特点1,已在生态毒理和鱼病学等研究中得到应用,显示了它作为实验鱼类的良好潜力。       

14种淡水鱼基因组大小变异的研究A

本文报道14种淡水鱼细胞核DNA含量的测定结果。这14种鱼DNA含量变异较大,从2.20pg到5.0pg不等,其中大多数种类具有较高的DNA含量。此外,我们还结合已有资料,讨论了迄今已研究的58种中国淡水鱼（主要为鲤科鱼）DNA含量的变异特征、意义及影响DNA含量变异的因素。     

植物基因组大小进化的研究进展

不同的真核生物之间基因组大小差异很大, 并与生物体复杂性不相关, 在基因组中存在大量的非编码DNA序列是造成这种差异的主要原因, 特别是转座子序列。文章综述了植物基因组大小差异以及引起这种差异的主要进化动力的最新研究进展。植物基因组多倍化和转座子积累是导致基因组增大的主要动力, 而同源不平等重组和非正规重组则是驱动基因组DNA丢失的潜在动力, 以制约基因组无限制地增大。文中还讨论了植物基因组大小进化方向, 即总体趋势是朝着增大的方向进化, 某些删除机制主要是削弱这种增大作用但不能逆转。     

昆虫基因组大小及其进化

基因组是物种遗传信息的集合,其大小是研究基因组进化、结构及功能的重要参数之一。本文介绍了测定基因组大小的方法,简述了基因组大小的进化假说及分子机制,综述了近年来昆虫基因组大小的研究进展,尤其是昆虫基因组大小变化的相关影响因子。总体而言,昆虫基因组大小变化是一个复杂的过程,与转座子的活性有密切的关系,是基因组序列丢失和获得两种过程平衡的结果。基因组大小的变化仍在不断地进化中,其对生物造成的影响是剧烈的,因此对昆虫的表型特征产生了重要的影响,但影响的程度和关系在不同的类群有明显的差异,表现出一定的随机性,目前尚未总结出明显的规律。昆虫是生物多样性最为丰富的动物类群,是研究基因组大小进化的最佳材料,随着越来越多的昆虫基因组被测序公开,对昆虫基因组数据进行深入分析,有利于破解基因组大小进化的"C值之谜",可为生物基因组大小的研究提供重要参考。     

利用流式细胞仪测定荚蒾属植物的基因组大小

荚蒾属 (Viburnum) 植物在园林中广泛用作观赏灌木,并且其具有优良园艺性状的杂交种在世界范围内越来越受欢迎。本研究利用流式细胞仪测定了14种荚蒾属植物的基因组大小。二倍体中基因组大小变化范围是255pg（陕西荚蒾）到426pg（琼花）。同时,琼花的核型也较不对称,这可能反应了它的育种历史。四倍体物种珊瑚树的基因组大小（762pg）是其他二倍体物种的两倍还多,这揭示了该属的多倍化在进化中可能并不遥远。该研究为荚蒾属细胞遗传学和分类学的深入研究奠定了基础,并为该属杂交育种提供有用的信息。     

利用流式细胞术测定大额牛基因组大小的研究

运用流式细胞术(FCM)测定大额牛基因组大小,并比较大额牛和普通牛基因组大小,为大额牛基因组学研究及其分类地位提供科学依据。分别提取人白细胞和鸡红细胞作为待选内参,用FCM测定经碘化丙啶(PI)染色的待选内参与大额牛白细胞的混合样品,通过比较大额牛与待选内参的荧光强度和DNA含量峰值的倍数关系,筛选出测定大额牛基因组大小最佳的标准内参,以此测定计算大额牛基因组大小;并比较大额牛和普通牛基因组大小关系。研究结果表明鸡红细胞是测定大额牛基因大小最佳的标准内参;以鸡红细胞(2C=2.5 pg)为参照,大额牛细胞核DNA含量C=2.780 9 pg,基因组大小为2.719 7×10~9 bp;大额牛与普通牛测定结果比较发现,大额牛基因组比普通牛基因组小。本研究筛选出测定大额牛基因组大小的最佳标准内参,并运用流式细胞术首次测定了大额牛基因组大小,证明了大额牛基因组较普通牛基因组小。本研究的结论将为后续大额牛De novo测序组装以及基因组学相关研究提供参考,也为确定大额牛进化提供科学依据。     

几种念珠菌DNA总含量的流式细胞分析

应用流式细胞术（ＦＣＭ）对处于稳定生长阶段的念珠菌属的７种８株念珠菌进行了ＤＮＡ总含量的流式细胞（ＦＣＭ）分析。这８株珠菌是：白念珠菌２株，热带念珠菌，克柔念珠菌，近平滑念珠菌，乳酒念珠菌，白念珠菌星形变种，即血清Ｂ型白念珠菌，季也蒙念珠菌各一株。应用ＥＢ一步插入法染色，用鸡红细胞（ＣＲＢＣ）作为内参标准进行ＤＮＡ总含量测定。分析结果表明：稳定生长阶段的组方图上，大多数念珠菌细胞处于ＤＮＡ合成周期     

慈姑基因组大小鉴定及核型分析

【目的】慈姑(Sagittaria sagittifolia L.)是中国重要的园艺经济作物,其基因组大小的鉴定和核型分析对慈姑基因组学研究及分子遗传学研究必不可少,现阶段慈姑的基因组大小、染色体形态的尚不明确,本研究以期为慈姑的育种和物种进化提供更多的细胞学参考。【方法】研究采用流式细胞技术、荧光原位杂交技术,并参考裂解液配方,探针配置,对慈姑品种‘紫金星’（ZJX）的根尖进行基因组大小进行鉴定,并对其染色体形态进行分析观察。【结果】（1）所测慈姑为二倍体植物,‘紫金星’慈姑基因组为16 Gb左右。（2）利用DAPI荧光染色,获得其染色体数目稳定,且与端粒荧光原位杂交的结果完全吻合,均为22条,染色体类型有中部着丝粒,近中部着丝粒以及近端部着丝,近端部着丝粒最多,且未发现随体;核型公式为：2n=2x=22=6m+4sm+12st,属于Stebbins类型中的3B型,核型不对称系数为99.13。【结论】慈姑基因组大小中等,核型极不对称,进化程度较高。     

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.     

Recombination and genome size

Summary Within complements the chiasma frequency per chromosome, which directly reflects the amount of recombination, is generally closely correlated with chromosome length, i.e. the chromosomal DNA content. The correlation does not apply when comparisons are made between the complements of different species. Analyses of results from three Angiosperm genera show a progressive decrease in the chiasma frequency per picogram of DNA with increase in nuclear DNA amount.     

Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

Estimation of the genome size of blue-green algae from DNA renaturation rates

The molecular weight of the genomes of the blue-green algaeAnacystis nidulans andAnabaena cylindrica have been estimated as 2.27×10⁹ and 2.47×10⁹ daltons respectively from the renaturation kinetics of DNA. Thus the genomes of these organisms are similar in size to that ofEscherichia coli K-12, (2.40×10⁹ daltons) measured by the same technique. No evidence was obtained of repeated sequences in the DNA of the two blue-green algae.     

Flow cytometric analysis of genome size variation in some Passiflora species

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

Evolution of genome size in Brassicaceae

BACKGROUND AND AIMS: Brassicaceae, with nearly 340 genera and more than 3350 species, anchors the low range of angiosperm genome sizes. The relatively narrow range of DNA content (0.16 pg < 1C < 1.95 pg) was maintained in spite of extensive chromosomal change. The aim of this study was to erect a cytological and molecular phylogenetic framework for a selected subset of the Brassicacae, and use this as a template to examine genome size evolution in Brassicaceae. METHODS: DNA contents were determined by flow cytometry and chromosomes were counted for 34 species of the family Brassicaceae and for ten Arabidopsis thaliana ecotypes. The amplified and sequenced ITS region for 23 taxa (plus six other taxa with known ITS sequences) were aligned and used to infer evolutionary relationship by parsimony analysis. KEY RESULTS: DNA content in the species studied ranged over 8-fold (1C = 0.16-1.31 pg), and 4.4-fold (1C = 0.16-0.71 pg) excluding allotetraploid Brassica species. The 1C DNA contents of ten Arabidopsis thaliana ecotypes showed little variation, ranging from 0.16 pg to 0.17 pg. CONCLUSIONS: The tree roots at an ancestral genome size of approximately 1x = 0.2 pg. Arabidopsis thaliana (1C = 0.16 pg; approximately 157 Mbp) has the smallest genome size in Brassicaceae studied here and apparently represents an evolutionary decrease in genome size. Two other branches that represent probable evolutionary decreases in genome size terminate in Lepidium virginicum and Brassica rapa. Branches in the phylogenetic tree that represent probable evolutionary increases in genome size terminate in Arabidopsis halleri, A. lyrata, Arabis hirsuta, Capsella rubella, Caulanthus heterophyllus, Crucihimalaya, Lepidium sativum, Sisymbrium and Thlaspi arvense. Branches within one clade containing Brassica were identified that represent two ancient ploidy events (2x to 4x and 4x to 6x) that were predicted from published comparative mapping studies.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Anthocyanin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry

BACKGROUND: Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics. METHODS: DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia). KEY RESULTS: There were large differences in PI staining (35-70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2.8-6.9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1.69-1.76 pg) than green leaves (1.81 pg). Chopping pea or poinsettia tissue in buffer with 0-200 microm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin. CONCLUSIONS: Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation.     

Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome

The discovery of classic parental imprinting came, at least in part, from the analysis of transgene expression in mice. It was noticed that some transgenes were only expressed following paternal transmission and that others sometimes showed differential patterns of methylation depending on the parent of origin. Here, we present evidence of a novel and more subtle form of parental imprinting by taking advantage of the highly sensitive detection of murine transgene expression afforded by flow cytometry. We have produced nine lines of transgenic mice carrying a GFP reporter linked to the human alpha-globin promoter and enhancer elements, which direct expression to erythroid cells. A high proportion of transgenic lines, four of the nine, display significantly lower levels of expression following maternal transmission. Both the percentage of expressing cells and the mean fluorescence in expressing cells are between 10% and 30% lower following maternal transmission. These effects are reversible upon passage through the opposite germline. This finding raises the possibility that differences in the epigenetic state of the maternal and paternal chromosomes in adult somatic cells are more widespread than was previously thought.