Variation In Chromosome Number Research Articles

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Comparative analysis of chromosome-level genomes provides insights into chromosomal evolution in Chiroptera.

Chiroptera (bats) presents a fascinating model due to its remarkable variation in chromosome numbers, which range from 14 to 62. This astonishing diversity makes bats an excellent subject for studying chromosome evolution. The black-bearded tomb bat (Taphozous melanopogon) occupies a pivotal phylogenetic position within Chiroptera, emphasizing its crucial role in the systematic examination of bat chromosome evolution. In this study, we present the first chromosome-level genome of T. melanopogon within the family Emballonuridae. Together with previously published genomes, we construct a strongly supported phylogenetic tree of bats, which supports that Emballonuridae forms a basal group within Yangochiroptera. Furthermore, we reconstruct ancestral karyotypes at key nodes along the bat phylogeny and conduct a synteny analysis among the genomes of 12 bat species. Our findings identified evolutionary breakpoint regions (EBRs) that are of particular interest. Notably, some bat genomes exhibit an enrichment of genes related to host defense against microbial pathogens within EBRs. Remarkably, one species possesses multiple copies of some β-defensin genes, while six other species have experienced the loss of some β-defensin genes due to EBRs. Furthermore, some olfactory receptor genes are located in EBRs of 12 species, 4 of which have a significant enrichment in sensory perception of smell. Together, our comparative genomic analysis underscores the potential link between chromosome rearrangements and the adaptation of bats to defend against microbial pathogens.

Genome-wide analysis tracks the emergence of intraspecific polyploids in Phragmites australis

Polyploidization plays an important role in plant speciation and adaptation. To address the role of polyploidization in grass diversification, we studied Phragmites australis, an invasive species with intraspecific variation in chromosome numbers ranging from 2n = 36 to 144. We utilized a combined analysis of ploidy estimation, phylogeny, population genetics and model simulations to investigate the evolution of P. australis. Using restriction site-associated DNA sequencing (RAD-seq), we conducted a genome-wide analysis of 88 individuals sourced from diverse populations worldwide, revealing the presence of six distinct intraspecific lineages with extensive genetic admixture. Each lineage was characterized by a specific ploidy level, predominantly tetraploid or octoploid, indicative of multiple independent polyploidization events. The population size of each lineage has declined moderately in history while remaining large, except for the North American native and the US Land types, which experienced constant population size contraction throughout their history. Our investigation did not identify direct association between polyploidization events and grass invasions. Nonetheless, we observed octoploid and hexaploid lineages at contact zones in Romania, Hungary, and South Africa, suggestively due to genomic conflicts arising from allotetraploid parental lineages.

Distinct patterns of satDNA distribution in holocentric chromosomes of spike-sedges (Eleocharis, Cyperaceae).

Eleocharis R. Br. (Cyperaceae) species are known for having holocentric chromosomes, which enable rapid karyotype differentiation. High intra- and interspecific variations in chromosome numbers and genome sizes are documented for different Eleocharis species, frequently accompanied by fluctuations in the repetitive DNA fraction. However, a lack of detailed analysis has hampered a better understanding of the interplay between holocentricity and repetitive DNA evolution in this genus. In our study, we confirmed the holocentricity of Eleocharis chromosomes by immunostaining against the kinetochore protein KNL1 and the cell-cycle dependent posttranslational modifications histone H2AThr121ph and H3S10ph. We further studied the composition and chromosomal distribution of the main satellite DNA repeats found in the newly sequenced species E. maculosa, E. geniculata, E. parodii, E. elegans, and E. montana. Five of the six satellites discovered were arranged in clusters, while EmaSAT14 was distributed irregularly along the chromatid length in a line-like manner. EmaSAT14 monomers were present in a few copies in few species across the Eleocharis phylogenetic tree. Nonetheless, they were accumulated within a restricted group of Maculosae series, subgenus Eleocharis. The data indicates that the amplification and line-like distribution of EmaSAT14 along chromatids may have occurred recently within a section of the genus.

Genomic evidence reveals three W-autosome fusions in Heliconius butterflies.

Sex chromosomes are evolutionarily labile in many animals and sometimes fuse with autosomes, creating so-called neo-sex chromosomes. Fusions between sex chromosomes and autosomes have been proposed to reduce sexual conflict and to promote adaptation and reproductive isolation among species. Recently, advances in genomics have fuelled the discovery of such fusions across the tree of life. Here, we discovered multiple fusions leading to neo-sex chromosomes in the sapho subclade of the classical adaptive radiation of Heliconius butterflies. Heliconius butterflies generally have 21 chromosomes with very high synteny. However, the five Heliconius species in the sapho subclade show large variation in chromosome number ranging from 21 to 60. We find that the W chromosome is fused with chromosome 4 in all of them. Two sister species pairs show subsequent fusions between the W and chromosomes 9 or 14, respectively. These fusions between autosomes and sex chromosomes make Heliconius butterflies an ideal system for studying the role of neo-sex chromosomes in adaptive radiations and the degeneration of sex chromosomes over time. Our findings emphasize the capability of short-read resequencing to detect genomic signatures of fusion events between sex chromosomes and autosomes even when sex chromosomes are not explicitly assembled.

Comparative genomics of the closely related fungal genera Cryptococcus and Kwoniella reveals karyotype dynamics and suggests evolutionary mechanisms of pathogenesis.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

Evolutionary patterns of variations in chromosome counts and genome sizes show positive correlations with taxonomic diversity in tropical gingers.

Cytogenetic traits such as an organism's chromosome number and genome size are taxonomically critical as they are instrumental in defining angiosperm diversity. Variations in these traits can be traced to evolutionary processes such as polyploidization, although geographic variations across cytogenetic traits remain underexplored. In the pantropical monocot family Zingiberaceae (~1500 species), cytogenetic traits have been well documented; however, the role of these traits in shaping taxonomic diversity and biogeographic patterns of gingers is not known. A time-calibrated Bayesian phylogenetic tree was constructed for 290 taxa covering three of the four subfamilies in Zingiberaceae. We tested models of chromosome number and genome size evolution within the family and whether lineage age, taxonomic diversity, and distributional range explain the variations in the cytogenetic traits. Tests were carried out at two taxonomic ranks: within Zingiberaceae and within genus Hedychium using correlations, generalized linear models and phylogenetic least square models. The most frequent changes in chromosome number within Zingiberaceae were noted to be demi-polyploidization and polyploidization (~57% of the time), followed by ascending dysploidy (~27%). The subfamily Zingiberoideae showed descending dysploidy at its base, while Alpinioideae showed polyploidization at its internal nodes. Although chromosome counts and genome sizes did not corroborate with each other, suggesting that they are not equivalent; higher chromosome number variations and higher genome size variations were associated with higher taxonomic diversity and wider biogeographic distribution. Within Zingiberaceae, multiple incidences of polyploidization were discovered, and cytogenetic events appear to have reduced the genome sizes and increased taxonomic diversity, distributional ranges and invasiveness.

AI/ML-derived mechanistically interpretable whole-genome biomarkers of patient survival in pre-treatment primary neuroblastoma tumors and whole blood.

10043 Background: Prediction, understanding, and management, of neuroblastoma (NBL) outcomes, from spontaneous regression to relapse and death, remain limited, and rely mostly on the International Neuroblastoma Staging System (INSS) stage, age, and the one-gene test for MYCN amplification. The entire multi-ome affects every aspect of the disease. But typical artificial intelligence and machine learning (AI/ML) cannot discover effective biomarkers from real-life, small-cohort, multi-omic, and noisy data. Methods: We use our AI/ML to identify two whole-genome biomarkers from open-source pre-treatment NBL patient-matched primary tumor and whole blood DNA copy numbers, and primary tumor mRNA expression. Our data-agnostic, unsupervised algorithms extend the mathematics that underlies quantum mechanics to overcome the limitations of typical AI/ML [1,2]. Our platform- and reference genome-agnostic biomarkers in astrocytoma, including glioblastoma, and lung, ovarian, and uterine adenocarcinomas, were mathematically discovered, and computationally and experimentally validated, repeatedly, in federated, imbalanced datasets from as few as 50–100 patients [3]. Results: We discover the biomarkers in “skinny” datasets of minimally preprocessed ≈3M-bin whole-genome sequences from tumors and blood of a set of 101 patients. Blindly, i.e., label-free, we separate both biomarkers from the normal X chromosome-number variation, demonstrating sex-agnostic learning. Combined, the biomarkers are statistically independent of all standard-of-care indicators, with the univariate Cox hazard ratio of 4.0 (log-rank P-value=2.3×10–5) within the 95% confidence intervals of the bivariate ratios. The risk that the tumor’s whole genome confers upon outcome, as is reflected by the bivariate ratios, is greater than that conferred by all standard-of-care indicators, including DNA ploidy, the Children’s Oncology Group (COG) risk, and the mitosis-karyorrhexis index (MKI), except for histopathology. We validate the biomarkers in ≈10K-bin target-capture sequences of a mutually-exclusive set of 419 patients, demonstrating generalizability as well as site-, platform-, and protocol-agnostic transfer learning. At 73–80% concordance, in both the tumor and blood profiles of both the discovery and validation sets, the biomarkers combined are more accurate than MYCN. We show that the biomarkers identify known and previously unrecognized disease mechanisms and druggable gene alterations, including co-amplification of MYCN with genes encoding for extra-embryonic transcripts, as well as the hijacking of embryonic development toward aneuploidy, which can spontaneously regress via embryonic self-correction. Conclusions: Our AI/ML is uniquely suited for personalized medicine. 1. doi: 10.1063/1.5099268 2. doi: 10.1073/pnas.0530258100 3. doi: 10.1063/1.5142559.

Complete telomere-to-telomere genomes uncover virulence evolution conferred by chromosome fusion in oomycete plant pathogens

Variations in chromosome number are occasionally observed among oomycetes, a group that includes many plant pathogens, but the emergence of such variations and their effects on genome and virulence evolution remain ambiguous. We generated complete telomere-to-telomere genome assemblies for Phytophthora sojae, Globisporangium ultimum, Pythium oligandrum, and G. spinosum. Reconstructing the karyotype of the most recent common ancestor in Peronosporales revealed that frequent chromosome fusion and fission drove changes in chromosome number. Centromeres enriched with Copia-like transposons may contribute to chromosome fusion and fission events. Chromosome fusion facilitated the emergence of pathogenicity genes and their adaptive evolution. Effectors tended to duplicate in the sub-telomere regions of fused chromosomes, which exhibited evolutionary features distinct to the non-fused chromosomes. By integrating ancestral genomic dynamics and structural predictions, we have identified secreted Ankyrin repeat-containing proteins (ANKs) as a novel class of effectors in P. sojae. Phylogenetic analysis and experiments further revealed that ANK is a specifically expanded effector family in oomycetes. These results revealed chromosome dynamics in oomycete plant pathogens, and provided novel insights into karyotype and effector evolution.

Chromosome stability of synthetic Triticum turgidum–Aegilops umbellulata hybrids

BackgroundUnreduced gamete formation during meiosis plays a critical role in natural polyploidization. However, the unreduced gamete formation mechanisms in Triticum turgidum–Aegilops umbellulata triploid F1 hybrid crosses and the chromsome numbers and compostions in T. turgidum–Ae. umbellulata F2 still not known.ResultsIn this study, 11 T.turgidum–Ae. umbellulata triploid F1 hybrid crosses were produced by distant hybridization. All of the triploid F1 hybrids had 21 chromosomes and two basic pathways of meiotic restitution, namely first-division restitution (FDR) and single-division meiosis (SDM). Only FDR was found in six of the 11 crosses, while both FDR and SDM occurred in the remaining five crosses. The chromosome numbers in the 127 selfed F2 seeds from the triploid F1 hybrid plants of 10 crosses (no F2 seeds for STU 16) varied from 35 to 43, and the proportions of euploid and aneuploid F2 plants were 49.61% and 50.39%, respectively. In the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes. The chromosome loss of the U genome was the highest (26.77%) among the three genomes, followed by that of the B (22.83%) and A (11.81%) genomes, and the chromosome gain for the A, B, and U genomes was 3.94%, 3.94%, and 1.57%, respectively. Of the 21 chromosomes, 7U (16.54%), 5 A (3.94%), and 1B (9.45%) had the highest loss frequency among the U, A, and B genomes. In addition to chromosome loss, seven chromosomes, namely 1 A, 3 A, 5 A, 6 A, 1B, 1U, and 6U, were gained in the aneuploids.ConclusionIn the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes, chromsomes, and crosses. In addition to variations in chromosome numbers, three types of chromosome translocations including 3UL·2AS, 6UL·1AL, and 4US·6AL were identified in the F2 plants. Furthermore, polymorphic fluorescence in situ hybridization karyotypes for all the U chromosomes were also identified in the F2 plants when compared with the Ae. umbellulata parents. These results provide useful information for our understanding the naturally occurred T. turgidum–Ae. umbellulata amphidiploids.

Diversity of genome size and chromosome number in homothallic and heterothallic strains of the Closterium peracerosum-strigosum-littorale complex (Desmidiales, Zygnematophyceae, Streptophyta).

The evolutionary transitions of mating systems between outcrossing and self-fertilization are often suggested to associate with the cytological and genomic changes, but the empirical reports are limited in multicellular organisms. Here we used the unicellular zygnematophycean algae, the Closterium peracerosum-strigosum-littorale (C. psl.) complex, to address whether genomic properties such as genome sizes and chromosome numbers are associated with mating system transitions between homothallism (self-fertility) and heterothallism (self-sterility). Phylogenetic analysis revealed the polyphyly of homothallic strains, suggesting multiple transitions between homothallism and heterothallism in the C. psl. complex. Flow cytometry analysis identified a more than 2-fold genome size variation, ranging from 0.53 to 1.42 Gbp, which was positively correlated with chromosome number variation between strains. Although we did not find consistent trends in genome size change and mating system transitions, the mean chromosome sizes tend to be smaller in homothallic strains than in their relative heterothallic strains. This result suggests that homothallic strains possibly have more fragmented chromosomes, which is consistent with the argument that self-fertilizing populations may tolerate more chromosomal rearrangements.

Facing climate change: Range dynamics and chromosome diversity in Hedeoma multiflora Benth., a South American aromatic-medicinal plant at risk

Climate change could significantly affect the geographic distribution of plant species. Hedeoma multiflora is a vulnerable medicinal and aromatic herb that distributes in the Pampa, Espinal and Chaco biogeographic provinces in austral South America. This integrated approach combines ecological models and cytogenetic evidence to assess the effects of climate change on this species. Species distribution modelling using the Maxent model was implemented under current climatic conditions and three future climate change scenarios, integrating data from three Global Climate Models. The most suitable areas span 68,557 km2, encompassing the Sierras Pampeanas in San Luis and Córdoba provinces, and the Tandilia and Ventania systems in Buenos Aires, Argentina. The primary variables influencing the models include elevation (500 to 2000 m.a.s.l.), annual mean temperature (10 to 17 °C), annual precipitation (500 to 900 mm) and precipitation seasonality (50 to 75%). While the results project an expansion in the potential distribution of the species, heterogeneous patterns of range shifts are predicted across the three mountain systems: expansion in Sierras Pampeanas, march in Ventania and retraction in the Tandilia system. Variations in chromosome numbers within four distinct localities were reported, indicating the presence of polyploidy. This could potentially provide adaptive advantages in response to changing climates. This plant lives in habitats that face human-induced alterations and insufficient area protected coverage, then we propose strategies for both in situ and ex situ conservation of this medicinal species in each area.

Comparative genomics reveals the dynamics of chromosome evolution in Lepidoptera

Chromosomes are a central unit of genome organization. One-tenth of all described species on Earth are butterflies and moths, the Lepidoptera, which generally possess 31 chromosomes. However, some species display dramatic variation in chromosome number. Here we analyse 210 chromosomally complete lepidopteran genomes and show that the chromosomes of extant lepidopterans are derived from 32 ancestral linkage groups, which we term Merian elements. Merian elements have remained largely intact through 250 million years of evolution and diversification. Against this stable background, eight lineages have undergone extensive reorganization either through numerous fissions or a combination of fusion and fission events. Outside these lineages, fusions are rare and fissions are rarer still. Fusions often involve small, repeat-rich Merian elements and the sex-linked element. Our results reveal the constraints on genome architecture in Lepidoptera and provide a deeper understanding of chromosomal rearrangements in eukaryotic genome evolution.

Cytogenetic Analysis of the Fish Genus Carassius Indicates Divergence, Fission, and Segmental Duplication as Drivers of Tandem Repeat and Microchromosome Evolution.

Fishes of the genus Carassius are useful experimental vertebrate models for the study of evolutionary biology and cytogenetics. Carassius demonstrates diverse biological characteristics, such as variation in ploidy levels and chromosome numbers, and presence of microchromosomes. Those Carassius polyploids with ≥150 chromosomes have microchromosomes, but the origin of microchromosomes, especially in European populations, is unknown. We used cytogenetics to study evolution of tandem repeats (U1 and U2 small nuclear DNAs and H3 histone) and microchromosomes in Carassius from the Czech Republic. We tested the hypotheses whether the number of tandem repeats was affected by polyploidization or divergence between species and what mechanism drives evolution of microchromosomes. Tandem repeats were found in tetraploid and hexaploid Carassius gibelio, and tetraploid Carassius auratus and Carassius carassius in conserved numbers, with the exception of U1 small nuclear DNA in C. auratus. This conservation indicates reduction and/or loss in the number of copies per locus in hexaploids and may have occurred by divergence rather than polyploidization. To study the evolution of microchromosomes, we used the whole microchromosome painting probe from hexaploid C. gibelio and hybridized it to tetraploid and hexaploid C. gibelio, and tetraploid C. auratus and C. carassius. Our results revealed variation in the number of microchromosomes in hexaploids and indicated that the evolution of the Carassius karyotype is governed by macrochromosome fissions followed by segmental duplication in pericentromeric areas. These are potential mechanisms responsible for the presence of microchromosomes in Carassius hexaploids. Differential efficacy of one or both of these mechanisms in different tetraploids could ensure variability in chromosome number in polyploids in general.

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

Long-Term Cryopreservation May Cause Genomic Instability and the Premature Senescence of Cells.

Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.

The interplay between climatic niche evolution, polyploidy and reproductive traits explains plant speciation in the Mediterranean Basin: a case study in Centaurium (Gentianaceae).

Speciation and diversification patterns in angiosperms are frequently shaped by niche evolution. Centaurium Hill is a Mediterranean genus with ca. 25 species, of which 60% are polyploids (tetra- and hexaploids), distributed mainly in the Mediterranean Basin and in areas with temperate and arid climates of Asia, Europe, North-Central Africa and North America. The evolutionary history of this genus has been studied using morphological, biogeographical and molecular approaches, but its climatic niche characterization and its relation with genome evolution (chromosome number and ploidy level) has not been addressed yet. Thus, this study aims to identify the role of the evolution of climatic niche, ploidy level, life cycle and floral traits in the diversification of Centaurium. Climatic niche characterization involved estimating present climate preferences using quantitative data and reconstructing ancestral niches to evaluate climatic niche shifts. The evolution of climatic niche towards selective optima determined by ploidy level (three ploidy levels) and different binary traits (polyploidy, floral size, floral display, herkogamy and life cycle) was addressed under the Ornstein-Uhlenbeck model. Chromosome number evolution was inferred using the ChromoSSE model, testing if changes are clado- or anagenetic. Chromosome number evolution and its link with cladogenesis, life cycle and floral traits was modeled on the phylogeny. The reconstruction of the ancestral niches shows that Centaurium originated in a mild climate and diversified to both humid and cold as well as to dry and warmer climates. Niche conservatism was estimated in the climatic niche of the ancestors, while the climatic niche of the current taxa experienced transitions from their ancestors' niche. Besides, the evolution of climatic niche towards multiple selective optima determined by the studied traits was supported, life cycle optima receiving the highest support. The reconstruction of chromosome number transitions shows that the rate of speciation process resulting from chromosomal changes (chromosomal cladogenesis) is similar to that of non-chromosomal cladogenesis. Additionally, dependent evolution of floral size, floral display and herkogamy with chromosome number variation was supported. In conclusion, polyploidization is a crucial process in the Mediterranean region that assisted speciation and diversification into new areas with different climates, entailing niche shifts and evolution of reproductive strategies.

Chromosomal evolution in Cryptangieae Benth. (Cyperaceae): Evidence of holocentrism and pseudomonads.

Cryptangieae has recently been revised based on morphology and molecular phylogeny, but cytogenetic data is still scarce. We conducted this study with the aim of investigating the occurrence of holocentric chromosomes and pseudomonads, as well as understanding the mode of chromosomal evolution in the tribe. We performed analyses of meiotic behavior, chromosome counts, and reconstruction of the ancestral state for the haploid number. We present novel cytogenetic data for eight potentially holocentric species: Cryptangium verticillatum, Krenakia junciforme, K. minarum, Lagenocarpus bracteosus, L. griseus, L. inversus, L. rigidus, and L. tenuifolius. Meiotic abnormalities were observed, with parallel spindles being particularly noteworthy. Intra-specific variations in chromosome number were not found, which may indicate an efficient genetic control for the elimination of abnormal nuclei. The inferred ancestral haploid number was n = 16, with dysploidy being the main evolutionary mechanism. At least five chromosomal fissions occurred in Krenakia (n = 21), followed by a further ascending dysploidy event in Lagenocarpus (n = 17). As proposed for Cyperaceae, it is possible that cladogenesis events in Cryptangieae were marked by numerical and structural chromosomal changes.

Effects of genome instability of parental CHO cell clones on chromosome number distribution and recombinant protein production in parent-derived subclones

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability.

Genetic enhancement of okra [Abelmoschus esculentus (L.) Moench] germplasm through wide hybridization.

The introgression of genetic material from one species to another through wide hybridization and repeated back-crossing, plays an important role in genetic modification and enriching the cultivated gene-pool with novel genetic variations. Okra (Abelmoschus esculentus [(L.) Moench)] is a popular vegetable crop with high dietary fibre and protein, rich in essential amino acids, lysine and tryptophan. The wild Abelmoschus genepool has many desirable traits like ornamental value, short internodal length, more number of productive branches, extended bearing, perennation tendency, reduced fruit length (more consumer preferred trait), high mucilage content (medicinal value), abiotic stress tolerances such as drought, high temperature and biotic stress resistances such as okra Yellow Vein Mosaic Virus (YVMV) and Enation Leaf Curl Virus (ELCV) diseases. The repeated use of elite breeding lines led to narrowing of the genetic base of the okra crop, one of the major factors attributed to breakdown of resistance/ tolerance to biotic stresses. YVMV and ELCV are the two major diseases, causing significant yield loss in okra. Hence, wide hybridization was attempted to transfer tolerance genes from wild species to the cultivated genepool to widen the genetic base. The screening of germplasm of wild Abelmoschus species at hotspots led to the identification of tolerant species (Abelmoschus pungens var. mizoramensis, A. enbeepeegeearensis, A. caillei, A. tetraphyllus and A. angulosus var. grandiflorus), which were further used in a wide-hybridization programme to generate interspecific hybrids with the cultivated okra. Presence of pre- and post-zygotic barriers to interspecific geneflow, differences in ploidy levels and genotype specific variations in chromosome numbers led to varying degrees of sterility in F1 plants of interspecific crosses. This was overcome by doubling the chromosome number of interspecific hybrids by applying Colchicine at the seedling stage. The 113 cross derivatives generated comprising amphidiploids in the F1 generation (30), F3 (14), one each in F2 and F4 generations, back cross generation in BC1F2 (03), BC1F3 (25), and BC2F3 (02), crosses between amphidiploids (27), multi-cross combinations (07) and inter-specific cross (between A. sagittifolius × A. moschatus subsp. moschatus) selfed derivatives at F8 generation (03) were characterized in the present study. Besides they were advanced through selfing and backcrossing. The amphidiploids were found to possess many desirable genes with a considerable magnitude of linkage drag. Majority of the wide cross derivatives had an intermediate fruit morphology and dominance of wild characters viz., hispid fruits, stem, leaves, tough fruit fibre, vigorous perennial growth habit and prolonged flowering and fruiting. The fruit morphology of three BC progenies exhibited a high morphological resemblance to the cultivated okra, confirming successful transfer of useful genes to the cultivated okra genepool. The detailed morphological characteristics of the various combinations of Abelmoschus amphidiploids and the genetic enhancement of the genepool achieved in this process is reported here.