CYTOLOGIA Vol. 79(2014) No. 4

This is the first report on standardized karyotype and idiogram analyses of Quoy's parrotfish, Scarus quoyi, from the Andaman Sea, Southern Thailand. Kidney cell samples were taken from three male and three female fish. Mitotic chromosome preparations were prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of Sc. quoyi was 2n=48, and the fundamental number (NF) was 74 in both males and females. The types of chromosomes were 4 large metacentric, 4 large submetacentric, 16 large acrocentric, 18 large telocentric, 2 medium acrocentric, 2 medium telocentric, and 2 small telocentric chromosomes. There were no irregularly sized chromosomes related to sex. The region adjacent to the short arm near the telomere of acrocentric chromosome pair 6 showed clearly observable nucleolar organizer regions/NORs. The karyotype formula for Sc. quoyi is as follows:
2n (diploid) 48=L₄^m+L₄^sm+L₁₆^a+L₁₈^t+M₂^a+M₂^t+S₂^t

We report the first chromosome analysis and chromosomal characteristics of nucleolar organizer regions/NORs in the tripletail wrasse (Cheilinus trilobatus) from the Andaman Sea, Southern Thailand. Kidney cell samples were taken from three male and three female fishes. The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of C. trilobatus was 2n=38, the fundamental number (NF) was 54 in both males and females. The types of chromosomes were 6 large metacentric, 4 medium metacentric, 2 medium submetacentric, 4 medium telocentric, 4 small submetacentric, and 18 small telocentric chromosomes. The region adjacent to the short arms near the telomeres of submetacentric chromosome pair 7 showed clearly observable NORs. There was no observation of irregularly sized chromosomes related to sex. The karyotype formula for C. trilobatus could be deduced as:
2n (diploid) 38=L₆^m+M₄^m+M₂^sm+M₄^t+S₄^sm+S₁₈^t

Chromosome analysis and morphometrics of the intermediate roundleaf bat (Hipposideros larvatus) from Northeast Thailand were studied. Bat chromosome preparations were conducted by squash technique from bone marrow and testis. Conventional staining, GTG-banding and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of H. larvatus was 2n=32, and the fundamental number (NF) was 66 in both males and females. The types of autosomes observed were 14 large metacentric, 2 large submetacentric, 4 medium metacentric, 4 medium submetacentric, 2 small metacentric, 2 small submetacentric, and 2 small telocentric chromosomes. The X-chromosome was a large submetacentric chromosome and the Y-chromosome was a small submetacentric chromosome. From the GTG-banding technique, the number of bands in H. larvatus was 159, and the location of each chromosome pair could be clearly differentiated. NORs are located at the secondary constriction near the centromere on the long arm of the large metacentric chromosome pair 8. We found that during metaphase I the homologous chromosomes showed synapsis, which can be defined as the 16 bivalents. Six external morphological characters were measured as well as 13 cranial and dental measurements. The karyotype formula of H. larvatus was as follows:
2n (44)=L₁₄^m+L₂^sm+M₄^m+M₄^sm+S₂^m+S₂^sm+S₂^t+sex-chromosomes

Chromosomal analyses of the common sun skink (Eutropis multifasciata) from mitotic and meiotic cell divisions were studied. Blood samples were taken from five male and five female skinks. Skink chromosome preparations were conducted by the squash technique from the bone marrow and testis. The chromosomes were stained by conventional staining and Ag-NOR banding techniques. The results showed that the diploid chromosome number of E. multifasciata was 2n=32, the fundamental number (NF) was 48 in both males and females. The types of chromosomes were present as 6 large metacentric, 2 large submetacentric, 6 small metacentric, 2 small submetacentric, 2 small telocentric macrochromosomes and 14 microchromosomes. There was no irregularly sized chromosome related to sex. We also observed distinctive nucleolar organizer regions (NORs) at the region adjacent to the short arms near the telomere of a pair of the largest metacentric chromosomes. We found that during diakinesis (prophase I) the homologous chromosomes showed synapsis, which can be defined as the 16 bivalents and 16 haploid chromosomes at metaphase II as diploid species. The karyotype formula of E. multifasciata is as stated:
2n (diploid) 32=L₆^m+L₂^sm+S₆^m+S₂^sm+S₂^t+14 microchromosomes

Vacuoles occupy 80–90% of a mature plant cell and mainly contribute to all types of cell expansion. Zeocin, an inducer of DNA double-strand breaks, causes cell expansion with endoreduplication. The vacuolar structure after zeocin treatment was examined in tobacco (Nicotiana tabacum) cultured cells expressing GFP fused to a vacuole membrane protein. We found that the genotoxic stress induced the cell expansion with subdivision of the vacuolar lumen by cytoplasmic strands. When a femtosecond laser was used to cut off the cytoplasmic strand, mitochondrial transport along the strand stopped. This suggested that in the elongated cells under the genotoxic stress, the transport of subcellular materials was activated for DNA repair within the damaged cell nucleus by the construction of a network of cytoplasmic strands in the vacuolar lumen.

Somatic embryogenesis is a relevant micropropagation technique in Carica papaya ‘Golden’ in view of the difficulties met in conventional seed propagation and the lack of an effective method for early sex determination in this trioecious species. Considering the interest in large-scale production of C. papaya seedlings, we adapted a somatic embryogenesis procedure associating liquid system and immature zygotic embryo explants. This protocol markedly contributed to an increase in the yield of friable embryogenic calli, resulting in a large number of normal somatic embryos at various developmental stages. From these embryos, plantlets were regenerated and evaluated for occurrence of somaclonal variation. Flow cytometry screening revealed diploid (88%), tetraploid (6%), and aneuploid (6%) plantlets. Owing to an interest in the mass propagation of diploid individuals, the karyotype of these plantlets was also analyzed. With this approach, diploidy (2X=18) was confirmed and no structural aberrations were found. Considering the results and the applicability of large-scale production of somatic embryos in C. papaya, the tissue culture protocols shown here could be used for commercial purposes. Moreover, the cytometric and cytogenetic analyses were crucial for a rapid, reliable and conclusive evaluation of the genetic stability of C. papaya plantlets regenerated by somatic embryogenesis.

An unconventional genetic resource for bread wheat improvement is the diploid D genome donor to bread wheat; Aegilops tauschii (2n=2x=14, DD). Bridge crossing the Triticum turgidum/Aegilops tauschii results in synthetic hexaploid (SH) wheats; the most common method of wheat improvement that efficiently utilizes the diversity of this diploid wheat progenitor of the primary gene pool. SH wheats exhibit “spring” or “winter” growth habits, depending on the parental durum germplasm. This study focuses on the molecular diversity among 58 winter synthetic hexaploid wheat (WSH) combinations that have been produced to facilitate winter wheat breeding programs. Genetic diversity and genome-wide linkage disequilibrium (LD) were investigated for all D-genome chromosomes using SSR markers. LD analysis based on 71 microsatellite loci represented a mean marker density of 7.1 cm. A total of 738 alleles ranging from 3 to 20 per locus were characterized in WSHs. The mean PIC and MI values were 0.61 and 3.41, ranging from 0.14 to 0.86 and 0.02 to 9.6, respectively. The analysis of molecular variance and Wright's fixation index further established significant genetic diversity among different clusters and durum groups leading to the conclusion that diversity within WSHs is well validated. The population structure also partially validated the cluster analysis by forming eight (K=6) sub-populations. The variable patterns of LD independent of the genetic distance (cm) in some cases provided critical information for these re-synthesized winter wheat genetic resources for their future deployment and association mapping studies. Molecular analyses, thus, have established ample diversity that is present within these winter SHs and generated information for effectively introducing new diversity from these unique genetic resources into the prevalent winter wheat germplasm.

Lipid droplets (LDs) form at the surface of the endoplasmic reticulum (ER) in cells of fungi and animals. In algae, however, they appear to form at the surfaces of both the ER and plastids. To study the universality of this process, we observed LD formation in the filamentous green alga Klebsormidium nitens. Each cell of this alga contains one large petal-like chloroplast. Observations of cells stained with dipyrrometheneboron difluoride (BODIPY) revealed that small LD spots formed at the edge of the chloroplast in young cells, and the size of the spots increased as the chloroplast grew. The LD spots were distributed evenly on the chloroplast, and consequently, were distributed equally between the daughter chloroplasts after binary fission. In non-dividing starved cells, large LDs formed as the chloroplast degraded and the chlorophyll concentration decreased, suggesting that components of the chloroplast were converted into LDs as the chloroplast degraded. To examine the spatial relationships among LDs, the ER, and the chloroplast, we examined cells by microscopy after dihexyloxcacarbocyanideiodide (DiOC₆) single-staining, BODIPY-DiOC₆ double-staining, and Nile red-DiOC₆ double-staining. ERs were elongated and arranged along the edge of the chloroplast in young cells. The LDs were associated with the ER, but were absent from the area between the ER and the chloroplast. These results suggest that the ER plays an important role in LD formation in young dividing cells in the green alga. The dynamics of the ER in starved cells were obscured by the large LDs that filled the cytoplasm.

In this study, the karyotype of the Chorthippus (Glyptobothrus) bornhalmi species (chromosome number, chromosome morphology and chromosome lengths) belonging to the Gomphocerinae subfamily of the Acrididae family was examined. As a result of these examinations, the species' number of chromosome was determined as 2n♂= 17 (X0). It was found that one pair of autosomal chromosome 3 (L₁–L₃) was submetacentric whereas five pairs (M₄–S₈) and X chromosome had an acrocentric structure. As a result of the counting carried out on five individuals, the mean chiasma frequency was found to be 15.36.

Biomonitoring programs are essential tools to evaluate the biological quality status of aquatic environments. Recently, several compounds with endocrine disruption ability have been included in the list of priority substances and therefore, implementation of biomarkers assessing the presence and the effects of such substances are required. In the present work we applied a battery of chemical and biological markers in order to study the effects of endocrine disruptors in four thicklip grey mullet (Chelon labrosus) populations from the Basque Country (Bay of Biscay) inhabiting differently polluted estuaries: Arriluze and Pasaia are marinas located in highly industrialized and densely populated areas, Plentzia is a leisure and touristic town and Gernika is located in the Biosphere Reserve of Urdaibai. Chemical analyses of fish bile were performed in order to determine the uptake of endocrine disruptors. Liver, gonad and brain samples were collected for the study of expression levels of genes associated with reproduction and development such as vtg, cyp19a1 and a2, er and rxr. Histological analysis of gonads was performed to identify possible gametogenic alterations such as intersex gonads. Results indicated clear pollution-dependent responses among four estuaries. Endocrine disruption effects were very marked in mullets from Gernika and Plentzia; these two populations showed high vtg gene expression levels in male mullets together with high alkylphenol metabolites in the bile. Bisphenol A was also present at high concentration in mullets from Gernika. cyp19a2 was upregulated in male mullets from Plentzia. Intersex fish were found in Gernika and Pasaia, the last ones showing high hormone metabolites in bile. The combination of chemical and biomarker approaches in biomonitoring programmes can be a valuable tool to be implemented within the framework of the new water policies.

Detailed male meiotic studies were made in Lindelofia longiflora (Royle ex Benth.) Baill. var. falconeri (Cl.) Brand (Family: Boraginaceae) from the cold deserts of Pangi Valley, Chamba District (Himachal Pradesh) in India. The meiotic course in some of the individuals was observed to be highly abnormal which include cytomixis involving chromatin transfer among meiocytes, spindle irregularities, chromatin stickiness and mutlipolar PMCs, resulting in abnormal sporads as dyads, triads, polyads and tetrads with micronuclei. The products of such sporads result in high pollen sterility (77%) and variable-sized pollen grains. As a consequence of cytomixis, hyperploid (73.04%), hypoploid (18.59%) and enucleated PMCs (8.37%) are resulted. The enucleated PMCs may degenerate during the later stages of meiosis while hypo- and hyperploid PMCs yielded pollen grains of variable sizes and sterile pollen grains. The phenomenon of cytomixis and associated meiotic irregularities seem to have been caused due to low temperature stress conditions prevailing in the area.

Seeds of Carthamus tinctorius L. (2n=24), an oil yielding plant, were treated with different doses (5, 10, 15, 20, 25 kR) of gamma rays. Three translocation heterozygotes were observed at meiotic division for 10- and 25-kR doses. The induced translocation heterozygotes showed a ring or chain of four chromosomes in most of the cells at diakinesis/metaphase I. The induced mutants showed unequal distribution at anaphase I, reduced vigour, delayed flowering, low flower number, low pollen fertility, and low seed sets as compared to control plants. Induction of permanent chromosomal structural changes may also sometime bring out favorable morphological variation. It is expected that the mutant, when established, could be used in further cytological and breeding programs.

The karyological characteristics of two Lilium species were investigated by aceto-ferric-hematoxylin staining. Chromosome characteristics, including the number and length of the chromosomes, length of their long and short arms, length of the total set of chromosomes, the arm ratio index and relative lengths of chromosome, were measured based on averages for five different metaphase cells. Both species are diploid (2n=2x=24). The karyotype of Lilium ledebourii consisted of 1 pair of metacentric, 4 pairs of submetacentric, 3 pairs of acrocentric and 4 pairs of subtelocentric chromosomes. The karyotype of Lilium longiflorum was comprised of 1 pair of metacentric, 4 pairs of acrocentric and 7 pairs of subtelocentric chromosomes. Chromosomes 5 and 7 in Lilium ledebourii and chromosomes 6 in Lilium longiflorum had a satellite. Karyotypes were classified as 3A by Stebbins classification.

In this research, Lallemantia iberica (Bieb.) Fisch. & Mey., L. canescens (L.) Fisch. & Mey. and L. peltata (L.) Fisch. & Mey. belonging to the Lallemantia Fisch. & C.A.Mey. genus (Lamiaceae) which is represented by three taxa growing naturally in Turkey, were studied. The somatic chromosome numbers observed in each taxa was 2n=14. The classification of chromosomes, the length of long and short arms, haploid chromosome length, arm ratio, centromeric index and relative chromosomal length were measured by Software Image Analyses (Bs200Pro). Also their karyotype asymmetry indices (TF%, As K%, Syi, Rec, A, A1 and A2) were calculated. According to the asymmetry index, L. peltata has a more symmetrical karyotype than L. canescens. This is the first study for account of chromosome counts and karyotypes for the studied taxa.

Genus Tricholepis DC. comprises about 18 species worldwide, of which India represents 10 species and one variety. Among the 10 species, 3 are endemic to the region, viz. Tricholepis amplexicaulis C. B. Clarke, T. glaberrima DC. and T. radicans DC. Somatic chromosome counts of T. amplexicaulis and karyotypic analysis of T. amplexicaulis and T. glaberrima have been reported for the first time in the present communication.

The objective of this study was to evaluate the genetic relationship between five Vasconcellea species (V. cauliflora, V. goudotiana, V. quercifolia, V. monoica, and V. cundinamarcensis) and Carica papaya based on their chromosome features. To do that, five metaphase plates showing well spread and condensed chromosomes were used for the chromosome measurements: the absolute chromosome length (CL) and the lengths of the long (LA) and short (SA) arms. Further, we estimated the ratio between the arms (r), the centromeric index (CI), and the total length of haploid set (TLH). The Total Form index (TF%), the Rec, and the SYi were estimated for symmetry/asymmetry of the karyotype. Most species show karyotype formulae of nine pairs of metacentric chromosomes (9m) except for V. goudotiana, which has five pairs of metacentric chromosomes and four pairs of sub-metacentric chromosomes (5m+4sm). V. goudotiana had the longest total length of haploid set, whereas V. quercifolia showed the shortest one. Based on the estimates of symmetry/asymmetry indexes, four species have symmetric karyotype; moreover, V. goudotiana has asymmetric karyotype because it showed the lowest values of Syi and TF% index. The similarity matrix of five parameters for the six species was employed and based on the dendogram four clusters were formed; the first included C. papaya and V. cundinamarcensis, the second cluster was formed by V. monoica and V. cauliflora, the third by V. quercifolia and fourth by V. goudotiana. So, based on chromosome features V. cundinamarcensis, V. cauliflora, and V. monoica are closer to papaya than V. goudotiana.

Gamete fusion or fertilization is one of the most important steps in sexual reproduction. HAP2/GCS1 is a potential gamete membrane fusion component. We analyzed whole genome sequences of two strains with opposite mating types (mt⁻ and mt⁺) in the marine macroalga Ulva compressa and found that identical genomic regions homologous with HAP2/GCS1 existed in their draft genome sequences. Although an RNA-Seq analysis revealed that a variety of transcripts was expressed from the two regions containing HAP2/GCS1 in both the gamete and gametophyte of the mt⁻ and mt⁺ genotypes, a putative transcript coding a protein containing a HAP2–GCS1 domain was identified only in the mt⁻ gamete. Immunofluorescence observations using an antibody against the putative HAP2/GCS1 demonstrated that the HAP2/GCS1 translational products localized to the cellular surface between the two flagella at a potential site for the mt⁻ mating structure. The antibody inhibited mating of the mt⁻ gamete with the mt⁺ gamete in U. compressa. Our data suggest that HAP2/GCS1 functions in the mt⁻ gamete and plays a role in gamete fusion. Moreover, the specific function of HAP2/GCS1 in the mt⁻ mating type may be regulated by an alternative splicing mechanism in U. compressa.