Chromatin live imaging integrated with programmable DNA binding proteins derived from genome editing methods opens up the era of spatio-temporal analyses of chromatin dynamics. The DNA binding proteins, including a transcription activator-like effector (TALE) or a nuclease-dead CRISPR-associated protein 9 (dCas9), can be freely designed to bind a specific DNA sequence. Instead of a nuclease for DNA cleavage, a fluorescent protein is fused to TALE or dCAS9 for visualization of chromatin. They enable us to observe the endogenous genomic loci without the fixation of cells, the denaturation of DNA for hybridization and the insertion of exogenous DNA sequences into targeted loci. Using these live imaging systems dependent on the interaction between cis endogenous DNA sequence and trans DNA binding proteins, we can analyze chromatin dynamics in living cells. Multicolor chromatin imaging based on different dCas9 proteins will be a powerful cytogenetical technique instead of multicolor FISH and chromosome painting.
In this study, the chromosome number and detailed morphometric properties of six endemic taxa belonging to five dicot families which grow naturally in Turkey were examined. The somatic chromosome number and karyotype formula of the taxa of Asteraceae family were determined to be 2n=18 and 8m+1sm in Tanacetum argenteum ssp. argenteum and 2n=18 and 7m+2sm+1B in Tanacetum densum ssp. sivasicum, respectively. The somatic chromosome number and karyotype formula were determined as 2n=32 and 10m+6sm in Fumana trisperma belonging to Cistaceae, 2n=14 and 5m+2sm in Onobrychis albiflora belonging to Fabaceae, 2n=14 and 2m+5sm in Salvia blepharochlaena belonging to Lamiaceae, and 2n=14 and 2m+5sm in Glaucium acutidentatum belonging to Papaveraceae family. The somatic chromosome numbers of six taxa collected from different localities in Turkey were counted for the first time, except for T. argenteum var. argenteum and Salvia blepharocleana.
Three species of Tacca J. R. & G. Forst., namely T. plantaginea, T. chantrieri and T. integrifolia, were investigated cytogenetically and at the molecular level to solve their taxonomic rank. The diploid chromosome number 2n=28 was found in T. plantaginea and T. integrifolia whereas 2n=22 was found in T. chantrieri. This is probably the first report on the diploid chromosome number of T. plantaginea and T. chantrieri around the world. The three species differed in respect of centromeric formulae such as 22m+6sm in T. plantaginea, 18m+4sm in T. chantrieri and 28m in T. integrifolia. In addition to regular 2n=22 chromosomes, four to eight small chromosomes were found in some cells of T. chantrieri suggesting the probable occurrence of B-chromosome. In T. integrifolia, a pair of satellites was found on the short arm, one in each member of pair VI, after orcein and CMA staining but not in DAPI, revealing the GC-rich nature. A wide range of CMA- and DAPI-positive bands was found in the metaphase chromosomes of the three species. DAPI-fluoresced chromosomes were frequent in these three species. Six primer combinations were applied for RAPD analysis in the three species of Tacca to find out their genomic relationship. The three species showed some unique bands useful as markers for each species. Conventional and fluorescent karyotype together with RAPD fingerprinting indicated that T. integrifolia is distantly related to T. plantaginea and T. chantrieri. Therefore, the compilation of the above cytological and molecular information will be very useful for authentic identification of the three Tacca species. This genetic information will help in the proper conservation of and to make patent for the three threatened Tacca species found in Bangladesh.
A total of 61 populations of A. indicum were collected from different regions of the North Indian Plains. All the collections showed n=21 as the gametic chromosome number with normal meiotic behavior in most of the populations. However, 23 populations showed abnormal meiosis with some of the following: laggards, secondary association of chromosomes, bridge formation, cytomixis, vagrants, early disjunction, interbivalent connections and multipolarity. The abnormalities manifested through the formation of monads, dyads, infertile pollens, etc. One population each from Jaipur and Udaipur districts of Rajasthan exhibited the presence of 0-1 B-chromosome along with 21 bivalents. This is the first report for B-chromosome in the species at world level.
Nine varieties of Cicer arietinum L. (chickpea) released by Bangladesh Agriculture Research Institute (BARI), viz. BC-1 (BARI Chola-1), BC-2, BC-3, BC-4, BC-5, BC-6, BC-7, BC-8 and BC-9, represented a broad spectrum of variation for several phenotypic and agronomic traits. For authentic characterization, these varieties were investigated cytogenetically by differential fluorescent banding. The nine chickpea varieties were found to possess 2n=16 metacentric chromosomes. A wide range of CMA-positive bands (5–20) was found in the metaphase chromosomes of the nine varieties. Two satellites were found in the first pair of only BC-2, BC-3, BC-4, BC-6, BC-8 and BC-9 after DAPI-staining. No satellite was found after CMA-staining. The differential staining property of satellites revealed the stain specific nature of these satellites. Entirely DAPI-fluoresced chromosomes were frequent in these varieties. The number, location and distributions of GC- and AT-rich repeats are specific for each variety. Some CMA- and DAPI-bands were so unique that these chromosomes could easily be used as marker chromosomes for the respective varieties. Fluorescent banding revealed the occurrence of genomic alteration within these varieties. Therefore, each variety could be characterized authentically by fluorescent banding analysis.
Male meiotic studies were performed on 14 wild accessions of Impatiens devendrae Pusalkar from the Western Himalayas. The species, which is endemic to the state of Uttarakhand, has been worked out for chromosome counts for the first time. The aim of the study was to analyse in detail the male meiotic course, microsporogenesis and pollen formation. All the scored accessions exhibited the same diploid chromosome number, n=7, as confirmed through male meiotic studies and pollen mitosis. The majority of the accessions exhibited normal meiotic course with regular seven bivalents formation, equal distribution of chromosomes during anaphases, regular sporads and high pollen fertility. However, some accessions showed the phenomenon of non-synchronous disjunction (early and late) of some bivalents, univalent chromosomes and unequal and scattered distribution of chromosomes during A-I leading to reduction in pollen fertility. As such the meiotic irregularities in these individuals of I. devendrae seem to be under the effect of some genetic factors.
This study presents a more detailed karyological analysis of seven Salvia L. taxa (S. ceratophylla, S. syriaca, S. palaestina, S. aethiopis, S. russellii, S. multicaulis, S. trichoclada) grown in Turkey. Also, this is the first chromosome number report for S. russellii and S. trichoclada. This study revealed that the chromosome numbers of the examined Salvia taxa were 2n=14, 18, 22 and 32. The Salvia taxa had median point (M), median (m), submedian (sm) and subterminal (st) chromosomes in the study. Furthermore, various karyotype asymmetry values, ploidy levels, karyotype formula, chromosome length range, total karyotype length, A1, A2 and Stebbins classification were determined in this study. On the other hand, Pearson correlations between the karyotype asymmetries of these Salvia taxa were determined and a dispersion diagram was formed by using A1 and A2. Furthermore, this study suggested that the examined Salvia taxa were 2A and only S. trichoclada was 3A.
The detailed meiotic behavior, chromosome number, morphological analysis and pollen fertility was studied in 10 populations of Melica persica belonging to family Poaceae from the cold desert region of Kinnaur. The collection and analysis of the species done on population basis showed a wide range of distribution with varied altitudinal habitats, and two distinct morphotypes-purplish red and creamy white colored spikelets. The collected morphotypes do not show any cytological variations and all the populations showed the same diploid chromosome number (2n=18 based on x=9). Two populations of white and one population of red morphotypes showed normal meiotic behavior while the rest of the seven populations showed a number of meiotic abnormalities including cytomixis, chromosome stickiness, unoriented bivalents, laggards, bridges and abnormal microsporogenesis in the form of dyad, leading to reduction in pollen fertility. In the present study, the existence of two morphotypes and the phenomenon of cytomixis have been reported for the first time in this grass species.
Every species has a unique karyotype, but certain genera have common karyptypes among species. The markers (chromosome length and centromere position) used in traditional karyotyping do not distinguish all chromosome pairs in the genus Pinus. However, the application of multi-probe fluorescence in situ hybridisation (FISH) procedures allowed exact karyotyping of 26 Pinus congeners. We used these new data to examine species relationships. The 35S rDNA and 5S rDNA, Arabidopsis-type telomere repeat sequences, and the proximal CMA band-specific repeat (PCSR) of P. densiflora were used as FISH probes for our analysis of chromosomes in 26 Pinus species. Each species had a unique FISH karyotype and most homologous chromosome pairs were identified. The FISH karyotypes were used to compare corresponding or homologous chromosomes among the species. Common or similar FISH signal patterns appeared in closely related species. Species that had inherited common FISH signal patterns were classified into four karyotype groups. We used cluster analyses to compare quantitative differences in FISH signals within these groups. The results of these analyses were consistent with recent systematic interpretations and resolved differences among existing taxonomic systems based on diverse methodologies. Our results indicate that FISH signal patterns reflect the history of species differentiation and that comparative FISH karyotyping has potential as an important tool for studying the taxonomy or phylogeny of Pinus.
Gemcitabine (GEM) is a chemotherapeutic agent with several dose limitation aspects. It was encapsulated in three microemulsion (ME) formulations with nanoparticles that differ in polarity: hydrophilic MEa, hydrophobic MEb and relatively neutral MEc. The apoptotic effect of the ME formulations was evaluated in the A549 non-small cell lung cancer cells, whereas the side effects of the formulas on the healthy cells were tested in the HFS human foreskin cells. The cell toxicity of all GEM-loaded-MEs (MEa+, MEb+ and MEc+) and free GEM solutions at concentrations of 1 and 10 µM was determined by using sulphorhodamine B (SRB) assay, while the mechanism of cell death was assessed by using ApopNexin FITC apoptosis detection kit and viewing the ultrastructure of treated A549 cells by using transmission electron microscope (TEM). It has been found that 10 µM of MEb+ (MEb10+) has the best antiproliferative and apoptotic effect compared to all of the ME formulations and GEM solutions. MEb10+ reduced the percentages of A549 cell viabilities to 11.15±1.4 whereas 10 µM of GEM (GEM10) decreased the percentages of A549 cell viabilities to 58.09±2.5. This study verified that ME formulations with hydrophobic droplets improved the therapeutic potential of GEM as an anticancer drug.
A coastal biennial herb, Lysimachia mauritiana, exhibited remarkable intraspecific karyotypic diversity, especially in the Ryukyu Archipelago of Japan (the Ryukyus). The species displayed five chromosome numbers (2n=16, 17, 18, 19, 20) and 18 cytotypes in the Ryukyus alone. Our serial investigations elucidated that: (1) 11 cytotypes on Takarajima Island (Is.) showed the highest intra- and inter-populational cytotype polymorphism in the Ryukyus, (2) a total of 15 cytotypes was recognized and several cytotypes coexisted in every locality on Amamioshima, Kakeromajima and Tokunoshima Is., and (3) closely located two Islands of Amamioshima and Tokunoshima had different dominant cytotypes, 16 (6m) and 18 (6m), respectively. In the present study, to explore whether a similar karyotypic polymorphism exists on their neighbor islands belonging to the Okinawa and the Daito Groups, a total of 610 plants from 51 localities on 10 islands were analyzed karyomorphologically and cytogeographically. As a result, five chromosome numbers (2n=16, 17, 18, 19, 20) and 13 cytotypes were recognized in the areas. Okinawajima Is. was divided into two areas according to cytotype distribution patterns. Northern and western areas facing the East China Sea showed intra- and inter-populational polymorphism in cytotype. By contrast, southern, eastern and northeastern areas facing the Pacific Ocean showed a single cytotype in a locality. In the Daito Group, 20 (4m) TS cytotype with a wide distribution in southern Taiwan was found in Japan for the first time. The cytotype probably descended from currents of southern Taiwan and/or the Philippines.
Few studies have aimed to develop physical chromosome mapping methods for cultivated strawberry (Fragaria×ananassa). Recently, linkage maps have been developed in this species using many single sequence repeat (SSR) markers. However, the accuracy of these needs to be evaluated because cultivated strawberries are allo-octoploids. This could be achieved by fluorescently labeling chromosomes that contain SSR markers on the same linkage group and observing whether other fluorescent signals can also be detected on the same chromosomes. The aim of this study was to develop a direct cycling primed in situ (C-PRINS) labeling technique for chromosome mapping of cultivated strawberry. To achieve this, we investigated the effect of the 1) maceration time for softening the root tips, 2) concentration of acetic acid for spreading chromosomes on the glass slide, 3) incubation time after slide preparation and before PRINS labeling, 4) number of PCR cycles for obtaining fluorescent signals, and 5) temperature accuracy for PCR. All of these factors were considered important for developing a chromosome labeling method. We found that a maceration time of 25 min was appropriate for obtaining many somatic cells with 56 chromosomes and that 45% acetic acid was effective for reducing the amount of damage to the chromosomes and obtaining clear chromosome images. A 72-h incubation time was appropriate for detecting chromosomes that had fluorescent signals, and the number of signals on the chromosomes increased with an increase in the number of PCR cycles. Finally, an aluminum tape treatment was effective for maintaining the PCR temperatures.
Detailed meiotic studies have been carried out in 11 accessions of Eremurus himalaicus Baker belonging to family Liliaceae—an endemic medicinal plant of the Northwestern Himalayas. E. himalaicus, due to its excessive exploitation for edible uses, is living under stress and has therefore been listed in the Red Data Book of Indian Plants as a ‘rare’ species. Considering x=7 as the basic chromosome number for the species, E. himalacus revealed the diploid chromosome count of 2n=14. Out of 11 populations worked out cytologically, 3 populations of the species were normal in their meiotic behavior while the rest of the populations showed abnormal meiotic behavior. The phenomenon of cytomixis, presence of two to five nucleoli and various other meiotic abnormalities were observed in the form of unoriented bivalents, chromosome stickiness, late disjunction, laggards and bridges in eight populations. Earlier studies were limited to the counting of chromosome number, so these meiotic abnormalities were reported for the first time in the studied species. Further, the microsporogenesis was also abnormal leading to the formation of monad, dyads, triads and abnormal tetrads with the fusion among tetrads which ultimately leads to reduced pollen fertility.
Scanning electron microscopy (SEM) is used to observe surface structures. The electron beam generally scans the cell surface at an acceleration voltage of 10–15 kV. We observed buried gold particles in cells at an acceleration voltage of 30 kV. In this paper, dim, opaque particles were observed on a leaf surface that was bombarded with gold particles using a particle gun. These gold particles were buried in epidermal cells and the outline of the particle was not clear compared to adhered particles on the leaf surface. A 1-µm hole (0.785 µm2 in area) made by a 1-µm gold particle was observed around the buried particle. We measured the areas of holes made by the passage of gold particles at 1 min (n=61), 2 min (n=56), 5 min (n=110), and 10 min (n=61) after bombardment using images obtained using a scanning electron microscope and ImageJ software. The size of the holes became smaller over time and was very small 10 min after bombardment. From these results, we reveal that the holes formed by gold particle bombardment are repaired after about 10 min.
We quantified C-band size variations of the No. 2 chromosomes in the Japanese house mice, Mus musculus, including wild-caught mice and their F1 offsprings produced by crosses between them. The sizes of C-bands were expressed by their relative lengths as a positively stained region / a negatively stained region by C-banding in each of the No. 2 homologue. In 11 of the 16 mice examined by one-way ANOVA, the relative lengths of the C-bands were statistically polymorphic in the No. 2 homologue with variable C-band sizes at p=0.05. On the other hand, the remaining five individuals carried slightly different relative lengths without significance, which is considered as a homomorphism. Usually, heterochromatin is thought to be evolutionally neutral without strong selections, and it is considered that the polymorphic state is a frequent phenomenon of the Japanese house mice without any cytogenetically deleterious effects. In addition, the mean of the difference between the relative lengths in the No. 2 homologue ranged from 0.046 to 0.210 in the 11 individuals showing polymorphic state and from 0.032 to 0.044 in the five showing homomorphic state. Thus, a mean index of approximately 0.045 is considered to be the boundary dividing polymorphic and homomorphic states.
Ultrastructural changes in the coral tissues and symbiotic zooxanthellae of the scleractinian coral, Pocillopora damicornis, after exposure to high temperature (HT), ultraviolet (UV) and far-red (FR) radiation were examined by transmission electron microscopy. In normal environmental conditions, the coral gastrodermis contained zero to two zooxanthellae and maintained well-defined cortical and intracellular structures. When corals were exposed to HT, UV and FR, the gastrodermal features changed remarkably depending on the duration of exposure. Treatment with HT for 15 min or exposure to UV and FR for 6 h induced the appearance of numerous, small, electron-dense granules in the gastrodermis. Autophagosomes and lysosomes in the gastrodermal cells also increased in number. The gastrodermal cells containing no zooxanthellae began to degrade and became vacuolated through the destruction and fragmentation of their cytoplasmic contents. Prolonged exposure to these stressors brought about the disintegration of the cells without zooxanthellae and led to the collapse of the gastrodermis. This resulted in the release of the other gastrodermal cells containing zooxanthellae from the gastrodermis to gastrovascular cavities. HT, UV and FR affected the gastrodermal cells without zooxanthellae more severely than the zooxanthellae themselves, although the thylakoids of the zooxanthellae were often destroyed when the coral was exposed to UV and FR. These results suggest that the coral receives the distinct stressors, HT, UV and FR, but has a common mechanism that promotes autolysis activities in the gastrodermis and discharges cells containing zooxanthellae.