Diploid Chromosome Research Articles

A detailed karyological investigation of three endemic Cobitis L. 1758 species (Teleostei, Cobitidae) in Anatolia, Türkiye.

Comparative cytogenetics is a vital approach for diagnosing chromosome abnormalities and identifying species-specific patterns. In this study, chromosomal analysis of three Anatolian endemic Cobitis species was performed. Conventional cytogenetic techniques such as Giemsa staining, C-banding and Ag-NOR staining were applied, followed by measurements of chromosome arm lengths including analysis of the measured data. The diploid chromosome number, 2n = 50, was determined for all three species. The karyotype formulas were as follows: four pairs of metacentric, five pairs of submetacentric, and 16 pairs of subtelo-telocentric chromosomes in Cobitis bilseli; 11 pairs of metacentric, seven pairs of submetacentric, and seven pairs of subtelo-telocentric chromosomes in C. fahireae; and four pairs of metacentric, four pairs of submetacentric, and 17 pairs of subtelo-telocentric chromosomes in C. turcica. Dark C-bands were observed on the pericentromeres of nearly all chromosomes in C. bilseli and C turcica, whereas light C-bands appeared on the pericentromeres of some chromosomes in C. fahireae. Silver-stained metaphases revealed signals on the short arm of a submetacentric chromosome pair in C. fahireae (each homologous chromosome carries one signal), while in C. bilseli and C. turcica, Ag-NOR signals were detected on the long arm of a single metacentric chromosome (only one homologous chromosome carries the signal, and the signal-carrying chromosome is the largest chromosome in the karyotype). This study provides new cytogenetic data consistent with the phylogenetic distances between the studied species, indicating pericentric inversions and/or translocations that govern the formation of Cobitis karyotypes.

Hybrid Origin of ×Leymotrigia bergrothii (Poaceae) as Revealed by Analysis of the Internal Transcribed Spacer ITS1 and trnL Sequences.

×Leymotrigia bergrothii is a presumed hybrid of Leymus arenarius and Elytrigia repens. This article investigates the hybrid origin and genome composition of this species. These plants are sterile, do not undergo pollination, and do not produce seeds; occasionally, underdeveloped stamens containing abortive pollen grains form in individual spikelets. The karyotype analysis of root meristem cells revealed a diploid chromosome number of 49 in ×L. bergrothii, reported here for the first time. Subsequently, we examined the intragenomic polymorphism of the transcribed spacer ITS1 in several species of Elytrigia, Elymus, Leymus, Hordeum, and Psathyrostachys, and compared the ribotype patterns of these species with those of ×L. bergrothii. It is shown that the St-ribotype variants found in Elytrigia repens and Elytrigia pseudocaesia, as well as the ribotypes of the La family, which dominate in the genome of Leymus arenarius, correspond to major ribotypes in ×L. bergrothii. The ribotypes of the St and La families are present in the nuclear genome of ×L. bergrothii in almost equal proportions. A comparison of intron and exon sequences of the trnL gene in the chloroplast DNA of Leymus arenarius, Elytrigia repens, and ×L. bergrothii showed that this region in ×L. bergrothii is identical or very close to that of Elytrigia repens, suggesting that Elytrigia repens was the cytoplasmic donor to ×L. bergrothii. Thus, our study confirms the hypothesis that this species represents a sterile first-generation hybrid of Leymus arenarius and Elytrigia repens, reproducing vegetatively.

Two species of Polycelis (Platyhelminthes, Tricladida, Planariidae) newly recorded for the Qinling Mountains and the Loess Plateau in China, with a comparative discussion on their karyotypes

The paper details new geographic records of two species of the genus Polycelis Ehrenberg,1831 (Platyhelminthes, Tricladida, Planariidae) for the Qinling Mountains and the Loess Plateau in China and provides redescriptions of these species based on an integrative taxonomic study involving morphology, karyology, and histology. This new information considerably expands our knowledge on these species, for which until now only limited data was available. The species Polycelis asiatica Selinova, 1985 is characterized by the following features: 63–80 eyes; sperm ducts that exhibit an intrabulbar knee-shaped bend towards the ventral surface before opening separately and symmetrically into the antero-dorsal portion of a large seminal vesicle; the latter is ventrally displaced, thus creating a narrow ventral lip on the penis papilla; dorsal wall of the seminal vesicle is provided with several well-developed folds; more or less bulbous penis papilla protrudes from the dorsal wall of the male atrium and is provided with a slender and flexible tip; chromosome complement diploid with 30 metacentric chromosomes, 8 submetacentric chromosomes, and 2 subtelocentric chromosomes. The species Polycelis eudendrocoeloides (Zabusova, 1929) is characterized by: 49–80 eyes; highly reduced and blunt penis, provided with a kind of flagellum that projects into the seminal vesicle; a relatively thick coat of circular and longitudinal muscles around the entire male atrium; generally, a very large intrabulbar seminal vesicle, extending from near the ventral body surface to occupy the major part of both the penis bulb and the dorso-ventral space of the body; diploid chromosome complement consisting of 36 metacentric chromosomes.

Karyotypes and chromosomal mapping of some repetitive DNAs in two stingless bee species (Apidae: Meliponini), with the description of a B chromosome in Plebeia genus.

Cytogenetic studies on stingless bees have significantly contributed to our understanding of karyotypic evolution and the composition of euchromatin and heterochromatin regions, including repetitive sequences. In this study, we performed classical cytogenetics, chromosomal banding, and mapping of some repetitive sequences in two stingless bee species, Frieseomelitta trichocerata and Plebeia poecilochroa. The species exhibit the typical diploid chromosome number of each genera, 2n=30 for Frieseomelitta and 2n=34 for Plebeia. Additionally, some individuals of P. poecilochroa presented a small heterochromatic B chromosome, showing a numeric variation of n=17 to 18 in males and 2n=34 to 35 in females. In both species heterochromatin is primarily distributed in the short arm and centromeric regions. Centromeric regions were found to be AT-rich in both species, while subterminal/terminal regions of the short arms of one and six chromosomes presented GC-rich sites in P. poecilochroa and F. trichocerata, respectively. The rDNA clusters mapped on two chromosome pairs in F. trichocerata, and in only one in P. poecilochroa. Microsatellites (GA)n, (GAG)n, and (CAA)n were predominantly mapped in euchromatic regions, while the telomeric motif (TTAGG)n mapped to the ends of most chromosomes, including the B chromosome of P. poecilochroa. The other repetitive probes used, including the rDNA clusters, do not label the B chromosome of P. poecilochroa. Our cytogenetic data highlight both similarities and differences when compared to other congeneric species, expanding the chromosomal data for both genera.

CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

Development of CGMS Systems in Pigeonpea with Special Reference to A2 Source of Cytoplasm

Background: Exploitation of hybrid vigour is quite possible in cross-pollinated crops. However, pigeonpea is a grain legume crop with a moderate level of cross-pollination (20-70%), with diploid (2n = 2×) chromosome number of 22 and genome size of 1C = 858 Mbp. Outcrossing mainly aided by insect pollinators. Commercialization of CMS based hybrid is constrained because of the labor intensiveness of seed production and concerns about seed purity. In pigeonpea, two dominant genes (Rf1 and Rf2) have been identified and reported by Saxena et al., (2011), which impart fertility restoration to the hybrid plants. The fertility restorer (Rf or Fr) genes in the nucleus suppress the male-sterile phenotype and allow the production of high yielding CGMS-based hybrids. Cytoplasmic male-sterile would effectively circumvent these constraints and revolutionize the hybrid seed industry. CGMS based hybrid breeding can be found more reliable to give productive hybrids in pigeonpea but need some expansion. Methods: In the present study, the objective was to develop cytoplasmic-genetic male-sterile (CGMS) lines in pigeonpea through wide hybridization involving conventional backcrossing. Five CGMS lines viz. BDN 2004-1A, BDN 2004-2A, BDN 2004-3A, BDN 2004-4A and BSMR 736A were developed from Cajanus scarabaeoides cytoplasmic background. This paper discusses development of A and B lines by using A2 CMS systems available for pigeonpea. Result: Our investigations on Development of CGMS systems in Pigeonpea with special reference to A2 source of cytoplasm have allowed us to diverse the male sterile lines based on Cajanus scarabaeoides (A2) cytoplasm in pigeonpea which can be used as marker for easy identification, however characterization of these mentioned five CGMS lines will also help in the predicting the performance of progenies in the different breeding programmes.

A chromosome phased diploid genome assembly of African hunting dog (Lycaon pictus).

The African hunting dog (Lycaon pictus, 2n=78) once ranged over most sub-Saharan ecosystems except its deserts and rainforests. However as a result of (still ongoing) population declines, today they remain only as small fragmented populations. Furthermore, the future of the species remains unclear, due to both anthropogenic pressure as well as interactions with domestic dogs, thus their preservation is a conservation priority. On the tree of life, the hunting dog is basal to Canis and Cuon and forms a crown group with them, making it a useful species for comparative genomic studies. Here, we present a diploid chromosome level assembly of an African hunting dog. Assembled according to VGP guidelines from a combination of PacBio HiFi reads and HiC data, it is phased at the level of individual chromosomes. The maternal (pseudo)haplotype (mat) of our assembly has a length of 2.38 Gbp, and 99.36 % of the sequence is encompassed by 39 chromosomal scaffolds. The rest is included in only 36 unplaced short scaffolds. At the contig level, mat consists of only 166 contigs with an N50 of 39 Mbp. BUSCO analysis showed 95.4 % completeness based on Сarnivora conservative genes (carnivora_odb10). When compared to other available genomes from subtribe Canina, the quality of the assembly is excellent, typically between the 1st and 3rd depending on the parameter used, and a significant improvement on previously published genomes for the species. We hope this assembly will play an important role in future conservation efforts and comparative studies of canid genomes.

The signal that stimulates mammalian embryo development.

Embryo development is stimulated by calcium (Ca2+) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a "toolkit", a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca2+ from the egg's intracellular stores which in mammals is followed by repetitive Ca2+ spikes known as Ca2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca2+ signal and describe the steps of the signal transduction mechanism that activates the egg's developmental program and turns it into an embryo.

Identification of a pathogenic variant and pre-implantation genetic testing for a Chinese family affected with split-hand/foot malformation.

Split-hand/foot malformation is a serious congenital limb malformation characterized by syndactyly and underdevelopment of the phalanges and metatarsals. In this study, we reported a case of a fetus with hand-foot cleft deformity. Whole exome and Sanger sequencing were used to filter out candidate gene mutation sites and provide pre-implantation genetic testing(PGT) for family members. Genetic testing results showed that there was a homozygous mutation c.786G>A (p.Trp262*) in the fetal WNT10B, and both parents were carriers of heterozygous mutations. PGT results showed that out of the two blastocysts, one was a heterozygous mutant and the other was a homozygous mutant. All the embryos had diploid chromosomes. The heterozygous embryo was transferred, and a singleton pregnancy was successfully achieved. This study suggests that homozygous mutations in WNT10B are the likely cause of hand-foot clefts in this family. For families with monogenic diseases, preimplantation genetic testing can effectively prevent the birth of an affected child only after identifying the pathogenic mutation.

Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae).

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Cytogenetic study of Meriz goat breeds in Iraqi Kurdistan region.

Goats are considered the leading farm animal that has a substantial role in the agricultural sector in the Kurdistan Region of Iraq. No cytological examination has been carried out on them. This experiment aims to identify the Karyotype of the local breeds of domestic goats. This experiment was conducted on the Karyotype and prepared the ideogram of Meriz goats. The determination of the relative length and centromeric index arm ratio of the chromosomes in the breed was achieved by the production of karyotypes. A total of (30)Meriz goats, consisting of (10) males and (20) females, were selected to collect blood samples for a short-term lymphocyte culture. The diploid chromosome count was observed to be (60), consisting of (29) pairs of acrocentric autosomes and one pair of allosomes, specifically the X and Y chromosomes. The acrocentric nature of the X-chromosome and the sub-metacentric nature of the Y-chromosome were identified through scientific investigation. The study observed a variation in the relative length of autosomal chromosomes in Meriz goats, with females ranging from 4.49% to 1.89% and males ranging from (4.53%) to (1.75%). The X-chromosome had a relative length of 3.96 in females, while the Y-chromosome displayed a relative length of (5.05). The findings of this karyological investigation suggest that the chromosomal composition seen in the Meriz goats under examination was within the expected range of normalcy. It is recommended that more cytogenetic analyses be conducted at the population level in order to identify individuals within the Meriz breed population who possesses numerical and/or structural chromosome abnormalities. This research is crucial for enhancing the efficiency of production and reproduction in this breed.

Multiple karyotype differences between populations of the Hoplias malabaricus (Teleostei; Characiformes), a species complex in the gray area of the speciation process.

Neotropical fishes exhibit remarkable karyotype diversity, whose evolution is poorly understood. Here, we studied genetic differences in 60 individuals, from 11 localities of one species, the wolf fish Hoplias malabaricus, from populations that include six different "karyomorphs". These differ in Y-X chromosome differentiation, and, in several cases, by fusions with autosomes that have resulted in multiple sex chromosomes. Other differences are also observed in diploid chromosome numbers and morphologies. In an attempt to start understanding how this diversity was generated, we analyzed within- and between-population differences in a genome-wide sequence data set. We detect clear genotype differences between karyomorphs. Even in sympatry, samples with different karyomorphs differ more in sequence than samples from allopatric populations of the same karyomorph, suggesting that they represent populations that are to some degree reproductively isolated. However, sequence divergence between populations with different karyomorphs is remarkably low, suggesting that chromosome rearrangements may have evolved during a brief evolutionary time. We suggest that the karyotypic differences probably evolved in allopatry, in small populations that would have allowed rapid fixation of rearrangements, and that they became sympatric after their differentiation. Further studies are needed to test whether the karyotype differences contribute to reproductive isolation detected between some H. malabaricus karyomorphs.

Karyological studies of four species of Lady’s Slipper Orchids (Paphiopedilum) collected in the Bogor Botanical Garden, Indonesia

Paphiopedilum is one of the most widely grown and hybridized of all orchid genera due to its distinctive flower morphology. This genus consists of 139 accepted species and is native to southern China to tropical Asia. Karyological studies on the genus Paphiopedilum have been reported by many cytologists in different countries. However, many Indonesian members of Paphiopedilum have remained comparatively limited in investigated cytologically. This study aimed to analyze karyological characters of four species of Paphiopedilum collected in the Bogor Botanical Garden, Indonesia, namely P. armeniacum, P. hirsutissimum, P. primulinum, and P. superbiens. Karyological studies were conducted by root tips squash method. The results showed that four species of Paphiopedilum have a basic chromosome number of x = 15. They are diploid with 2n=30, instead of the common diploid chromosome number of Paphiopedilum (2n = 26). Paphiopedilum hirsutissimum and P. primulinum possess 16 m + 14 sm chromosome formulae. Whereas Paphiopedilum armeniacum and P. superbiens revealed 16 m + 14 sm and 18 m + 12 sm chromosome formulae, respectively.A Robertsonian change in chromosome number generated by the fission of chromosomes would best explain the origin of the new diploid chromosome number and karyotypes of these species.

P-520 Analyses of mosaic embryos reveal critical regions on human chromosome 11 that involve embryo implantation

Abstract Study question Why some mosaic embryos can be implanted, whereas others cannot? What are the secrets hidden in the human genome that control embryo implantation? Summary answer By data-mining genomic information from PGT-A results of 109 mosaic embryos, we have defined the critical regions in chr11 that play essential roles in implantation. What is known already Embryo implantation is usually a bottleneck step for a successful IVF treatment. What factors control the embryo implantation process has been pursued for many years, and the answers remain largely unknown. PGT-A is a widely applied procedure to identify embryos with normal diploid chromosomes to increase the clinical pregnancy rate per embryo transfer. However, some poor responder patients may only have mosaic embryos that can be used for transfer. We thus investigate the critical regions of the human chromosomes that are important for controlling embryo implantation. Study design, size, duration One hundred and nine mosaic blastocysts were analyzed based on PGT-A assay from 94 patients (ages ranging from 30 to 49 years old, mean age = 38.9 years) undergoing IVF between November 2020 and March 2022. The impact of chromosomal regions on implantation was explored by analyzing the sequencing results together with the pregnancy outcome of mosaic embryos. Additional published outcome data of 1000 mosaic embryo transfers was used to confirm our findings. Participants/materials, setting, methods Pregnant outcomes were followed, including hCG examination, sac formation, and live birth. Whole genome data analysis was performed using individual PGT-A results. Different types of chromosome abnormalities were calculated, and heatmap and clustering analysis were used to define the critical chromosome regions for embryo implantation. In addition, gene set enrichment and pathway analyses were conducted to reveal the functions of genes within the critical regions. Main results and the role of chance Among the transferred 109 mosaic embryos, the clinical outcomes of hCG positive, sac formation, and live birth rates were 45.8%, 42.2%, and 32.1%, respectively. There were 74 (67.8%) and 35 (32.1%) embryos carrying single and multiple mosaic chromosomes, respectively, resulting in 182 chromosomal mosaicism events. The trisomy mosaicism (133/182, 73.0%) occurred with higher frequency than monosomy mosaicism (49/182, 26.9%). Using the heatmap and clustering analysis, our data showed clustered monosomy mosaicism in non-pregnant embryos, especially for chr2, chr4, chr11, chr19, and chr22. Further refinement using segmental mosaic embryos has identified the critical regions, ranging from 10M to 40M in size on different chromosomes that are involved in hundreds of protein-coding genes and known dosage-sensitive genes. Importantly, functional analyses of the genes within the critical regions of chromosome 11 revealed enrichments in embryo invasion and angiogenesis, and suggest that loss of the chromosomal copy of these regions may result in implantation failure. Limitations, reasons for caution It’s still preliminary. More data is required to further confirm the conclusion. Wider implications of the findings Our study provides insights into the chromosomal regions involved in human embryo implantation and will improve the embryo prioritizing strategy in future IVF clinics. Importantly it is the first time the genomics information and gene-mapping strategy have been applied in mosaic embryos to uncover mechanisms underlying human embryo implantation. Trial registration number not applicable

Karyological variation of Clethrionomys glareolus (Mammalia: Rodentia) from Türkiye

Conventionally stained, C- banded and AgNOR stained karyotypes of Clethrionomys glareolus was studied from Samsun, Türkiye. Diploid chromosome number of a female C. glareolus specimen was found as 2n = 56, NFa = 56, and NF = 58. The karyotype consisted of a pair of small metacentric and 26 pairs of acrocentric chromosomes in different size. The X chromosomes were large acrocentrics. Similar to the result of past karyotype study on C. glareolus, the C-positive and C-negative heterochromatin blocks observed in the karyotype. As a difference from the results of a previous study, NORs were localized on the six acrocentric chromosome pairs instead seven pairs. Thus, a chromosomal variation among C. glareolus populations was determined due to variability in the number of NOR bearing autosomes. In this way, it was contributed to the determination of intraspecific variations of this species and to Türkiye's biodiversity at the level of genetic diversity.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV)

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

Revision of chromosome complement of an oriental bug Macrocheraia grandis (Gray, 1832) (Heteroptera: Pyrrhocoroidea: Largidae)

ABSTRACT Cytogenetic data of Macrocheraia grandis (Gray, 1832) have been a subject of much controversy as different diploid chromosome complements have been reported by different workers. In contrast to earlier studies claiming X0 and X1X20 sex mechanism in males, our observations suggest the existence of multiple X and Y chromosomes. On the basis of an exhaustive study of 457 well spread different stages, examination of the entire male meiotic cycle up to telophase II and study of the spermatogonial and oogonial complement we decided the chromosome complement to be 2n (male) = 12A+2m+XmYn, with m+n = 3, 5 or 7 and 2n (female) = 12A+2m+XmXn with m+n = 2, 3, 6 or 8 where m and n are the number of maternal and paternal sex chromosomes. Our data are further supplemented with banding techniques. Besides, the presence of a conspicuous autosomal bivalent, DAPI bright microchromosomes and presence of remnant nucleolar bodies up to diplotene are worth mentioning features of the species.

Karyological analyses in several Algerian populations of six species of the genus Vicia L. (Fabaceae)

As part of the evaluation and valorization of plant genetic resources of fodder and pastoral interest in Algeria, seventeen (17) natural populations belonging to six (06) species of the genus Vicia (Vicia sativa, Vicia disperma, Vicia monardii, Vicia ohchroleuca, Vicia onobrychioides and Vicia lutea), originated from different ecological regions in the North-Eastern of the country, were considered. The populations have been the subject of mitotic and meiotic studies. Haploid and diploid numbers and chromosome measurements were determined. Original results were observed for the first time in some species. In fact, chromosome counts have revealed some new chromosome numbers. The first number (2n=14, n=7) was observed in the endemic subspecies of Algeria, V. ochroleuca subsp. atlantica and in the species Vicia onobrychioides. The second number (n=6) was observed in the species Vicia disperma. Within Vicia monardii, the three new chromosome numbers, previously observed only in mitosis by our research team, were confirmed for the first time through the present meiosis study (n=6, 7 and 8), indicating that they are A type chromosomes. The base number x=7 is the most frequently observed number in the six Vicia species. The observed chromosome numbers would be related to some ecological factors (altitude, rainfall) of the origin environment of the populations. Chromosome measurements and established karyotypes were determined for the first time in V. monardii, V. onobrychioides and V. ochroleuca subsp. atlantica. Chromosome size and karyotype formula are variable among the studied species and subspecies. Karyotypes vary from symmetrical to asymmetrical and the intrachromosomal asymmetry is higher than interchromosomal one. The new cytogenetic data would contribute to a better understanding of the evolution mechanism of the species in the genus Vicia L.

First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand

The first chromosome study of Trigonostigma somphongsi (Meinken,1958) from Thailand. Specimens were collected from Pak Phli District, Nakhornnayok Province, central Thailand. The mitotic chromosomes were directly prepared from kidney tissues of two males and two females. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results shown that diploid chromosome number of T. somphongsi was 2n=50, the fundamental numbers (NF) were 92 in both male and female. The types chromosomes consisted of 10 metacentric, 20 submetacentric, 12 acrocentric and 8 telocentric chromosomes. The result also exhibited that the interstitial nucleolar organizer regions (NORs) were clearly observed at the short arm of chromosome pair 2. The karyotype formula could be deduced as: 2n (diploid) 50=10m+20sm+12a+8t.