RAPID DETERMINATION OF NUCLEAR DNA AMOUNTS AND PLOIDY LEVELS IN GERMPLASMS OF ASPARAGUS USING FLOW CYTOMETRY

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of 11 Salix species and 5 hybrids. One hundred and sixty nine individuals were studied including 159 individuals from a sequence of 32 communities along a stretch of river in France and 10 individuals from French and English collections for comparison. Isolated nuclei were stained with propidium iodide. FCM was a significantly more practical and rapid technique than that of establishing the karyotype to survey many samples of Salix for variation in ploidy. The 2C DNA amounts for diploid species ranged from 0.76 to 0.98 pg, and tetraploid values ranged from 1.62 to 1.80 pg. The DNA values were consistent with the known ploidy levels. With the exception of a doubtful Salix xquercifolia, ploidy levels and DNA amounts of hybrids were intermediate compared with those of their parents. Intraspecific variation of nuclear DNA values including instrumental variation was low (i.e., 6-11% at the same ploidy level). FCM appeared to be an accurate tool for determination of Salix triploid hybrids. However, it remains limited concerning hybrids from crosses between species of the same ploidy level. Results suggest that natural hybridization might not be frequent in the communities studied, although they have been subject to disturbance. Previous overestimates of hybridization frequency in willows were probably due to misinterpretation of the effects of the environment on Salix spp. morphology; however, the extent and mechanisms of introgression in the genus remain to be further investigated. Key words: flow cytometry, Salix, hybridization, nuclear DNA content, riparian vegetation, disturbance.

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Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)

Genes ◽

10.3390/genes12121950 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1950

Author(s):

Guadalupe Palomino ◽

Javier Martínez-Ramón ◽

Verónica Cepeda-Cornejo ◽

Miriam Ladd-Otero ◽

Patricia Romero ◽

...

Keyword(s):

Flow Cytometry ◽

Chromosome Number ◽

Ploidy Level ◽

Dna Content ◽

Chromosome Numbers ◽

Nuclear Dna ◽

Nuclear Dna Content ◽

Root Tips ◽

Ploidy Levels ◽

Dna Amounts

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

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Application of Flow Cytometry for Rapid Determination of Ploidy Levels in Asparagus (Asparagus officinalis L.)

Journal of the Faculty of Agriculture, Kyushu University ◽

10.5109/24253 ◽

1998 ◽

Vol 43 (1/2) ◽

pp. 83-88

Author(s):

Yukio Ozaki ◽

Kumiko Narikiyo ◽

Michikazu Hiramatsu ◽

Kenji Ureshino ◽

Hiroshi Okubo

Keyword(s):

Flow Cytometry ◽

Rapid Determination ◽

Asparagus Officinalis ◽

Ploidy Levels

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Ploidy level determination of Hedera (Araliaceae) with an emphasis on discussable species (Hedera hibernica)

Caryologia ◽

10.36253/caryologia-881 ◽

2021 ◽

Vol 74 (1) ◽

pp. 109-116

Author(s):

Fahimeh Fallah ◽

Farrokh Ghahremaninejad

Keyword(s):

Flow Cytometry ◽

Genome Size ◽

Nuclear Dna ◽

Petroselinum Crispum ◽

New Taxon ◽

Internal Reference ◽

Ploidy Levels ◽

Internal Reference Standard ◽

Significant Difference

Genome size is a helpful tool for circumscribing taxa at diverse taxonomic degrees (mostly species) and resolving intricate low-level taxonomies. The correct genome size in Hedera (Araliaceae) has long been discussed, and the ploidy levels of some taxa are still unclear. Twelve accessions of Hedera were measured via flow cytometry. Flow cytometry is a relatively rapid, inexpensive, and credible tool. Fresh leaves of Hedera samples and internal reference standard parsley (Petroselinum crispum) were stained with propidium iodide (PI). Flow cytometry measurements showed that for the accessions of 2CV (3.09 - 6.40 pg), the lowest amount of nuclear DNA was 3.09 pg for Hedera crebrescens (So), while the highest amount was 6.40 pg for H. hibernica “Hamilton,” representing a statistically significant difference. According to this study, the new taxon (H. crebrescens) is a diploid, though this taxon was previously considered H. hibernica (tetraploid).

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Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.

Genes ◽

10.3390/genes12070957 ◽

2021 ◽

Vol 12 (7) ◽

pp. 957

Author(s):

Paulina Tomaszewska ◽

Till K. Pellny ◽

Luis M. Hernández ◽

Rowan A. C. Mitchell ◽

Valheria Castiblanco ◽

...

Keyword(s):

Flow Cytometry ◽

Germplasm Collection ◽

Forage Grass ◽

Breeding Programs ◽

Robust Identification ◽

Ploidy Levels ◽

Sustainable Improvement ◽

Tropical Forage ◽

Relative Differences

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

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Diversity in ploidy levels and nuclear DNA amounts in Korean Miscanthus species

Euphytica ◽

10.1007/s10681-013-0910-6 ◽

2013 ◽

Vol 193 (3) ◽

pp. 317-326 ◽

Cited By ~ 13

Author(s):

Youn-Ho Moon ◽

Young-Lok Cha ◽

Yong-Hwan Choi ◽

Young-Mi Yoon ◽

Bon-Cheol Koo ◽

...

Keyword(s):

Nuclear Dna ◽

Ploidy Levels ◽

Dna Amounts

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Intraoperative flow cytometry analysis of glioma tissue for rapid determination of tumor presence and its histopathological grade

Journal of Neurosurgery ◽

10.3171/2013.1.jns12681 ◽

2013 ◽

Vol 118 (6) ◽

pp. 1232-1238 ◽

Cited By ~ 28

Author(s):

Takahiro Shioyama ◽

Yoshihiro Muragaki ◽

Takashi Maruyama ◽

Takashi Komori ◽

Hiroshi Iseki

Keyword(s):

Flow Cytometry ◽

Predictive Value ◽

Rapid Determination ◽

Characteristic Curve ◽

Biopsy Sample ◽

Surgical Biopsy ◽

Diagnostic Efficacy ◽

Histopathological Grade ◽

Number Of Cells

Object Intraoperative histopathological investigation plays an important role during surgery for gliomas. To facilitate the rapid characterization of resected tissue, an original technique of intraoperative flow cytometry (iFC) was established. The objective in this study was evaluation of this technique's efficacy for rapidly determining tumor presence in the surgical biopsy sample and WHO histopathological grade of the neoplasm. Methods In total, 328 separate biopsy specimens obtained during the resection of 81 intracranial gliomas were analyzed with iFC. The evaluated malignancy index (MI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. The duration of iFC in all cases was approximately 10 minutes. Each sample was additionally investigated histopathologically on frozen and permanent formalin-fixed paraffin-embedded tissue sections. The latter process was used as a “gold standard” control for evaluation of the diagnostic efficacy of iFC analysis. Results The MI differed significantly between neoplastic and perilesional brain tissue (25.3% ± 22.0% vs 4.6% ± 2.6%, p < 0.01). Receiver operating characteristic curve analysis revealed a corresponding area under the curve value of 0.941. The optimal cutoff level of the MI for identification of tumor in the biopsy specimen was 6.8%, which provided 0.88 sensitivity, 0.88 specificity, 0.97 positive predictive value, 0.60 negative predictive value, and 0.88 diagnostic accuracy. Additionally, the MI showed a significant association with WHO histopathological grades of glioma (p < 0.01), but its values in Grade II, III, and IV tumors overlapped prominently and were on average 13.3% ± 11.0%, 35.0% ± 21.8%, and 46.6% ± 23.1%, respectively. Conclusions Results of this study demonstrate that iFC with the determination of the MI may be feasible for rapidly determining glioma presence in a surgical biopsy sample.

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Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-10046-3 ◽

2019 ◽

Vol 103 (20) ◽

pp. 8619-8629 ◽

Cited By ~ 4

Author(s):

Christian Opitz ◽

Grit Schade ◽

Silvan Kaufmann ◽

Marco Di Berardino ◽

Marcel Ottiger ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Cell Cultures ◽

Rapid Determination ◽

Eukaryotic Cell ◽

Harvest Time ◽

Optimal Harvest

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Non-invasive ploidy determination in live fish by measuring erythrocyte size in capillaries

Acta Ichthyologica Et Piscatoria ◽

10.3897/aiep.51.65718 ◽

2021 ◽

Vol 51 (3) ◽

pp. 275-280

Author(s):

Karel Halačka ◽

Karel Janko ◽

Lukáš Vetešník

Keyword(s):

Flow Cytometry ◽

Rapid Determination ◽

Minimum Size ◽

Gibel Carp ◽

Small Fish ◽

Reliable Determination ◽

Live Fish ◽

Blood Smears ◽

The Individual

Information about ploidy is important in both commercial and conservation aquaculture and fish research. Unfortunately, methods for its determination, such as karyology, determination of the amount of DNA in a cell using microdensitometry or flow cytometry and/or measuring erythrocytes in a blood smear can be stressful or even destructive. Some of these methods are also limited by the relatively large minimum size of the individual being measured. The aim of this study was to test a new low-stress method of determining ploidy by measuring the size of erythrocytes in the capillaries of a fish, including small individuals. First, we examined diploid and triploid loach (Cobitis sp.) and gibel carp, Carassius gibelio (Bloch, 1782), using flow cytometry and blood smears, with these results being used as a control. Subsequently, we measured the size of erythrocytes in the caudal fin capillaries of anesthetized fishes of known ploidy under a light microscope. For both the loaches and gibel carp, direct observation of the mean erythrocyte size in epithelial fin capillaries provided a consistent and reliable determination of ploidy when compared with the controls based on flow cytometry and blood smears. This new method allows for rapid determination of ploidy in living small fish, where collection of tissue using other methods may cause excessive stress or damage. The method outlined here simply requires the measurement of erythrocytes directly in the bloodstream of a live fish, thereby making it possible to determine ploidy without the need for blood sampling. The method described is sufficiently efficient, less demanding on equipment than many other procedures, can be used by relatively inexperienced personnel and has benefits as regards animal welfare, which is especially important for fish production facilities or when dealing with rare or endangered species.

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NUCLEAR DNA CONTENT IN BLUEBERRY DETERMINED BY FLOW CYTOMETRY

HortScience ◽

10.21273/hortsci.27.6.580e ◽

1992 ◽

Vol 27 (6) ◽

pp. 580e-580

Author(s):

Rodomiro Ortiz ◽

D.E. Costich ◽

T.P. Meagher ◽

N. Vorsa

Keyword(s):

Flow Cytometry ◽

Dna Content ◽

Nuclear Dna ◽

Nuclear Dna Content ◽

Haploid Genome ◽

Dna Flow Cytometry ◽

Dna Amount ◽

Ploidy Levels ◽

Polyploid Evolution ◽

Haploid Genome Size

DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.

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