Determination Of Nuclear DNA Content Research Articles

Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry

Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C - DNA content of winter-hardy Hibiscus (H. syriacus ‘Saejamyung’, H. sinosyriacus ‘Seobong’, H. moscheutos ‘Luna Red’ and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range in length of somatic chromosomes in H. syriacus, H. sinosyriacus, H. moscheutos, and H. paramutabilis is 2.66–7.06, 3.18–7.31, 2.91–5.23, and 4.75–7.60, respectively. The 2C - DNA content of H. syriacus, H. sinosyriacus, and H. paramutabilis are very similar, the amount was 4.06, 4.11, and 4.18 pg, respectively whereas, H. moscheutos has nearly half and that amount was 2.06 pg. These findings will contribute to the detailed cytogenetic assessment of Hibiscus and thus benefit plant breeding in this genus.

Determination of nuclear DNA content, ploidy, and FISH location of ribosomal DNA in Hibiscus hamabo

Hibiscus hamabo Sieb.et Zucc, a deciduous shrub or tree, is an important semi-mangrove plant with golden yellow flowers. The plant is also cultivated for its fiber and grown as an ornamental. It is halophytic and considered one of the best afforestation trees used in reclamation programs in urban and coastal areas high in salt content. However, very little work has been done on its genetics; therefore, this study was undertaken to determine the genome size, the chromosome numbers, and rDNA (18S rDNA and 5S rDNA) loci in H. hamabo using flow cytometry, protoplast spread technique, and oligonucleotide fluorescence in situ hybridization (FISH), respectively. H. hamabo was found to have a 2C-value of 4.06 + 0.08 pg (3973.08 + 78.90 Mbp) and a chromosome number of 2n = 92. We observed four 18S loci and one 5S rDNA locus on five different pairs of homologous chromosomes. The observed FISH signal intensities for the 18S rDNA varied from chromosome to chromosome, indicating that each locus had different rDNA repeat copy numbers. The pericentromeric regions of each chromosome observed were highly heterochromatic. The findings of this study will benefit future cytogenetics and genomic investigations as well as lay the groundwork for more efficient breeding programs aimed at developing improved H. hamabo hybrids.

Determination of nuclear DNA content and ploidy of some Bromus L. germplasm by flow cytometery

Determination of nuclear DNA content and ploidy of some Bromus L. germplasm by flow cytometery

Determination of nuclear DNA content and ploidy levels of oat (Avena spp.) accessions belongs to different species

Genom büyüklüğü biyoloji, genetik, taksonomi ve evrim çalışmaları için son derece yararlı bir ölçüttür. Bu ölçüt türlere özel olduğundan, tür teşhisine ve gen bankalarında korunan genetik materyalin etiket bilgilerinin hızlı bir şekilde teyit edilebilmesine imkân sağlamaktadır. Bu çalışmada flow sitometri ile 13 farklı Avena türüne ait 64 aksesyonun ortalama genom büyüklüklerinin ve ploidi seviyelerinin belirlenmesi hedeflenmiştir. Analiz edilen A. brevis, A. hirtula, A. longiglumis, A. nuda, A. strigosa, A. ventricosa, A. abyssinica, A. barbata, A. murphyi, A. vaviloviana, A. fatua, A. sativa ve A. sterilis türlerine ait aksesyonların ortalama çekirdek DNA içeriklerinin 8.58 ile 26.54 pg/2C arasında değiştiği belirlenmiştir. Avena aksesyonlarının çekirdek DNA içerikleri arasındaki farklılık istatistiki olarak önemli bulunmuş ve aksesyonların ploidi düzeylerinin diploid ile hekzaploid arasında değiştiği saptanmıştır. Analizlerden elde edilen sonuçlar USDA-NSGC gen bankasında saklanan bu aksesyonlardan bazılarının etiket bilgilerinin doğru olmadığını ortaya çıkarmıştır. Literatürde mevcut çalışmaların sonuçlarından farklı olarak, incelenen dokuz A. brevis aksesyonunun DNA içeriklerinin 12.21–12.61 pg/2C aralığında olduğu tespit edilmiştir. Benzer şekilde, USDA-GRIN sisteminde mevcut tek A. hirtula aksesyonunda daha önce yapılmış çalışmaların aksine çekirdek DNA miktarı 16.16 pg/2C olarak bulunmuştur.

Determination of Nuclear DNA Content and Ploidy of Hypericum perforatum L. Accessions Collected From Western Turkey

Abstract Hypericum perforatum L. (St John’s Wort) is a medicinal plant that produces pharmaceutically important compounds with antidepressive, anticancer and antiviral activities. H. perforatum is a facultative apomictic species as it has the ability to reproduce with multiple reproduction mechanisms affecting genetic structure and chemical composition of the plants. The objective of this study was to determine nuclear DNA content and ploidy of H. perforatum L. plants growing naturally in the flora of Turkey. The seeds of 39 Hypericum perforatum L. accessions collected from 23 different locations in Turkey were used in the study. Nuclear DNA content of three different seedlings for each of the 39 H. perforatum accessions was determined using flow cytometry. Based on the results of flow cytometric analysis, nuclear DNA content of the accessions varied between 0.8 - 2.57 pg2C -1 . Nuclear DNA content differences observed among H. perforatum accessions were statistically significant (P< 0.01) and the Duncan test revealed that the accessions formed three clearly distinguishable main groups. Mean nuclear DNA contents of each group were 0.80 pg2C -1 , 1.58 pg2C -1 (1.36-1.73 pg2C -1 ) and 2.38 pg2C -1 (2.15-2.57 pg2C -1 ). Ploidy levels were determined by correlating nuclear DNA content of accessions with the number of their mitotic chromosomes by counting chromosome number of one plant from each group. Findings suggest that H. perforatum accessions used in this study had the following three different ploidy levels: diploid, tetraploid and hexaploid. The percentage of diploid, tetraploid and hexaploid accessions was 2.2, 86.4 and 11.4% respectively.

Phylogenetic and cytogenetic relationships among species of Oxalis section Articulatae (Oxalidaceae)

Oxalis section Articulatae Knuth (Oxalidaceae) comprises a group of five species with a restricted distribution in the southern part of South America. To assess the current circumscription of section Articulatae and the evolutionary mechanisms involved in its diversification, we used three plastid DNA sequences and the nuclear ribosomal transcribed spacer (ITS) to build a molecular phylogeny. A cytogenetic analysis of the group including chromosome number, heterochromatin distribution and determination of nuclear DNA content was also performed. Cytologically, all species presented symmetrical karyotypes except for O. linarantha with a bimodal karyotype although it maintained the same haploid number as the other species (n = 7). Nuclear DNA contents in the Articulatae species are among the lowest for the genus. The O. linarantha 2C value represents the minimum value known for Oxalis, extending the genome size range from 0.58 to 41.10 pg, representing approximately a 70-fold difference, and one of the highest ranges known for an angiosperm genus. Molecular, chromosomal and morphological data suggest that section Articulatae should be redefined to include only those species with rhizomatous stems while the tunicate-bulbous species O. linarantha should be placed elsewhere. Different ploidy levels were found, ranging from 2x to 7x. Diploids and polyploids showed a geographic structure; diploids are widely distributed, while polyploids are found in the uplands of eastern Uruguay and southern Brazil. Haplotype sharing and chromosome data suggest that at least some of section Articulatae polyploids may be the result of interspecific hybridization and appear to have originated very recently.

Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction

Certain cellular processes are sensitive to changes in gene dosage. Aneuploidy is deleterious because of an imbalance of gene dosage on a chromosomal scale. Identification, classification and characterization of aneuploidy are therefore important for molecular, population and medical genetics and for a deeper understanding of the mechanisms underlying dosage sensitivity. Notwithstanding recent progress in genomic technologies, limited means are available for detecting and classifying changes in chromosome dose. The development of an inexpensive and scalable karyotyping method would allow rapid detection and characterization of both simple and complex aneuploid types. In addition to the problem of karyotyping, genomic and molecular genetic studies of aneuploids and polyploids are complicated by multiple heterozygous combinations possible at loci present in more than two copies. Quantitative scoring of allele genotypes would enable large-scale population genetic experiments in polyploids, and permit genetic analyses on bulked populations in diploid species. Here, we demonstrate that quantitative fluorescent-polymerase chain reaction (QF-PCR) can be used to simultaneously genotype and karyotype aneuploid and polyploid Arabidopsis thaliana. Comparison of QF-PCR with flow cytometric determination of nuclear DNA content indicated near perfect agreement between the methods, but complete karyotype resolution was only possible using QF-PCR. A complex karyotype, determined by QF-PCR, was validated by comparative genomic hybridization to microarrays. Finally, we screened the progeny of tetraploid individuals and found that more than 25% were aneuploid and that our artificially induced tetraploid strain produced fewer aneuploid individuals than a tetraploid strain isolated from nature.

Mitotic instability in wheat×Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern

To evaluate the mitotic stability of Triticum aestivum x Thinopyrum ponticum derivatives (BC(2)F(7) and BC(2)F(5) doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring' and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.

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.

Variation in Ploidy Levels and Reproductive Pathways Among Flowering Crabapples

Information on ploidy levels is extremely valuable for use in plant breeding programs. Fertility, crossability, and heritability of traits are all influenced by ploidy levels. Knowledge of reproductive pathways, including occurrence of apomixes, pseudogamy, and formation of unreduced gametes can also be important information for developing breeding strategies. Although ploidy level can be determined by counting chromosomes, flow cytometry provides a reliable and much faster means for determination of nuclear DNA content and associated ploidy level. Measurement of ploidy levels of seeds (embryo and endosperm) can also provide useful insights into reproductive pathways. The objective of this study was to determine the approximate genome size, estimated ploidy level, and range of reproductive pathways of a diverse collection of flowering crapbapples (Malus spp.). Genome sizes were calculated as nuclear DNA content for unreduced tissue (2C). Results from the taxa included in our survey showed DNA contents ranging from 1.52 to 1.82 for diploids, 2.40 to 2.62 for triploids, and 3.36 to 3.74 pg/2C for tetraploids. Based on these ranges, we identified 43 diploid, 10 triploid, and 4 tetraploid crabapple taxa in this collection. Results from open pollinated seeds and seedlings demonstrated a variety of reproductive pathways including apomixes and unreduced gametes. This research provides information on ploidy levels and reproductive pathways of flowering crabapples and will allow for more systematic and efficient progress in the development of improved cultivars.

Genome multiplication is a generalised phenomenon in placentomal and interplacentomal trophoblast giant cells in cattle

The frequency of polyploidisation in bovine binucleate trophoblast giant cells (TGC) from placentomes (PL) and the interplacentomal allantochorion (AL) of six male fetuses with a crown–rump length between 3.5 and 103 cm was determined by in situ hybridisation with a chromosome-7-specific probe, using a probe specific for the Y chromosome to distinguish between maternal and fetal nuclei. The results showed that polyploid nuclei were essentially always of fetal origin. The frequency of tetraploid nuclei varied between 3% and 15% in both the placentomal and interplacentomal samples, with mean frequencies of 8.8% and 10.0% respectively. Octoploid nuclei were observed with a mean frequency of 1.1% in the interplacentomal samples, but were absent in samples from placentomes. Subsequent determination of nuclear DNA content by cytophotometric measurement of Feulgen-stained nuclei revealed that the frequency of nuclei with an 8C DNA content was several fold higher (AL 5.4%; PL 7.8%) than the frequency of octoploidy, suggesting that tetraploid TGC cells are arrested in the G2 phase of the cell cycle.

Regulation of bud dormancy by manipulation of ABA in isolated buds of Rosa hybrida cultured in vitro

In vitro cultures showed that the proximal buds isolated from a rose (Rosa hybrida L. cv. Ruidriko Vivaldi®) stem were endodormant. Growth and a high percentage of bud break could be observed when cultures were treated with fluridone, an inhibitor of carotenoid synthesis. Flow cytometry determination of nuclear DNA content revealed that cell cycle activity of endodormant buds was arrested in the G 1 phase. Upon culture, the large decrease in bud ABA content was responsible for the progress from G1 to G2 phase whatever the culture medium. However, in control culture, neither cell division nor leaf primordium initiation could be observed and cells appeared stably arrested in G2 . By contrast, with fluridone, an additional ABA decrease was observed resulting from an inhibition of its synthesis inside the bud. New leaf primordia were initiated and many figures of mitosis could be observed, indicating that intense activity of cell division occurred after DNA replication. Therefore, the results indicate that, as long as ABA was synthesized inside the buds, cell cycle was arrested in G2 phase and buds remained dormant. Continued in situ ABA biosynthesis appears, therefore, to be required for the maintenance of bud dormancy.

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120.

Production of Interspecific Hybrids of Persimmon by Protoplast Fusion

Interspecific hybrids between Diospyros glandulosa (2n = 2x = 30) and D. kaki cv. Jiro (2n = 6x = 90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using modified KM8p medium. Relative nuclear DNA contents of calli derived from fusion-treated protoplasts were determined by flow cytometry. One-hundred-forty-nine of 166 calli obtained had the nuclear DNA content of the sum of those of D. glandulosa and D. kaki cv. Jiro. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and they appeared to be interspecific somatic hybrid calli. Shoots were regenerated from 63 of the 149 interspecific hybrid calli. PCR-RFLP of chloroplast DNA analysis, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained nuclear genome from both the parents but only chloroplast genome from D. glandulosa. Microscopic observation of root tip cells confirmed that somatic chromosome numbers of the interspecific hybrids were 2n = 8x = 120.

Determination of Nuclear DNA Content and Ploidy Level in Rubus by Flow Cytometry

Knowledge of the chromosome number in Rubus would be valuable when planning crosses and identifying plants, etc., however, preparation of tissue for microscopic evaluation and chromosome counting is difficult and time-consuming. Flow cytometry offers a more-efficient approach to this task. DNA flow cytometry was used to determine the nuclear DNA content in 22 Rubus genotypes. The genotypes represented a range of reported chromosome numbers from 2x to 12x. Six of the genotypes were representatives of Rubus ursinus, which is reported to have both 8x and 12x forms. Samples of nuclei were prepared from leaf discs of newly emerged and mature leaves following published protocols with some modifications. The DNA content was estimated by comparison of the fluorescence of Rubus nuclei with an internal DNA standard. There was an increase in nuclear DNA content concurrent with the increase in chromosome number. In these studies DNA flow cytometry could differentiate genotypes that differed by 2x, such as 6x and 8x, but could not reliably distinguish genotypes that differed by 1x, such as 7x vs. 8x or 6x. Aneuploids cannot be differentiated at this time.

Flow cytometric analysis does not reliably differentiate benign from malignant phaeochromocytoma.

The differentiation of benign from malignant phaeochromocytoma is difficult. We have examined whether the use of flow cytometric determination of nuclear DNA content would be useful as a predictor of malignant behaviour in patients with phaeochromocytoma as some previous studies had suggested that a diploid cytometric DNA pattern indicated benign disease. DNA flow cytometry was performed on phaeochromocytoma tissue from 36 patients (19 female, 17 male; mean age at presentation 39.5 years). The results were correlated with clinical outcome after prolonged follow-up. DNA histograms were constructed following nuclear suspension analysis. Of 26 patients followed up for more than 5 years after initial removal of primary phaeochromocytoma, three have died from malignant recurrence. In these patients a diploid DNA cytometric pattern was observed in two and an aneuploid pattern in one. Twenty-one patients are still alive. DNA cytometry showed a diploid pattern in the one patient who developed recurrent phaeochromocytoma 4 years after removal of a primary tumour. In this study, three of nine patients with an apparently benign diploid cytometric pattern subsequently developed recurrent disease. Routine use of DNA flow cytometry did not reliably differentiate benign from malignant phaeochromocytoma. Prolonged clinical and biochemical follow-up is still necessary for all patients with this condition.

Genome size, fluorochrome banding, and karyotype evolution in some Hypochoeris species.

Four South American and two European species of Hypochoeris (Asteraceae) were studied using fluorochrome banding, and genome size was determined by flow cytometry, in order to obtain information about microevolution in this genus and about its primary origin. Fluorochrome banding patterns showed GC-rich repeated sequences, particularly around the nucleolar organizer regions. Few differences appeared among the South American species. Nevertheless, determination of nuclear DNA content and base composition revealed significant differences among these species. The phylogenetic position of Hypochoeris robertia, which has the smallest DNA content, is discussed with regard to chromosome evolution in this genus.

Analysis of correlation between nuclear DNA content, chromosome number, and flowering capacity of asymmetric somatic hybrids of diploid Solanum brevidens and (di)haploid S. tuberosum.

Selected stable standard Solanum genotypes and two populations of asymmetric somatic hybrids between Solanum brevidens (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24) were analyzed to test the resolution of flow cytometric determination of nuclear DNA content (2C value) in terms of a loss or gain of chromosomes, and to analyze the correlation between the 2C value and the chromosome number. Correlation was high within the standard genotypes (r=0.99, P<0.001). However, the correlation was low within the two populations of somatic hybrids (r=0.54 and 0.65, 0.01<P<0.05). Highly chimeric , hybrids were readily detected by flow cytometry, whereas microscopic chromosome observations failed to detect them. Data were analyzed regarding their potential usefulness in predicting the flowering capacity. The 2C values deviating from those of the euploid standard genotypes were highly associated with abnormal morphology and lack of flowering. Flowering capacity did not correlate with chromosome number. These results suggest that flow cytometry can be utilized for initial selection of somatic hybrids.

Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum)

Flow cytometric determination of nuclear DNA contents in embryos of dry, fully matured pepper seeds revealed only 2C signals. Therefore, pepper belongs to those species in which the quiescent embryo arrests nuclear division in the presynthetic G1 phase. In whole seeds, two other peaks were observed which corresponded with the 3C and 6C DNA content and originated from the triploid endosperm. After 36 to 48 h of imbibition in water (48 to 60 h before visible germination), a rise in the 4C signal was observed in the root tip of the embryo.Osmotic preconditioning (priming) of pepper seeds for 7, 14 or 21 days in polyethylene glycol (PEG) or KNO3+ K3PO4 considerably reduced the time to 50%; germination (t50) and the mean germination time, and the effect was proportional to the duration of the priming treatments. An induction of the 4C signal was firstly observed after 10 days osmopriming in either PEG or KNO3+ K3PO4. For both priming methods, a positive correlation was found between the efficiency of the treatment to reduce the mean germination time, and the induction of DNA synthesis, measured as the amount of 4C versus 2C, at 12 h before radicle protrusion. Apart from the quantitative effect, there was also a temporal influence of priming on DNA synthetic activity. When primed seeds were subsequently imbibed in water, the induction of DNA synthesis started about 12 h earlier than in untreated seeds. This was also found for seeds primed for 7 days, when no induction of DNA synthesis had yet occurred. Apparently, during priming of pepper seeds there is a lag period of 9 days in which physiological processes other than DNA synthesis can take place. These processes sustain a rapid inception of nuclear replicative activity upon imbibition. By comparing the relative nuclear DNA contents of dry, imbibed and primed seeds, we provide information about the relation between the percentage of cells in seeds which have entered the replication stage of nuclear division, and germination performance.

Attempts to improve strain A-101 of Yarrowia lipolytica for citric acid production from n-paraffins

Citric and isocitric acid production were measured in strain A-101 of Yarrowia lipolytica. Product yield on n-hexadecane, including substrate used for cell growth, was in the range of 1·08–1·44 g total acid/g n-hexadecane. The citric acid fraction of total acids synthesized by a particular subculture was in the range 54–64%. Mutants of Y. lipolytica incapable of growth on citrate were isolated at low frequency and were extremely unstable. The difficulty was due to ploidy of our strain as confirmed by determination of nuclear DNA content, average cell diameter, and UV inactivation kinetics. Some mutants were also isolated that excreted predominantly isocitric acid when grown on n-hexadecane.