Doleel, J., Greilhuber, J. & Suda, J. Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2, 2233-2244

Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, Olomouc, Czech Republic.
Nature Protocol (Impact Factor: 9.67). 02/2007; 2(9):2233-44. DOI: 10.1038/nprot.2007.310
Source: PubMed


Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

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Available from: Jaroslav Dolezel
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    • "Ploidy levels were inferred on the basis of the DNA content measured in plants with known (counted here) chromosome numbers. Glycine max 'Polanka' (2C = 2.50 pg; Doležel et al., 1994 ) was used as an internal standard in each measurement, following the best practice of this method ( Doležel et al., 2007 ). In the initial analyses, two to six plants per population were pooled before measurements . "
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    ABSTRACT: • Contact zones between diploids and their autopolyploid descendants represent a unique evolutionary venue for studying polyploid establishment, cytotype coexistence, and interactions. Here, we examine cytotype coexistence in a diploid-tetraploid contact zone of a perennial herb, Cardamine amara, located north of the Alps by assessing cytotype spatial patterns, ecological divergence, and genetic variation and structure.• Flow cytometry was applied to screen DNA ploidy levels in 302 populations (3296 individuals) and the genetic variation of a selection of 25 populations was examined using microsatellite and AFLP markers. Environmental (landscape and climatic) data were analyzed to assess ecological differentiation between the cytotypes.• A parapatric distribution of the cytotypes with a relatively wide (over 100 km in some regions) secondary contact zone was identified. Mixed-ploidy populations, documented for the first time in this species, as well as triploid individuals were found along the diploid-tetraploid borderline. Different climatic requirements of the two main cytotypes were revealed, mirrored in their altitudinal separation. The tetraploids were genetically differentiated from both the diploids and the modeled, in silico autotetraploid genotypes, in accordance with the assumed polyploid origin and spread linked to past glaciations, and largely independent evolution in allopatry.• The observed spatial and genetic patterns likely reflect the evolutionary and colonization history of the two cytotypes and have been maintained by multiple factors such as ecological divergence, limited gene flow between the cytotypes, and the restricted dispersal capacity. © 2015 Botanical Society of America, Inc.
    Full-text · Article · Aug 2015 · American Journal of Botany
    • "The standard plant material Zea mays cv. 'CE-777' was kindly provided by Dr. Jaroslav Doležel, Institute of Experimental Botany, Olomouc, Czech Republic with reported 2C value of 5.43 pg (Doležel et al., 2007). Nuclei suspensions of sample and standard materials were simultaneously analyzed with a BD FACS Verse Flow Cytometer with a 488 nm solid state laser (50 mW). "
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    ABSTRACT: Basic cytogenetic investigation of economically important members of Cucurbitaceae is a prerequisite to understand their genetic architecture. The study reports detailed chromosome morphometric analyses with fluorescence banding technique, differential distribution of heterochromatin, flow cytometric nuclear DNA contents and meiotic behavior of the native populations of Benincasa hispida, Luffa cylindrica and Trichosanthes dioica in India. In spite of frequent vegetative propagation, different populations of these cultivated cucurbits show regular genetic processes evident from stable chromosome counts, nuclear DNA sizes, ploidy levels and regular meiotic behavior. Distal CMA+ve signals have been scored in two pairs of chromosomes in B. hispida and four pairs of chromosomes in L. cylindrica, representing the GC rich heterochromatic portions in genomes. The presence of terminal DAPI+ve bands in the chromosomes of male and female populations of T. dioica indicates the predominance of AT rich heterochromatin. In addition to DAPI bands, terminal CMA+ve bands in the female plants of T. dioica depict the chromosomal signs of sexual differentiation in this species. The existence of rod bivalent in meiosis of male plants facilitates further approach to characterize the genetics of chromosomal evolution since gross similarity in genome size and karyotypic features of male and female plants advocates genic control of sexual dimorphism in T. dioica.
    No preview · Article · Aug 2015 · Scientia Horticulturae
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    • "The methodology generally followed Suda and Trávníček (2006) and Glycine max 'Polanka' (2C = 2.50 pg; Dolě zel et al. 2007 "
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    ABSTRACT: Vaccinium oxycoccos s.l. is a complex of diploid and polyploid plants. The taxonomic relationship between the cytotypes is uncertain, with conflicting treatments in recent revisions. To clarify this situation, we investigated the relationships among ploidy, morphology, and genetic diversity in this group. We collected samples from a 1000 km transect in eastern Canada. We used flow cytometry to determine DNA ploidy, completed a morphometric analysis of flowering stems, and assessed genetic diversity using AFLPs. Diploids only occurred growing in mixed populations with tetraploids. There were statistically significant morphological differences between ploidies; however, tetraploid variation encompasses the diploid range for most characters. AFLP data demonstrate that the tetraploids have undergone genetic divergence since their formation, obscuring whether they are auto -or allo-polyploids. Our results agree with previous work using isozymes, which revealed genetic divergence of diploids and tetraploids in North America; and morphometry, which demonstrated clear distinctions between diploids and tetraploids in Europe. We found that diploids and tetraploids co-occur much more frequently than previously recognized, which may explain the conflicting treatment of this group by North American taxonomists. We recommend recognizing diploids and tetraploids as distinct species. The distribution of the two species in North America suggests two hypotheses regarding the successful establishment of the tetraploid: the tetraploids' success is due to their capacity to exploit novel habitats outside the range of the diploid; or the tetraploid has in fact already out-competed the diploid in large areas of its former range, limiting the diploid to the far north. While we cannot rule out ongoing gene flow between diploids and tetraploids, it is likely a rare phenomenon in this group.
    Full-text · Article · Jun 2015 · Botany
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