CYTOLOGIA Vol. 82(2017) No. 3

Hi-C (high-resolution chromosome conformation capture) provides the genome-wide interactions of all genomic regions. Hi-C can identify topologically associating domains and enhancer-promoter loops. Recently, Hi-C data could be acquired from only a single cell or a single-nucleus. These new approaches enable us to identify the character of individual cells. Moreover, Hi-C is also applicable for the construction of long-range scaffolding of chromosome-scale de novo genome assembly. The chromosome-scale assembly with Hi-C enables us to reveal the spatial chromatin organization in repetitive sequence-rich genomes and validate chromosome rearrangements including chromosome fusions and translocations. In the future, Hi-C will contribute to the determination of an optimal strategy in cancer therapy.

Standardized karyotype and idiogram of Indian hog deer (Hyelaphus porcinus) at Khon Kaen Zoo, Thailand was explored. Blood samples were taken from two male and two female deer. After standard whole blood T-lymphocytes were cultured at 37°C for 72 h in presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, GTG-, high-resolution and Ag-NOR banding techniques were applied to stain the chromosome. The results show that the diploid chromosome number of H. porcinus was 2n=68, the fundamental number (NF) was 70 in both males and females. The types of autosomes observed were 6 large telocentric, 18 medium telocentric and 42 small telocentric chromosomes. The X chromosome was large metacentric chromosome and Y chromosome was small submetacentric chromosome. The GTG-banding and high-resolution techniques showed that the numbers of bands and locations in H. porcinus are 239 and 301, respectively, and each chromosome pair could be clearly differentiated. In addition, the subtelomeric q-arm of chromosome pairs 1 and 2 showed clearly observable nucleolar organizer regions (NORs). Our results are the first reports of GTG-, high-resolution and Ag-NOR banding techniques on this species. The karyotype formula of H. porcinus is as follows:

Meiotic studies were carried out in 14 wild accessions of Lavatera cachemiriana, an important medicinal herb, collected from various localities of Kashmir Himalaya. The selected plant has anti-cancer and anti-bacterial properties and could prove beneficial in phyto-pharmacological research for the discovery of new therapeutic drugs. At present, male meiosis, microsporogenesis and pollen grain studies have been carried out in various populations of L. cachemiriana collected from different altitudinal zones of Kashmir Himalaya. All the presently worked out accessions share the same meiotic chromosome number n=21. The meiotic course in all the populations is found to be abnormal. The meiotic irregularities like stickiness, interbivalent connections, unoriented bivalents, laggards, bridges, non-synchronous disjunction (early and late disjunction), and formation of micronuclei leading to abnormal microsporogenesis were investigated for the first time for the species that existed at diploid level (2n=42). Consequently, variable sized fertile pollen grains and considerable amount of sterile pollens were the result. The abnormal meiosis coupled with pollen sterility has great influence on the reproductive potential and survival of this endangered species.

Three mulberry genotypes viz., MR₂, C₇₆₃ and Morus macroura were analyzed for the stomatal frequency and karyotypic attributes. MR₂ and C₇₆₃ were found to be diploids with somatic chromosome number of 2n=28, while variety Morus cathyana revealed tetraploid chromosome number of 2n=56. Somatic chromosomes measured 1.26 to 2.86 µm in length. Karyotypes of these taxa are symmetric. Only metacentric and sub-metacentric chromosomes are found in the complement. Stomatal frequency was found to be high in diploid mulberry varieties compared to tetraploid variety.

Physochlaina praealta (family: Solanaceae), a widely distributed species of Indian cold deserts, is studied for male meiotic studies. We here report for the first time the existence of intraspecific polyploidy (2x, 4x) in the species. Both the 2x and 4x individuals show perfectly normal meiotic behavior and nearly 100% fertile pollen grains. The 4x plants show allopolyploid like meiotic behavior characterized by normal chromosome pairing and regular segregation of chromosomes. The 2x and 4x plants grow under similar type of habitats but the 4x individuals were noticed to be more pubescent. The 4x plants can also be differentiated from the 2x on the basis of micro-characters like trichome (size, density), stomata (size and frequency) and pollen grain size which increases with ploidy.

Nematode infection of plant roots is a paradigm of host–parasite interactions. Although nematodes can be labeled with fluorescent dyes, migration of the worms into the deep regions of host roots makes them difficult to track. Here we report the use of two fluorescent dyes, FM4-64 and SYBR green I, to intensely label the soybean cyst nematode (SCN) Heterodera glycines for one week in host plants. Continuous monitoring of the labeled SCN juveniles was achieved with two-photon microscopy. Additionally, we developed a transient transformation system consisting of the non-model leguminous plant (fabaceous) roots, Astragalus sinicus and Agrobacterium rhizogenes to observe the cellular structures of the plant during SCN infection. By the combined use of fluorescent dyes and two-photon microscopy, clear images of infecting SCNs were obtained even in deep regions of A. sinicus roots. The fluorescent labeling described herein can also be used in detailed monitoring of the infection processes of other non-model nematodes, as well as the associated morphological changes in the host plant roots.

Polo-like kinase (PLK) is a serine/threonine kinase involved in the formation of the mitotic spindle and the maturation of centrosomes. PLKs are highly conserved in the animal and fungi kingdoms but have disappeared in land plants. Plant PLK orthologs have been found in green algae, red algae and a filamentous terrestrial alga, Klebsormidium flaccidum, which can adapt to both fresh water and land environments. Our evolutionary analyses suggest that plants evolutionally lost the PLK ortholog before the divergence of land plants. Our localization analyses of the PLK ortholog in Cyanidioschyzon merolae demonstrated that it is localized in spindle poles and on mitochondrial polar microtubules, suggesting that plant ancestral PLK functions in the formation of mitotic spindles like animal and fungus PLK orthologs.

Dry seeds (moisture content: 17.50%) of Lathyrus sativus L. (Family: Fabaceae, common name—grass pea) are exposed (doses: 0.25, 0.50 and 1.00 µg mL⁻¹, duration: 3 and 6 h) to chemically synthesized cadmium sulphide and copper oxide nanoparticle (NP) treatments for assessment of genotoxic potentiality. The objective of the work is to foresee whether the NPs possess effective mutagenic potentiality. Result is significant in the direction of underlined objective.

On the basis of meiotic studies carried out presently on Inula grandiflora from Malana Valley in Kullu district, Himachal Pradesh, we here report the existence of cytomixis and intraspecific diploid (n=8) and tetraploid (n=16) cytotypes. The 4x individuals grow much taller and possess larger sized leaves and capitula. Stomata and pollen grain characters could also be employed in the segregation of the two cytotypes. The plants of 4x cytotype showed normal bivalent formation, regular microsporogenesis and high pollen fertility. Meanwhile, the diploid individuals which also grow under the same climatic conditions depicted the phenomenon of cytomixis involving chromatin transfer among two to eight meiocytes at different stages of meiosis. The PMCs involved in cytomixis showed various meiotic irregularities leading into reduction in some pollen fertility. Although the inter-cellular nuclear migration has been confined only to diploid individuals, which grow under the same environmental conditions, cytomixis seems to be a natural phenomenon controlled by some genes as suggested by earlier workers.

In this work, the effects of potassium nitrate (KNO₃) on the seed germination, seedling growth (radicle length, radicle number and fresh weight), mitotic activity and chromosomal aberrations of Allium cepa L. germinated under both normal conditions and salt stress were studied. The final germination percentages, radicle lengths, radical numbers and fresh weights of onion seeds germinated in the medium with KNO₃ alone were statistically the same as ones of the control seeds germinated in distilled water medium. In addition, the mitotic index in root tip meristems of A. cepa seeds germinated in the medium with KNO₃ alone demonstrated a decrease according to ones of the control seeds germinated in distilled water medium, while their frequency of chromosomal aberrations showed an increase according to the control. On the other hand, salt stress considerably inhibited the seed germination and seedling growth of A. cepa. Furthermore, it markedly decreased the mitotic index in root tip meristems of the seeds and increased the number of chromosomal aberrations. The detrimental effects of salt on the seed germination, seedling growth, mitotic activity and chromosomal aberrations was dramatically alleviated in varying degrees by KNO₃ application.

The cytological screening of gamma ray treated plants revealed few desynaptic plants where an enhanced frequency of univalents were found at metaphase I along with few bivalents. The desynaptic plants were obtained at 200 Gy dose of gamma rays and were characterised as weak and medium strong type. The univalents remained unpaired till later meiotic stages and resulted in the unequal separation of chromosomes at anaphase I. The unequal separation of chromosomes as a consequence of desynapsis lead to the formation of micronuclei and gametes with imbalanced chromosomes, thus affecting the post-meiotic stages also. The desynaptic plants possess very few pods and acquire high pollen inviability. The study suggests the ability of gamma rays in the creation of male sterile lines in Phaseolus vulgaris L. The gamma rays either act on the genes responsible for synapsis and chiasma formation or disrupt the synaptonemal complex affecting chiasmate dissociation. The desynaptic mutants could be used as a potential source for gathering information on chiasma maintenance mechanism as well as to study the genetic mechanism and consequences of male sterility in plants. Further, the male sterile lines could also be profitably utilized in hybridization breeding programmes to produce hybrid seeds.

A cytological ‘plant type’ showing impairment of homologues (desynapsis) has been spotted in M₂ population of Nigella sativa L. (black cumin) following exposure of dry seeds (moisture content 5.00%) to cadmium sulphide nanoparticle (CdS-NPs) treatments (0.25 µg mL⁻¹, 3 h). Present investigation highlights the effective potentiality of CdS-NPs in inducing desynaptic mutant, a pioneer report of its type.

Hedeoma multiflorum (Lamiaceae) commonly known as “tomillito de las sierras” is a native aromatic herb used in folk medicine for its stimulant and digestive properties. Plants are collected in the wild; their uncontrolled exploitation exceeds their rate of natural regeneration leading to the risk of genetic erosion. The aims of this work were to determine the chromosome number of three populations of Hedeoma multiflorum native to Córdoba (Argentina) and characterize them from the meiotic point of view. Most of the somatic cells observed showed the chromosome number 2n=72. In relation to their meiotic behavior, irregularities as presence of univalent, laggards, micronucleus and occurrence of cytomixis were observed. The characterization of the native germoplasm of this valuable aromatic and medicinal species is fundamental to achieve its sustainable handling and conservation.

Capsaicinoids are pungent components that are produced only in the genus Capsicum. These functional compounds are produced in chili pepper fruits and have health benefits for humans. A high capsaicinoid level is one of the major parameters determining the commercial quality and health-promoting properties of chili peppers. Several reports have indicated that capsaicinoids are produced in the interlocular septum of chili pepper fruits. Microscopic analyses show that there are morphological changes in epidermal cells in the interlocular septum during the capsaicinoid-biosynthesis stages of fruit development. Epidermal cells containing capsaicinoids in the interlocular septum had an elongated shape. Recently, it was reported that capsaicinoids and transcripts of genes encoding enzymes involved in capsaicinoid biosynthesis were present in the fruit pericarp of extremely pungent chili cultivars. However, the morphological changes in epidermal cells in pericarp tissue had not been reported. In this study, microscopic analyses of the fruit of Capsicum chinense cv. ‘Bhut Jolokia,’ an extremely pungent chili pepper variety, revealed elongated epidermal cells secreting capsaicinoids on the inside surface of the pericarp.

Hybrids between A genome diploid plants from the Chinese and Korean populations of the Scilla scilloides complex were found. Molecular cytogenetic examination and genome size measurement showed that the hybrids were composed of two distinguishable A genomes. Genomic in situ hybridization (GISH) using mixed probes of both parental genomic DNAs could distinguish the chromosomes of each A genome in the chromosome spreads of hybrid plants. This suggests that the A genome plants of the two populations differ at the genomic DNA level. The genome size of the Korean population was 1.2–1.3 times larger than that of the Gansu population according to the area of chromosome set and fluorescence intensity in flow cytometry (FCM). Despite these cytological differences, the hybrids showed normal chromosome pairing in meiosis of pollen mother cells (PMCs), suggesting that the genomes of these two populations still belong to the same genome in terms of classical cytogenetics. Despite these genomes belonging to the same A genome, molecular signs of genome differentiation in the A genome were also found in the 5S rDNA spacer region. Southern blot hybridization of this spacer region from several populations showed that the three A genome populations were distinct from one another. Additionally, in a sequence analysis of a part of matK gene and ITS1, the three A genome populations showed population-specific sequence variations. These results suggest that the A genome populations of S. scilloides is just in the process of genome differentiation.

Acremonium sclerotigenum (Bionectriaceae, Hypocreales, Sordariomycetes, Ascomycota) is a soil fungus found worldwide as an air and food contaminant. This fungus produces the antibiotic ascofuranone, a promising drug candidate against African trypanosomiasis; however, the details of ascofuranone biosynthesis have not yet been elucidated. Genetic manipulation techniques would greatly facilitate identification of the genes from ascofuranone biosynthetic pathway, but these techniques have not yet been established for this organism. Protoplast generation is a required step of the polyethylene glycol-mediated transformation of fungi. In this study, we aimed to establish a protocol for the isolation of protoplasts from A. sclerotigenum strain F-1392. The cell wall was digested with a commercial enzyme, Yatalase, to promote protoplast release. Spherical cells that were sensitive to osmotic shock were recovered only when the mycelia were pretreated with dithiothreitol. As a result, 2.0×10⁷ protoplasts were obtained from a 40-mL liquid culture of A. sclerotigenum; the regeneration efficiency was estimated as 17%. This study comprises the first step toward development of genetic manipulation techniques in A. sclerotigenum, allowing future genetic dissection of this important microorganism.

2C-values of nine species of the genus Drosera, and two monotypic genera Aldrovanda and Dionaea were estimated to provide an overview of the genome diversity and chromosome differentiation in the Droseraceae. The measured DNA contents of all species used in this study ranged over nine-fold from 2C=0.63 pg in D. burmannii to 5.67 pg in D. anglica. In the genus Drosera, even though the polyploid species were excluded, the difference of the 2C DNA contents among diploid species was still high, ranging 4.3-fold from 0.66 pg in D. spatulata to 2.85 pg in D. intermedia. In subgenus Drosera, especially the polyploidal group, two chromosome types were identified by means of their size; this therefore made it possible to discriminate two groups of the genomes: one group was of a smaller genome size (S genome group) consisting of a total of 10 small-sized chromosomes (x=10s), and the other group was of a larger genome size (M genome group) consisting of a total of 10 middle-sized chromosomes (x=10 m). The Cx-value of the S genome group was less than 0.4 pg (ca. 400 Mbp). On the other hand, the Cx-value of the M genome group showed a range of 1.3–1.5 pg (1270–1470 Mbp). Moreover, the 2C DNA content of the hexaploid species D. tokaiensis (2n=6x=20 m+40s, 2C=3.57 pg), which originated from naturally occurring interspecific hybridization event between D. rotundifolia (2n=2x=20 m, 2C=2.73 pg) and the tetraploid D. spatulata (2n=4x=40s, 2C=1.38 pg), was less (86.9%) than the sum of their putative parental species.

Carex L. is one of the most species-rich genera in the flora of Korea with about 157 taxa. Somatic or meiotic chromosome numbers of eight Carex taxa from the Korean Peninsula are reported, including first counts for C. macrandrolepis H. Lév. (n=37II) and C. splendentissima U. Kang & J. M. Chung (2n=12). Furthermore, there are first chromosomal investigations from Korea populations: C. bostrychostigma Maxim. (n=22II), C. capricornis Meinsh. ex Maxim. (n=35II), and C. breviculmis R. Br. (n=33II). None of the chromosomes has distinct primary constrictions. Carex sect. Mitratae exhibits high variation in chromosome numbers with aneuploidy (chromosome number increases with genomic duplication) and/or agmatoploidy (chromosome number increases without genomic duplication) whereas C. sect. Siderostictae shows polyploidy. Chromosome dynamics have played an important role in Carex species diversity in the Korean Peninsula.