2C DNA Research Articles

Genetic variability in Erianthus arundinaceus accessions native to Japan based on nuclear DNA content and simple sequence repeat markers

Erianthus arundinaceus, part of the Saccharum complex, is increasingly important as a bioenergy crop and a potential resource for sugarcane improvement. Germplasm is widely distributed in the tropical zone in Southeast Asia and southern China, and has been found in the subtropical and temperate zones in Japan. Here, we investigated the 2C DNA content and simple sequence repeat (SSR) polymorphisms in E. arundinaceus accessions native to Japan. To estimate DNA content, flow cytometric analysis was performed on accessions collected from three climatic zones (temperate, subtropical, and tropical) in Japan and Indonesia. DNA content ranged from 7.42 to 8.10 pg (mean 7.62 pg), and all examined accessions fell into two groups reflecting collection location: the temperate zone in Japan (mean 8.06 pg) and the subtropical and tropical zones in Japan and Indonesia (mean 7.56 pg). Although DNA content differed significantly between the accessions from temperate and tropical/subtropical zones, chromosome number was inferred to be identical in all accessions. A phylogenetic tree of 42 accessions in the Saccharum complex based on detected fragments from 31 SSR primer pairs classified Erianthus and related genera into well-defined distinct groups. Thus, both 2C DNA content and phylogenetic analysis subdivided the E. arundinaceus accessions into two groups, suggesting that some of the Japanese accessions had different genetic characteristics from other accessions from Japan and Indonesia.

The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture

Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.

Abstract 5205: Poly(ADP-ribose) polymerase inhibitors induce β-radiosensitization through an altered selection of DNA double-strand break repair pathways

Abstract Background: Systematically reviewing published studies on a broad range of potential radiosensitizers, we previously reported that poly(ADP-ribose) polymerase inhibitors (PARPi) may have a unique radiosensitizing effect to enhance β-components of the linear-quadratic model. In contrast, many other radiosensitizing agents predominantly modified α-components. The aim of this study is to elucidate underlying mechanisms of “β-radiosensitization”. Methods and Results: We first confirmed that treatments with PARPi PJ34, olaparib, or veliparib two hours prior to and 48 hours after 10 Gy of ionizing radiation (IR) potentiated radiation-induced cell death in human cancer cells HCT116, HT29, H460 and A549 using the colony-forming assay. Tumor regrowth assay with 3-dimensional multicellular spheroids of HCT116 and H460 cells showed that PJ34 significantly delayed tumor regrowth when combined with single dose of 10 Gy but not in tumors treated with 10 Gy in 5 fractions. Similar results were found using HCT116 tumor xenograft model in mice. We next examined what cellular events may underlie the radiosensitization using PARPi PJ34 in HCT116 cells. Kinetics of DNA damage responses were first measured by phosphorylation of histone H2AX (γH2AX). PJ34 did not change an initial increase of γH2AX following 10 Gy IR, but γH2AX levels decreased faster in PJ34-treated cells than control cells. Immunocytochemical analysis of Rad51 showed that PJ34 reduced Rad51 foci formations up to 6 hours following 10 Gy IR. IR-induced chromosomal aberrations were quantitated by the C-banding of metaphase spreads. More dicentric chromosomes were found in PJ34-treated cells than the control. These data suggested that PARPi did not inhibit total DNA double-strand break (dsb) repair but altered the selection in DNA dsb repair pathways with increased end-joining type(s) of (mis)repair compensating for reduced homologous recombination repair. Cell cycle profiles showed sustained 4C DNA cell peaks in cells treated with PJ34 up to 72 hours following 10 Gy IR. Cell cycle markers (cyclin B1 and phosphorylated histone H3) indicated that the 4C DNA peaks of the PJ34- treated cells included substantial amount of G1 tetraploid cells in addition to G2- or M-phase diploid cells. Additionally, senescence-associated β-galactosidase staining showed that PJ34 promoted radiation-induced premature senescence, while smaller IR-induced sub-G1 populations were found in PJ34-treated cells than the control. Conclusion: PARPi increased IR-induced chromosomal aberrations through the altered selection in DNA dsb repair pathways. The increased quadratic misrepair enhanced β-components of IR-induced cell death mediated by the increased formation of G1 tetraploid cells and premature senescence. DNA dsb repair pathway selection may be a novel and effective target to elicit β-radiosensitization. Citation Format: Yuji Seo, Keita Yoshizaki, Keisuke Tamari, Yutaka Takahashi, Keisuke Otani, Masahiko Koizumi, Kazuhiko Ogawa. Poly(ADP-ribose) polymerase inhibitors induce β-radiosensitization through an altered selection of DNA double-strand break repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2017-5205

Physiological, cellular and molecular aspects of the desiccation tolerance in Anadenanthera colubrina seeds during germination.

During germination, orthodox seeds become gradually intolerant to desiccation, and for this reason, they are a good model for recalcitrance studies. In the present work, physiological, biochemical, and ultrastructural aspects of the desiccation tolerance were characterized during the germination process of Anadenanthera colubrina seeds. The seeds were imbibed during zero (control), 2, 8, 12 (no germinated seeds), and 18 hours (germinated seeds with 1 mm protruded radicle); then they were dried for 72 hours, rehydrated and evaluated for survivorship. Along the imbibition, cytometric and ultrastructural analysis were performed, besides the extraction of the heat-stable proteins. Posteriorly to imbibition and drying, the evaluation of ultrastructural damages was performed. Desiccation tolerance was fully lost after root protrusion. There was no increase in 4C DNA content after the loss of desiccation tolerance. Ultrastructural characteristics of cells from 1mm roots resembled those found in the recalcitrant seeds, in both hydrated and dehydrated states. The loss of desiccation tolerance coincided with the reduction of heat-stable proteins.

Determination of DNA content and relative 2C genome sizes of some promising commercial varieties of sugarcane using flow cytometer

&lt;p&gt;In the present study, 2C DNA content and the genome sizes (in picograms-pg and megabase pairs-Mbp respectively) of 19 promising commercial varieties of sugarcane, the derivatives of man-made interspecific hybrids between cultivated and wild species were analyzed using flow cytometry. In this work, 2C nuclear DNA content was determined. Knowing the 2C nuclear DNA content, the unknown chromosome numbers of the varieties could be predicted. Large differences (65 % variation) in DNA content (2C) of 19 varieties were detected, ranging, from 3.80 pg to 10.96 pg, which corresponds to a genome size ranging from 3724.00 Mbp to 10740.80 Mbp due to the variation of ploidy level and are considered the most complex genomes among crop plants. However, the relationship between chromosome number and genome size was highly significant (P &amp;lt; 0.001). In the present study, internode diameter, Sugar juice content and cane yield/ha are also positively correlated with DNA content. The estimated genome sizes would also yield information critical for sugarcane breeding and genome sequencing programs. &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Keywords&lt;/strong&gt;&lt;strong&gt;: &lt;/strong&gt;Genome size, Sugarcane varieties, Flow cytometry, DNA content.&lt;/p&gt;

The largest fungal genome discovered in Jafnea semitosta

Jafnea semitosta is an ascomycete (Pyronemataceae, Pezizales) originating from North America and spreading uncommonly in Europe. Its genome size was measured via flow cytometry of fruiting bodies from five localities in the Czech and Slovak Republic. The nuclear 1C DNA content was estimated at 3.706 ± 0.011 pg (~3.625 ± 0.011 Gbp) which represents the highest value ever reported for fungi and 100× higher than the average. Generally, the genome inflation in fungi appears to be driven mainly by proliferation of repetitive sequences, but polyploidy should also be considered in further studies on this greatly unexplored topic.

Variability of stem morphology in Lycopodium clavatum (Lycopodiaceae) is not related to ploidy level

Background and aims – Based on pedicel length, three morphological forms of Lycopodium clavatum L. are recognized. It has been previously reported that these are correlated with the ploidy level. The present investigation compares for the first time in Poland the diversity in pedicel length with the corresponding nuclear DNA amount and the ploidy level of L. clavatum. We aimed to find correlation between the morphotypes and the ploidy level in the Polish population of L. clavatum.Methods – Samples of shoots were taken from two clumps of L. clavatum. The ploidy level and 2C DNA content analysis was combined with pedicel length measurements. The ploidy level and genome size estimation were performed using flow cytometry.Key results – We found the occurrence of two morphological types of generative stems. The morphotype A produced distinct long pedicels (10.8–43.3 mm). The morphotype B had visible, but very short pedicels (4.5–9.3 mm). We tested the already existing hypothesis, according to which the A and B morphotypes represent respectively diploid and triploid ploidy level. Surprisingly, we did not find such a correlation. Regardless of the morphotype, all the studied plants were diploid, with 5.04–5.13 pg/2C nuclear DNA content.Conclusions – The occurrence of stems of different morphology in L. clavatum was not linked to the ploidy level. It is not excluded that the morphotypes may be genetically determined, but in the studied material, the ploidy level is certainly not at the origin of their differentiation.

Reproductive biology of the native forage grass Trichloris crinita (Poaceae, Chloridoideae).

Trichloris crinita is a perennial forage grass species native to arid regions of the American continent. Due to its extensive area of distribution, good forage quality and resistance to drought and grazing, this species is widely utilised as forage and for revegetation purposes in environments with low water availability. Despite its importance, genetic improvement of T.crinita has been very limited, partly as consequence of the lack of knowledge on its mode of reproduction. In the present work, we studied the reproductive biology of T.crinita by means of embryological analyses, flow cytometric seed screen (FCSS), self-compatibility tests and progeny testing with morphological and molecular markers. Cytological analyses revealed embryo sacs with eight nuclei and of Polygonum type for all T.crinita accessions analysed. FCSS histograms exhibited two clear peaks corresponding to 2C and 3C DNA content, indicating embryo sacs of sexual origin. Controlled pollination experiments designed to evaluate seed set (%) demonstrated that T.crinita is self-compatible, whereas results from morphological and simple sequence repeat (SSR) marker analysis of progeny revealed lack of outcrossing. Together, these results indicate that T.crinita reproduces sexually. It is a self-compatible and autogamous species. It is expected that these data will have a positive impact in the genetics and breeding of this species, and therefore contribute to its proper utilisation in arid regions.

Models of amphibian myogenesis - the case of Bombina variegata.

Several different models of myogenesis describing early stages of amphibian paraxial myotomal myogenesis are known. Myoblasts of Xenopus laevis and Hymenochirus boettgeri change their position from perpendicular to parallel, in relation to axial organs, and differentiate into mononucleate myotubes. In Bombina variegate the myotomal myoblasts change their shape from round to elongate and then differentiate into mononuclear, morphologically mature myotubes. In Pelobates fuscus and Triturus vulgaris, myoblasts fuse into multinuclear myotubes. Mono- and multinucleate myotubes achieve morphological maturity during the differentiation process. During myogenesis of B. variegata, the nuclei of mononucleate, differentiating myotubes contain a tetraploid quantity of DNA (4C DNA). The stable quantity of DNA is confirmed by lack of 3H-thimidine incorporation into myotube nuclei. This outcome is a proof that myoblasts withdraw from the cell cycle in the G2 phase. The further development of myotomal myotubes involves myoblasts of mesenchymal origin. These myoblasts fuse with myotubes in X. laevis and B. variegate in the G1 phase. Secondary muscle fibres in amphibian myotomes have only mesenchymal origin. Mesenchymal myoblasts fuse into multinucleated myotubes. Myofibril development in the differentiating myotube and lack of DNA replication confirm the classical paradigm of myogenesis. Mesenchymal myoblasts are taking part in the myogenesis of musculus rectus abdominis and limb muscles. The mesenchymal cells in the myogenesis process show one model of myogenesis, which is a classical model of myogenesis. The mesenchymal cells probably come from dermatome.

Nuclear 2C DNA and genome size analysis in somatic embryo regenerated gladiolus plants using flow cytometry

Gladiolus is a valuable asexually propagated plant of horticultural importance. Here, in the present communication, in vitro embryogenesis protocol and 2C DNA content of embryo regenerated plant are described. Callus was first induced from corms on 2, 4-D and NAA + BAP amended MS medium and the frequency was maximum (75.12%) in 0.5 mg/l NAA + 0.5 mg/l BAP added medium. The callus differentiated into embryos on 2, 4-D at variable numbers (3.13-5.32/callus mass); the addition of 1.0 mg/l BAP and 0.25 mg/l NAA was found very efficient in proliferating embryos (7.99/callus mass). Direct somatic embryos were also formed on corm surfaces on 2, 4-D (0.5-1.0 mg/l) amended medium at varying numbers. The embryos did not progress to maturity in same induction medium, so other PGR treatments containing GA3 and ABA were added. The amendment of GA3 was more responsive compared to ABA and 0.5-1.0 mg/l of GA3 was identified as best effective treatment. The embryos showed a maximum of 62.15% maturity in 0.5 mg/l GA3 added medium after 8th of culture. On BAP containing medium the mature embryos converted into plantlets and highest germination (42.65%) was noticed on 0.5 mg/l BAP added medium. The 2C DNA content of regenerated plant was measured by flow cytometry and was noted to be 1.34 pg. The somatic embryo derived plantlets are true-to-type, stable and grew normally in outdoor conditions, genome size is identical to corm derived gladiolus plants. This is the first flow cytometric DNA analysis description in somatic embryo regenerated gladiolus plant.

How genome size variation is linked with evolution within Chenopodium sensu lato

The significance of the wide interspecific variation in nuclear genome size of angiosperms is still not fully understood. It has been repeatedly suggested, however, that genome size can impose phenotypic constraints on plant development, phenology and ecological performance. We analysed nuclear genome size variation in diploid and polyploid species of Chenopodium s. lat. within a phylogenetic framework using flow cytometry of 456 accessions from 175 populations of 49 species: 32 diploids, 12 tetraploids, 4 hexaploids, and one decaploid. To this end, we also determined chromosome numbers and did an ITS-based phylogenetic reconstruction to analyze genome size variation using the phylogenetic generalized least-squares approach (PGLS). Results of these experiments can be summarized as follows. (1) We report the first chromosome counts for five Chenopodium species. (2) Flow cytometry determined that 2C and 1Cx DNA values differed up to 7.83- and 3.60-fold, respectively, with the lowest 1Cx value for C. schraderianum (0.412pg) [excluding tetraploid (2n=4x=32)C. ambrosioides with x=8 and 1Cx DNA content 0.279pg] and the highest for C. californicum (1.484pg). (3) Our extended phylogeny confirms the existence of previously recognized basic evolutionary lineages while underscoring the need to further increase taxon sampling for a full understanding of relationships in Chenopodioideae. (4) Our analysis of genome size evolution estimated the ancestral genome size of Chenopodium s. lat. at 0.541pg/1Cx. In addition, the data revealed a correlation between 1Cx DNA content and ploidy level. Moreover, the PGLS approach indicated that the genome size variation (i) followed the random walk model, indicating no unambiguous trend towards genome size increase or decrease; (ii) was correlated with phylogeny (λ=0.987); (iii) evolved gradually (κ=2.256); and (vi) occurred rather late after speciation, which can be attributed to species-specific adaptation (δ=3.000). (5) There are indications that several ecological traits were significantly associated with 2C DNA content. While mean plant height, maximum plant height, fruit diameter and life form were positively correlated with genome size, the species’ continent of origin showed no correlation. In summary, the strong phylogenetic signal detected in genome size of Chenopodium indicates that its genome size variation is significantly associated with phylogenetic divergence. We herein suggest a pattern of species-specific adaptations in the evolution of Chenopodium genome size.

Intraspecific variations in cardamom (Elettaria cardamomum Maton): assessment of genomic diversity by flow cytometry, cytological studies and ISSR analysis.

Background The main goal of the work was to analyse intraspecific variation in Elettaria cardamomum Maton (cardamom) using genome size, cytological studies and molecular marker data. Nuclear DNA content and molecular marker details furnish data on genome size and genetic diversity respectively among the studied accessions and both complement each other for evolutionary and taxonomic studies.ResultsThe relative 2C genome size and total number of base pairs of cardamom was determined through flow cytometric analysis using propidium iodide staining. The nuclear DNA content was estimated in various sections of the species representing individuals from wild and cultivar genotypes following Zea mays L. CE-777 (2C = 5.43 pg) as internal reference standard. Chromosome number from growing root tip was examined following standard protocols. Twenty-six ISSR primers that generated polymorphic bands were used for genetic diversity analysis of the thirty accessions of cardamom. Estimated nuclear 2C DNA content ranged from 2.57 to 3.22 pg demonstrating 1.25-fold variation. The mean amount of 2C nuclear DNA of the cardamom was calculated as 2.87 pg which is equivalent of 2806 Mbp as the diploid genome size. The chromosome number was found to be 2n = 48. Among the thirty accessions of cardamom studied using ISSR markers, C53 (feral from Bonacaud) showed a very prominent level of genetic diversity and was lowest for C96 (Avinash-I, a released variety from Indian Institute of Spices Research, Kozhikode).ConclusionThese analyses revealed the existence of genetic variability within the studied cardamom accessions. The plant specimens also differed significantly in their genome size. However, the genetic variability parameters did not show any correlation with genome size.

RDNA mapping, heterochromatin characterization and AT/GC content of Agapanthus africanus (L.) Hoffmanns (Agapanthaceae).

Agapanthus (Agapanthaceae) has 10 species described. However, most taxonomists differ respect to this number because the great phenotypic plasticity of the species. The cytogenetic has been an important tool to aid the plant taxon identification, and to date, all taxa of Agapanthus L'Héritier studied cytologically, presented 2n = 30. Although the species possess large chromosomes, the group is karyologically little explored. This work aimed to increase the cytogenetic knowledge of Agapanthus africanus (L.) Hoffmanns by utilization of chromosome banding techniques with DAPI / CMA3 and Fluorescent in situ Hybridization (FISH). In addition, flow cytometry was used for determination of DNA content and the percentage of AT / GC nitrogenous bases. Plants studied showed 2n = 30 chromosomes, ranging from 4.34 - 8.55 µm, with the karyotype formulae (KF) = 10m + 5sm. Through FISH, one 45S rDNA signal was observed proximally to centromere of the chromosome 7, while for 5S rDNA sites we observed one signal proximally to centromere of chromosome 9. The 2C DNA content estimated for the species was 2C = 24.4 with 59% of AT and 41% of GC. Our data allowed important upgrade for biology and cytotaxonomy of Agapanthus africanus (L.) Hoffmanns.

A First Glimpse of Wild Lupin Karyotype Variation As Revealed by Comparative Cytogenetic Mapping.

Insight into plant genomes at the cytomolecular level provides useful information about their karyotype structure, enabling inferences about taxonomic relationships and evolutionary origins. The Old World lupins (OWL) demonstrate a high level of genomic diversification involving variation in chromosome numbers (2n = 32–52), basic chromosome numbers (x = 5–7, 9, 13) and in nuclear genome size (2C DNA = 0.97–2.68 pg). Lupins comprise both crop and wild species and provide an intriguing system to study karyotype evolution. In order to investigate lupin chromosome structure, heterologous FISH was used. Sixteen BACs that had been generated as chromosome markers for the reference species, Lupinus angustifolius, were used to identify chromosomes in the wild species and explore karyotype variation. While all “single-locus” in L. angustifolius, in the wild lupins these clones proved to be “single-locus,” “single-locus” with additional signals, “repetitive” or had no detectable BAC-FISH signal. The diverse distribution of the clones in the targeted genomes suggests a complex evolution history, which possibly involved multiple chromosomal changes such as fusions/fissions and repetitive sequence amplification. Twelve BACs were sequenced and we found numerous transposable elements including DNA transposons as well as LTR and non-LTR retrotransposons with varying quantity and composition among the different lupin species. However, at this preliminary stage, no correlation was observed between the pattern of BAC-FISH signals and the repeat content in particular BACs. Here, we describe the first BAC-based chromosome-specific markers for the wild species: L. cosentinii, L. cryptanthus, L. pilosus, L. micranthus and one New World lupin, L. multiflorus. These BACs could constitute the basis for an assignment of the chromosomal and genetic maps of other lupins, e.g., L. albus and L. luteus. Moreover, we identified karyotype variation that helps illustrate the relationships between the lupins and the extensive cytological diversity within this group. In this study we premise that lupin genomes underwent at least two rounds of fusion and fission events resulting in the reduction in chromosome number from 2n = 52 through 2n = 40 to 2n = 32, followed by chromosome number increment to 2n = 42.

Nuclear DNA content variation within the genus Daucus (Apiaceae) determined by flow cytometry

The genus Daucus (Apiaceae) comprises species from around the world, covering a wide climatic range, and showing great morphological plasticity. Both cultivated and wild forms are described within the genus. The aim of the present study was to estimate the genome size variability in the collection of diploid Daucus species differing in chromosome number (2n=18, 20 or 22) and originating from various regions of the world. In total, the 2C DNA content in 19 accessions of nine wild Daucus species, as well as in 22 wild and 26 cultivated accessions of Daucus carota L. was measured by flow cytometry. The 2C DNA content varied over 3-fold, from 0.920pg in D. carota subsp. maximus (2n=18, Spain) to 3.019pg in D. littoralis (2n=20, Israel). The interspecific genome size differences within wild Daucus species were pronounced, e.g. D. carota (2n=18, Greece) possessed 0.940pg/2C DNA, D. broteri (2n=20, Cyprus) 2.218pg/2C and D. montevidensis (2n=22, South America) 1.295pg/2C. In the group of 22-chromosome Daucus species, high similarity in nuclear DNA content among different accessions of one species was observed, in contrast to the 20-chromosome Daucus species, suggesting possible taxonomical misclassifications of accessions within the latter group. The nuclear DNA content of non-cultivated forms of D. carota varied from 0.920pg/2C in subspecies maximus (Spain) to 1.154pg/2C in subspecies halophilus (Portugal), which reflects 20% difference. In turn, in the group of cultivated carrots we observed a high homogeneity among the studied accessions, the 2C DNA content ranged between 0.950pg (‘Kuroda type II’, China; ‘Imperial Long Scarlet’, Japan; ‘Koral’, Poland) and 0.977pg (‘Cape Market’, South Africa). The mean 2C value calculated for both wild and cultivated forms of D. carota amounted 0.973pg.

Taxonomic remarks on the genus Sternbergia L. (Amaryllidaceae) in Turkey based on leaf anatomy, karyosystematic analysis and nuclear DNA content

Anatomy of leaf cross sections, karyological studies, and nuclear DNA content analysis by flow cytometry were carried out on the six Sternbergia species from Turkey: S. candida (endemic to the country), S. clusiana, S. colchiciflora, S. lutea, S. sicula and S. vernalis. In karyological analysis, number and morphology of the somatic chromosomes, the ploidy level and karyotype formula of the specimens used in the study were investigated in detail. The measurements of somatic chromosomes were calculated with formula of the relative variation in chromosome lenght (CVCL) and mean centromeric asymmetry (MCA). According to the results of this study, there are two different basic chromosome numbers. i.e. x = 10 (S. candida, S. clusiana, S. colchiciflora and S. vernalis) and x = 11 (S. lutea and S. sicula). Results of karyotype analysis corroborated results of previous studies. The smallest chromosome length was measured as 3.05 μm in S. clusiana while the longest length of 20.89 μm was observed in S. sicula. This is the first study reporting nuclear DNA content results for Sternbergia. Based on results of the nuclear DNA content analysis, there is a great variation within the genus in the interspecific level as 2C DNA content varies between 54.03 and 80.42 pg among the species investigated in the study. Anatomical features of the leaves were good taxonomic markers, especially concerning their palisade cells, ribs, cuticle, laticifer canals, vascular bundles, crystals, stomatal index, and stomata structure. Analytical drawings were presented. Six noteworthy leaf characters were quantified, organized in a data matrix, and analysed with UPGMA methods. In conclusion, cytological characteristics of the Sternbergia species together with their leaf features proved to be quite useful in identification and therefore can provide more clarity for taxonomic revision of the genus.

Nuclear DNA Content and Ploidy Level of Apple Cultivars Including Polish ones in Relation to Some Morphological Traits

Abstract Apple species and cultivars differ in nuclear (2C) DNA content and ploidy level. The majority of these genotypes are diploids, but there are some triploids and a few tetraploids. Nuclear DNA content is a specific feature and its flow cytometric evaluation can be helpful in differentiating taxa. For many apple genotypes – including all the Polish ones, these characteristics are not known. 2C DNA was evaluated in relation to leaf, flower, fruit, pollen grain and stomata sizes as well as to the flowering time for seventy genotypes (including 46 Polish cultivars) gathered in the gene bank of the Research Institute of Horticulture, Skierniewice, Poland. For standard cultivars with the known chromosome number, 2C value was 1.71 pg for diploid cultivar ‘Alwa’ (2n=2x=34), 2.55 pg for triploid ‘Boskoop’ (3x=51), and 3.37 pg for tetraploid genome (4x=68) of mixoploid ‘McIntosh 2x+4x’. In 61 cultivars (including 41 Polish ones), the nuclear DNA content ranged from 1.58 to 1.78 pg indicating their diploid chromosome number. Five cultivars were identified as triploids (‘Bursztówka Polska’, ‘Pagacz’, ‘Rapa Zielona’, ‘Rarytas Śląski’ and ‘Witos’) owing to their nuclear DNA amount ranging between 2.42 and 2.58 pg. Leaf, flower, fruit, stomata and pollen grain sizes were on average significantly larger in triploids. Thus, in 3x plants the mean leaf surface was 49.1 cm2, flower diameter – 52.4 mm, fruit weight – 204.7 g, stomata length – 32.1 μm and pollen grain diameter – 33.7 μm, whereas in diploids – 36.0 cm2, 46.1 mm, 162.7 g, 28.4 μm and 30.7 μm, respectively. Pollen grain viability was on average significantly higher in diploids (75.6%), compared to triploids (22%). These results confirm that in apple, as in many other plant species, the higher ploidy level of triploids is generally associated with increased sizes of pollen grains, stomata, flowers, fruits and leaves but decreased pollen viability. No clear correlation between ploidy level and flowering time was found. In the case of mixoploid apple genotypes possessing diploid and tetraploid genomes, some phenotype observation is helpful in describing the ploidy level of the histogenic layers, L1 and L2. Small stomata sizes (similar to diploid) indicate diploid L1 and larger leaf sizes, compared to diploid counterparts, show tetraploid L2. The results will be used for breeding, in which it is important to determine maternal and paternal genotypes as well as the direction of the crossing that is of great importance in obtaining seeds and materials for further selection.

Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA). By analysis of the duration of the fluorescence signal as well as the intensity of the DNA-related signal, it is possible to discriminate between cells in M/G1, S, and G2 This protocol describes how to prepare cells for flow cytometry and analyze them. We also describe the application of barcoding for more accurate comparison of samples.

The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed “progressively partial endoreplication” (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical “horseshoe” pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

The Small Nuclear Genomes of Selaginella Are Associated with a Low Rate of Genome Size Evolution.

The haploid nuclear genome size (1C DNA) of vascular land plants varies over several orders of magnitude. Much of this observed diversity in genome size is due to the proliferation and deletion of transposable elements. To date, all vascular land plant lineages with extremely small nuclear genomes represent recently derived states, having ancestors with much larger genome sizes. The Selaginellaceae represent an ancient lineage with extremely small genomes. It is unclear how small nuclear genomes evolved in Selaginella. We compared the rates of nuclear genome size evolution in Selaginella and major vascular plant clades in a comparative phylogenetic framework. For the analyses, we collected 29 new flow cytometry estimates of haploid genome size in Selaginella to augment publicly available data. Selaginella possess some of the smallest known haploid nuclear genome sizes, as well as the lowest rate of genome size evolution observed across all vascular land plants included in our analyses. Additionally, our analyses provide strong support for a history of haploid nuclear genome size stasis in Selaginella. Our results indicate that Selaginella, similar to other early diverging lineages of vascular land plants, has relatively low rates of genome size evolution. Further, our analyses highlight that a rapid transition to a small genome size is only one route to an extremely small genome.