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。     

毛竹基因组大小测定

毛竹(Phyllostachys edulis)属禾本科(Poaceae)竹亚科(Bambusoideae)刚竹属(Phyllostachys), 是我国分布和栽培面积最大的经济竹种, 有着广泛的开发前景。本实验以水稻(Oryza sativa)为内标, 用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定, 通过比较水稻与毛竹样品峰值的倍数关系, 计算出毛竹的基因组大小。对24组样品进行重复测试, 测得毛竹基因组大小为 2 075.025±13.08 Mb, 即2 C DNA含量为4.24 pg(以1 pg DNA = 0.978×109 bp计算)。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

毛竹基因组大小测定

毛竹（Phyllostachys edulis）属禾本科（Poaceae）竹亚科（Bambusoideae）刚竹属（Phyllostachys）,是我国分布和栽培面积最大的经济竹种,有着广泛的开发前景。本实验以水稻（Oryza sativa）为内标,用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定,通过比较水稻与毛竹样品峰值的倍数关系,计算出毛竹的基因组大小。对24组样品进行重复测试,测得毛竹基因组大小为2075.025±13.08Mb,即2C DNA含量为4.24pg（以1pg DNA=0.978×10^9bp计算）。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

五节芒基因组大小测定

五节芒(Miscanthus floridulus)属于禾本科(Poaceae)芒属(Miscanthus Andersson),被认为是一种开发潜力巨大的生物质能源植物。本研究以水稻日本晴(Oryza sativa L.var.Nipponbare)为内标,采用流式细胞仪测定6份采自中国不同地区的五节芒基因组大小。结果首次确定了五节芒的基因组大小平均为2596.59 Mb,即2 C DNA含量为5.31 pg(以1 pg=978 Mb计算)。     

长江江豚基因组大小测定

本研究采用流式细胞术,以公鸡(Gallusdomesticus)红血细胞DNA含量为标准,测定了23头长江江豚(Neophocaenaphocaenoidesasiaeorientali)的基因组大小(或称C值)。实验过程中采用了保存在3中不同条件下的长江江豚的全血样品,用3种不同的方法提取白细胞。为了获得本实验所用的公鸡红血细胞DNA含量的准确值,首先以人(Homosapiens)的C值为标准,对其进行了校正。然后其C值(2C=2.35pg)用于长江江豚的基因组大小测定。结果发现:长江江豚的单倍体DNA含量为3.27pg/C,由此得出其基因组大小为3.17×109bp;雌性和雄性的C值分别为3.25pg和3.29pg,野外长江江豚和豢养长江江豚的C值分别为3.30pg和3.05pg。对雌雄个体之间以及不同生长条件下长江江豚的C值应用独立样本t检验分析,发现:1)不同性别的长江江豚基因组大小之间没有明显的差异;2)豢养条件下的长江江豚和野生长江江豚之间的基因组大小有明显差异,豢养条件下的长江江豚的基因组明显小于野生条件下的长江江豚。据此推测必要环境因子的变化可能会对长江江豚的基因组DNA含量造成影响。     

基于流式细胞术和K-mer分析的银缕梅属(Parrotia C.A.Mey.)植物基因组大小测定

金缕梅科(Hamamelidaceae)银缕梅属(Parrotia C.A.Mey.)仅包含银缕梅(Parrotia subaequalis(H.T.Chang)R.M.Hao&H.T.Wei)和波斯铁木(Parrotia persica(DC.)C.A.Mey.)两种落叶阔叶乔木,其中银缕梅是我国华东地区特有的Ⅰ级濒危珍稀保护植物,属东亚第三纪孑遗成分;其姊妹种波斯铁木则间断分布于伊朗北部,属北极第三纪孑遗植物类群。本研究首次利用流式细胞术和K-mer分析方法对银缕梅属两姊妹种的基因组大小进行了测定,建立和优化了以萝卜(Raphanus sativus L.‘Saxa’)为内标、WPB(Woody plant buffer)为细胞核解离液的两种植物单倍体基因组的DNA含量(DNA C值)流式测定的适宜体系,旨在为金缕梅科银缕梅属植物的全基因组测序、基因组学研究、种质资源开发和利用以及物种保育等提供前期基础数据参考;同时也可为金缕梅科其他属、种的基因组大小测定提供借鉴。主要研究结果如下:(1)通过流式测定银缕梅基因组大小约为971.45±13.91 Mb,波斯铁木基因组大小约为890.52±24.69 Mb;(2)K-mer分析估测银缕梅基因组大小为951.70 Mb,杂合率为1.740%,重复序列比例为77.50%;波斯铁木基因组大小为858.50 Mb,杂合率为0.695%,重复序列占74.30%;(3)银缕梅属于高杂合和高重复基因组,波斯铁木则属于微杂合和高重复基因组。本研究的结果为银缕梅属植物后续基于DNA三代高通量测序技术的全基因组测序、组装及去冗余处理等工作提供了重要的数据参考。     

昆虫基因组及其大小

昆虫基因组大小是由于基因组各种重复序列在扩增、缺失和分化过程中所致的数量差异造成的。这些差异使得昆虫不同类群间、种间和同种的不同种群间表现出基因组大小的不同。目前有59种昆虫已经列入基因组测序计划, 其中6种昆虫（黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、家蚕Bombyx mori、意大利蜜蜂Apis mellifera、埃及伊蚊Aedes aegypti和赤拟谷盗Tribolium castaneum）的全基因组序列已经报道。有725种昆虫的基因组大小得到了估计, 大小在0.09~16.93 pg （88~16 558 Mb）之间。本文还介绍了昆虫基因组大小的估计方法, 讨论了昆虫基因组大小的变化及其意义。     

海三棱藨草及其近缘种基因组大小的测定

基因组大小是物种基因组的重要特征,通常用DNA C值来衡量,能够用于快速判断基因组倍性,并为分类学与进化生物学提供重要依据。海三棱藨草(Scirpus mariqueter)是长江口和杭州湾具有重要生态意义的标志性物种,被认为是扁秆藨草(S. planiculmis)和藨草(S. triqueter)的杂交种,因染色体小而难以准确确定倍性。近年来,部分研究者指出该物种的分类和命名存在疑点。该研究通过基因组Survey分析检测海三棱藨草样本CJ1的基因组特征,测序深度约为120 ×,并以绿豆(Vigna radiata)为参考标准,利用流式细胞术测定了海三棱藨草及其同域近缘种扁秆藨草和藨草以及海三棱藨草和扁秆藨草的杂交F1共13个样本的DNA C值和相对倍性。结果表明:(1)基因组Survey分析测得CJ1的基因组大小为244.12 Mbp,杂合率为0.68%,重复序列比例为42.38%,GC含量为37.25%。(2)流式细胞术测得来自不同区域的海三棱藨草各样本的基因组倍性相同,1C值在234.87 ～ 242.5 Mbp之间,其中CJ1的基因组大小与基因组Survey检测结果高度一致。(3)扁秆藨草的1C值在251.77 ～ 264.13 Mbp之间,藨草1C值为537.33 Mbp。根据上述基因组大小,认为海三棱藨草不可能是这两者的杂交种。该研究补充了海三棱藨草及其近缘种的基因组特征,为后续全基因组测序奠定基础,同时也否定了海三棱藨草起源于扁杆藨草和藨草杂交的假说。     

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。     

Sympatric genome size variation and hybridization of four oak species as determined by flow cytometry genome size variation and hybridization

The Quercus species serve as a powerful model for studying introgression in relation to species boundaries and adaptive processes. Coexistence of distant relatives, or lack of coexistence of closely relative oak species, introgression may play a role. In the current study, four closely related oak species were found in Zijinshan, China. We generated a comprehensive genome size (GS) database for 120 individuals of four species using flow cytometry‐based approaches. We examined GS variability within and among the species and hybridization events among the four species. The mean GSs of Q. acutissima, Q. variabilis, Q. fabri, and Q. serrata var. brevipetiolata were estimated to be 1.87, 1.92, 1.97, and 1.97 pg, respectively. The intraspecific and interspecific variations of GS observed among the four oak species indicated adaptation to the environment. Hybridization occurred both within and between the sections. A hybrid offspring was produced from Q. fabri and Q. variabilis, which belonged to different sections. The GS evolutionary pattern for hybrid species was expansion. Hybridization between the sections may be affected by habitat disturbance. This study increases our understanding of the evolution of GS in Quercus and will help establish guidelines for the ecological protection of oak trees.     

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.     

Transposable elements and the evolution of genome size in eukaryotes

It is generally accepted that the wide variation in genome size observed among eukaryotic species is more closely correlated with the amount of repetitive DNA than with the number of coding genes. Major types of repetitive DNA include transposable elements, satellite DNAs, simple sequences and tandem repeats, but reliable estimates of the relative contributions of these various types to total genome size have been hard to obtain. With the advent of genome sequencing, such information is starting to become available, but no firm conclusions can yet be made from the limited data currently available. Here, the ways in which transposable elements contribute both directly and indirectly to genome size variation are explored. Limited evidence is provided to support the existence of an approximately linear relationship between total transposable element DNA and genome size. Copy numbers per family are low and globally constrained in small genomes, but vary widely in large genomes. Thus, the partial release of transposable element copy number constraints appears to be a major characteristic of large genomes.     

Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation

BACKGROUND AND AIMS: In microdensitometry and flow cytometry, estimation of nuclear DNA content in a sample requires a standard with a known nuclear DNA content. It is assumed that dye accessibility to DNA is the same in the sample and standard nuclei. Stoichiometric error arises when dye accessibility is not proportional between the sample and standard. The aim of the present study was to compare the effects of standardization (external-internal) on nuclear fluorescence of two Coffea species and petunia when temperature increases, and the consequences on genome size estimation. METHODS: Two coffee tree taxa, C. liberica subsp dewevrei (DEW) and C. pseudozanguebarieae (PSE), and Petunia hybrida were grown in a glasshouse in Montpellier, France. Nuclei were extracted by leaf chopping and at least 2 h after nuclei extraction they were stained with propidium iodide for approx. 3 min just before cytometer processing. In the first experiment, effects of heat treatment were observed in mixed (DEW + petunia) and unmixed extracts (petunia and DEW in separate extracts). Nine temperature treatments were carried out (21, 45, 55, 60, 65, 70, 75, 80 and 85 degrees C). In a second experiment, effects of heating on within-species genome size variations were investigated in DEW and PSE. Two temperatures (21 and 70 degrees C) were selected as representative of the maximal range of chromatin decondensation. KEY RESULTS AND CONCLUSIONS: In coffee trees, sample and standard nuclei reacted differently to temperature according to the type of standardization (pseudo-internal vs. external). Cytosolic compounds released in the filtrate would modify chromatin sensitivity to decondensation. Consequently, the 'genome size' estimate depended on the temperature. Similarly, intraspecific variations in genome size changed between estimations at 21 degrees C and 70 degrees C. Consequences are discussed and stoichiometric error detection methods are proposed, along with proposals for minimizing them.     

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.     

Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size

Endopolyploidy is a systemic feature in seed plants. A negative correlation between genome size and endopolyploidization has been claimed previously, assuming that a minimum amount of DNA, necessary for certain cell functions, has to be acquired by endopolyploidization of the corresponding cells in plants with small genomes. This assumption was based on the analysis of only a limited set of data from few species. In the present study the endopolyploidization of several organs of 54 seed plant species belonging to two gymnosperm and 14 angiosperm families was investigated. The results revealed a low negative correlation between genome size and endopolyploidization. However, differences between the families, between the different organs of a given species and between the different life‐cycle types with regard to endopolyploidization became obvious. A three‐way analysis of variance with covariate to quantify the impact of the different factors on the extent of endopolyploidization suggested that taxonomic position is the major factor determining the degree of endopolyploidy within a species, while life cycle, genome size and organ type have a minor but also significant effect on endopolyploidization. The comparison of habitats of 16 investigated Central European species implies that endopolyploidization represents a mean to accelerate the growth of plant species in niches, which require and support fast development.     

Evolution of genome size and chromosome number in the carnivorous plant genus Genlisea (Lentibulariaceae), with a new estimate of the minimum genome size in angiosperms

MethodsNuclear genome sizes were measured from cultivated plant material for a comprehensive sampling of taxa, including nearly half of all species of Genlisea and representing all major lineages. Flow cytometric measurements were conducted in parallel in two laboratories in order to compare the consistency of different methods and controls. Chromosome counts were performed for the majority of taxa, comparing different staining techniques for the ultrasmall chromosomes.ConclusionsGenlisea is an ideal candidate model organism for the understanding of genome reduction as the genus includes species with both relatively large (∼1700 Mbp) and ultrasmall (∼61 Mbp) genomes. This comparative, phylogeny-based analysis of genome sizes and karyotypes in Genlisea provides essential data for selection of suitable species for comparative whole-genome analyses, as well as for further studies on both the molecular and cytogenetic basis of genome reduction in plants.     

Genomic variation in Pekin duck populations developed in three different countries as revealed by whole‐genome data

It is well known that both British and American Pekin ducks originated from China. However, the populations differ substantially in production performance, but the genetic changes involved are still poorly understood. Herein, we sequenced 24 individual Pekin ducks (eight from each population) with an average sequencing depth of more than 45× for each population (mean coverage of 6.29 per individual). Among these populations from three different countries, we identified a large number of SNPs and indels as well as many unique population variants, which can be used as population‐specific molecular markers. Genomic comparisons among the three duck populations revealed many candidate genes as well as pathways and Gene Ontology categories that are putatively associated with meat yield in the British population, growth in the American population and brain development in all three populations. These findings will enable a better understanding of the artificial selection history of Pekin ducks and provide a valuable resource for future research on the breeding of this species.     

The considerable genome size variation of Hordeum species (poaceae) is linked to phylogeny, life form, ecology, and speciation rates

Genome size variation in plants is thought to be correlatedwith cytological, physiological, or ecological characters. However,conclusions drawn in several studies were often contradictory.To analyze nuclear genome size evolution in a phylogenetic framework,DNA contents of 134 accessions, representing all but one speciesof the barley genus Hordeum L., were measured by flow cytometry.The 2C DNA contents were in a range from 6.85 to 10.67 pg indiploids (2n = 14) and reached up to 29.85 pg in hexaploid species(2n = 42). The smallest genomes were found in taxa from theNew World, which became secondarily annual, whereas the largestdiploid genomes occur in Eurasian annuals. Genome sizes of polyploidtaxa equaled mostly the added sizes of their proposed progenitorsor were slightly (1% to 5%) smaller. The analysis of ancestralgenome sizes on the base of the phylogeny of the genus revealedlineages with decreasing and with increasing genome sizes. Correlationsof intraspecific genome size variation with the length of vegetationperiod were found in H. marinum populations from Western Europebut were not significant within two species from South America.On a higher taxonomical level (i.e., for species groups or theentire genus), environmental correlations were absent. Thiscould mostly be attributed to the superimposition of life-formchanges and phylogenetic constraints, which conceal ecogeographicalcorrelations.