流式细胞仪鉴定猕猴桃倍性技术研究

利用流式细胞仪(Flow cytometer,FCM)对不同倍性猕猴桃DNA的相对含量进行测定,建立一套简易、快速的鉴定猕猴桃倍性方法。以二倍体"红阳"猕猴桃为对照,‘81-5'、‘皖翠'猕猴桃为试材,通过筛选最优鉴定叶片部位;改良解离液配方;提高滤膜的目数;过滤次数;增加离心次数等优化鉴定技术。结果表明,露地栽培条件下,茎尖下初展叶是倍性鉴定的最佳部位;两次离心;解离液中2%PVP-30;500目滤膜抽滤两次可以有效解决仪器堵管现象,有效去除杂质峰并获得清晰样品峰。通过优化后的鉴定技术可实现同时测定2个以上倍性的猕猴桃混合样品。     

基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。     

猕猴桃倍性混合居群基因组遗传和表观遗传变异

植物倍性混合居群的形成和维系常伴随着明显的基因组遗传及表观遗传变异。利用AFLP和MSAP两种分子标记探讨了中华猕猴桃复合体(Actinidia chinensis)倍性混合居群的遗传变异和结构及其基因组甲基化变异方式。结果表明, 该倍性混合居群具有较高的遗传和表观遗传多样性, 但两者之间没有明显的相关性。种群的遗传多样性与海拔呈显著的负相关(P<0.05), 但表观遗传多样性与海拔不具显著相关性。AMOVA分析显示, 主要的遗传和表观遗传分化出现在倍性小种内部(97.65% vs 99.84%, P<0.05); 同时, AFLP邻接聚类分析显示二者存在一定程度的倍性相关性, MSAP分析则未显示有明显的倍性相关性。进一步研究发现, 中华猕猴桃居群的总甲基化程度为24.86%, 且多倍体具有更多的甲基化位点变异。该研究结果为深入探讨猕猴桃倍性混合居群的形成和维系机制奠定了基础。     

DNA流式细胞术在植物遗传及育种中的应用

DNA流式细胞术是一种分析和测定分离细胞核DNA含量的方法。该方法的关键技术包括制备完整细胞核悬浮液、核酸荧光染料染色、根据相对荧光密度(DNA含量)对细胞核进行分类。由于样品制备及分析方便快捷,该技术已成为植物倍性鉴定、基因组大小测定、生殖途径鉴定等研究的重要工具。该文在对DNA流式细胞术基本原理简要介绍的基础上,对其在植物遗传和育种中的应用进行了综述。     

小鼠冷冻胚胎和精子SNP遗传鉴定方法的建立

目的建立小鼠冷冻胚胎和精子SNP(single nucleotide polymorphism)分型方法,用于冷冻胚胎和精子快速遗传鉴定方案。方法以中科院上海实验动物中心(国家啮齿类实验动物种子中心上海分中心)提供的小鼠冷冻胚胎和精子为样本,采用全基因组扩增技术和PCR-LDR分型技术建立小鼠冷冻物SNP遗传鉴定方法。结果全基因组扩增技术能大幅度增加冷冻胚胎样本的DNA总量;PCR-LDR分型方法适用于小鼠全基因组45个SNPs的分型;分型确定C57BL/6,BALB/c,FVB/NJ等胚胎和精子各10种近交系,SNP位点信息与测序结果一致;小鼠冷冻胚胎个数与SNPs检出个数成正比,当胚胎数达到12以上时SNP检出率100%。结论实现近交系小鼠冷冻胚胎和精子快速SNP基因分型及遗传质量鉴定。     

雌核发育草鱼及亲本倍性研究

应用流式细胞术(now cytometry, FCM)和红细胞(核)体积测量法对雌核发育草鱼、普通草鱼、湘江野鲤染色体倍性进行比较研究,检测获得的子代雌核发育草鱼倍性情况.结果表明雌核发育草鱼与普通草鱼红细胞及细胞核大小无显著差异,且DNA含量一致,湘江野鲤红细胞和细胞核体积是雌核发育草鱼、普通草鱼的两倍,DNA含量是草鱼的两倍.证实人工雌核发育草鱼具有与普通草鱼相同的染色体倍性.     

四倍体不结球白菜的诱导及染色体倍性鉴定

用不同浓度秋水仙素处理子叶期不结球白菜生长点对其进行染色体倍性操作,根据形态解剖学、细胞学特征和流式细胞仪进行倍性鉴定.结果表明,浓度为0.2%的秋水仙素处理4次的效果最好,四倍体诱变率为8.42%.与二倍体相比,四倍体植株叶片、花器官、气孔等均表现巨大性;气孔密度和结实率降低;抽薹较晚.用流式细胞仪进行倍性鉴定,对照DNA相对含量为100,疑似株为200,表明是四倍体;疑似株有2个值与对照的比值约为1和2,表明是嵌合体(2x 4x).流式细胞仪鉴定结果与染色体计数法鉴定结果一致,表明流式细胞仪可以较准确地检测不结球白菜突变株倍性.     

猕猴桃属种间体细胞杂种

利用PEG融合方法,分别进行了中华猕猴桃(Actinidia chinensis var.chinensis)(2n=2x=58)子叶愈伤组织来源的原生质体与美味猕猴桃(A.deliciosa var.deiciosa)(2n=6x=174)子叶愈伤组织原生质体、以及狗枣猕猴桃(A.kolomikta)(2n=2x=58)叶肉原生质体种间原生质体融合。结果表明:中华猕猴桃与美味猕猴桃融合的1个克隆和中华猕猴桃与狗枣猕猴桃融合的4个克隆的RAPD谱带分别具有双亲特异的DNA谱带;经流式细胞仪分析,前者细胞核倍性推测为8倍体,后者细胞核为3倍体、4倍体和5倍体。初步鉴定这5个克隆是猕猴桃属种间体细胞杂种。     

猕猴桃属十个种的染色体倍性鉴定

猕猴桃属(Actinidia)植物全世界有66种,约有118个种下分类单位(也有新的划分方法将其划分为54种21变种),其中大部分为中国特有。在猕猴桃杂交育种中,不同倍性之间选配不当会出现杂交失败、后代不育等现象,因此倍性鉴定是猕猴桃常规育种亲本选择的前提条件之一。但到目前为止,不少猕猴桃种或亚种的染色体倍性研究并不十分清楚,因而限制了这些资源的进一步开发利用。该研究针对广西植物研究所猕猴桃种质资源圃收集的目前倍性尚不明确的白萼、白花柱果、二色花、临桂、卵圆叶、桃花、宛田、长果、融水和五瓣猕猴桃等10个种类的猕猴桃,使用酸解法制备染色体标本,通过显微镜观察确定其倍性。这10个种类大多为广西特有,其中蕴藏着独特的优良园艺性状,具有很高的生产和开发价值。该研究结果表明这10个种类猕猴桃的染色体倍性均为二倍体(2n=2x=58)。该研究结果进一步丰富了猕猴桃种质资源多样性数据库,为这些猕猴桃资源的合理开发利用奠定了基础。     

基于PLP-LDR-HRCA策略进行微量DNA分析——rs17750303位点分型

增强PCR和全基因组扩增是当前微量DNA分析的主要策略,但是,由于DNA模板量过少,受随机效应影响显著,往往不能得到可靠的DNA分型结果.本文提出一种新的检验策略:PLP-LDR-HRCA,尝试微量DNA检材的SNPs分型研究.选择rs17750303位点,并设计等位基因特异性锁式探针,采用连接酶检测反应来识别等位基因,而后采用超分支滚环扩增反应来放大检测信号.结果表明,PLP-LDR-HRCA反应特异性好,灵敏度高,能够直接鉴别微量基因组DNA模板中待测SNP位点,rs17750303纯合型样品(AA型或CC型)和杂合型样品(AC型)准确分型所需最少模板量分别为20pg和30pg.对于增强PCR和全基因组扩增技术不能有效检验的微量检材,PLP-LDR-PCR策略独具优势,可能具有较大的开发价值.     

Influence of sample size on image cytometry of DNA ploidy measurements

OBJECTIVE: To assess the number of nuclei required for significant image cytometry DNA ploidy measurements on one archival case of breast cancer. STUDY DESIGN: From one case of aneuploid DNA breast cancer, 18 subsets made up of 152-1,524 for the whole population of undamaged nuclei and made up of 74-735 epithelial nuclei had DNA measured. DNA ploidy type and five DNA ploidy indices, allowing DNA ploidy histogram interpretation were evaluated on each population. RESULTS: Three hundred nuclei were always sufficient for DNA typing, whereas reliable results for DNA ploidy indices required at least 750 nuclei. CONCLUSION: To DNA measure the above number of nuclei, fully automated image cytometry DNA ploidy measurements are required.     

Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata

During in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors

The correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q-banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4-18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated. The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.     

Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column

Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However, a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals.     

Flow cytometry analysis of DNA ploidy levels and protein profiles distinguish between populations of Lumbriculus (Annelida: Clitellata)

Variations in DNA ploidy have been observed in Lumbriculus, a freshwater annelid, as well as in other clitellates. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect the protein expression, reproductive behavior, and response to stressors. Ploidy is typically determined by chromosome spreads, a time‐consuming and inefficient method. We adapted flow cytometry protocols used on vertebrates and plants to determine the ploidy levels in different populations of Lumbriculus, including a laboratory strain (Environmental Protection Agency), a commercial strain (Aquatic Foods), and worms collected from natural habitats. To isolate nuclei, worms were homogenized, filtered to remove cell debris, and centrifuged through Optiprep? density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed that Lumbriculus from natural habitats in Minnesota and Iowa were diploid, with an estimated genome size of 2.7 pg. Populations from natural habitats in California and Oregon were highly polyploid, as were the laboratory and commercial strains. Chromosome spreads verified the high ploidy levels indicated by flow cytometry results, but also suggested that flow cytometry may be underestimating the DNA content levels. Staining of nuclei with diamidino‐2‐phenylindole indicated that this may be due to high levels of heterochromatin in nuclei from polyploid forms of Lumbriculus. To further compare the populations, proteins in worm homogenates were subjected to isoelectrofocusing gel electrophoresis. Distinct protein profiles were seen; one was shared in common by the diploid worms, the other was characteristic of polyploid populations. Diploid worms could also be distinguished from polyploid worms based on differences in hemoglobin linker proteins. The results further support taxonomic classification of the diploid and polyploid forms of Lumbriculus as distinct species.     

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.     

利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。