Low-pass whole genome sequencing as a cost-effective alternative to chromosomal microarray analysis for low- and middle-income countries.

Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal microarray analysis (CMA), the sequencing-based approach provides a similar resolution of CNV detection at a lower cost. In this study, we assessed the efficiency and reliability of LP-WGS as a more affordable alternative to CMA. A total of 1363 patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies were enrolled. Those patients were referred from 15 nonprofit organizations and university centers located in different states in Brazil. The analysis of LP-WGS at 1x coverage (>50kb) revealed a positive testing result in 22% of the cases (304/1363), in which 219 and 85 correspond to pathogenic/likely pathogenic (P/LP) CNVs and variants of uncertain significance (VUS), respectively. The 16% (219/1363) diagnostic yield observed in our cohort is comparable to the 15%-20% reported for CMA in the literature. The use of commercial software, as demonstrated in this study, simplifies the implementation of the test in clinical settings. Particularly for countries like Brazil, where the cost of CMA presents a substantial barrier to most of the population, LP-WGS emerges as a cost-effective alternative for investigating copy number changes in cytogenetics.

Effects of a S-metolachlor based herbicide on two plant models: Zea mays L. and Lactuca sativa L.

Corn is the second most cultivated crop in Brazil, the number-one country in pesticide consumption. Chemical control of weeds is performed using herbicides such as S-metolachlor with pre- and post-emergence action and thus the toxicity of herbicides constitutes a matter of great concern. The present investigation aimed to examine the effects of an S-metolachlor-based herbicide on Lactuca sativa L. (lettuce) and Zea mays L. (maize) utilizing various bioassays. The test solutions were prepared from commercial products containing the active ingredient. Seeds from the plant models were exposed in petri dishes and maintained under biochemical oxygen demand (BOD) at 24°C. Distilled water was negative and aluminium positive control. Macroscopic analyses (germination and growth) were conducted for both plant species, and microscopic analysis (cell cycle and chromosomal alterations) were performed for L. sativa root tip cells. Detrimental interference of S-metolachlor-based herbicide was noted with lettuce for all parameters tested reducing plant germination by over 50% and the germination speed by over 45% and showing a significant decrease in mitotic index, from 16.25% to 9,28% even on the lowest concentration tested. In maize, there was no significant interference in plant germination; however, speed of germination was significantly hampered, reaching a 51.22% reduction for the highest concentration tested. Data demonstrated that the herbicide was toxic as evidenced by its phyto- and cytotoxicity in L. sativa L. and Z. mays L.

LBDNet interlaboratory comparison for the dicentric chromosome assay by digitized image analysis applying weighted robust statistical methods.

PURPOSE: This interlaboratory comparison was conducted to evaluate the performance of the Latin-American Biodosimetry Network (LBDNet) in analyzing digitized images for scoring dicentric chromosomes from in vitro irradiated blood samples. The exercise also assessed the use of weighted robust algorithms to compensate the uneven expertise among the participating laboratories. METHODS: Three sets of coded images obtained through the dicentric chromosome assay from blood samples irradiated at 1.5 Gy (sample A) and 4 Gy (sample B), as well as a non-irradiated whole blood sample (sample C), were shared among LBDNet laboratories. The images were captured using the Metafer4 platform coupled with the AutoCapt module. The laboratories were requested to perform triage scoring, conventional scoring, and dose estimation. The dose estimation was carried out using either their laboratory calibration curve or a common calibration curve. A comparative statistical analysis was conducted using a weighted robust Hampel algorithm and z score to compensate for uneven expertise in dicentric analysis and dose assessment among all laboratories. RESULTS: Out of twelve laboratories, one had unsatisfactory estimated doses at 0 Gy, and two had unsatisfactory estimated doses at 1.5 Gy when using their own calibration curve and triage scoring mode. However, all doses were satisfactory at 4 Gy. Six laboratories had estimated doses within 95% uncertainty limits at 0 Gy, seven at 1.5 Gy, and four at 4 Gy. While the mean dose for sample C was significantly biased using robust algorithms, applying weights to compensate for the laboratory's analysis expertise reduced the bias by half. The bias from delivered doses was only notable for sample C. Using the common calibration curve for dose estimation reduced the standard deviation (s*) estimated by robust methods for all three samples. CONCLUSIONS: The results underscore the significance of performing interlaboratory comparison exercises that involve digitized and electronically transmitted images, even when analyzing non-irradiated samples. In situations where the participating laboratories possess different levels of proficiency, it may prove essential to employ weighted robust algorithms to achieve precise outcomes.

Integrating Genetic and Cytogenetic Data: A Diversity Study of Astyanax and Psalidodon (Characidae) Species from the Paraná River Basin.

Astyanax is one of the most specious fish groups in the Neotropical region, with many cryptic species, which represents a challenge for correct identification through traditional taxonomic methods. Psalidodon is a recently resurrected genus group of species previously belonging to Astyanax, specifically those with extensive chromosomal variation of the A. scabripinnis and fasciatus complexes. In the present study, the mitochondrial genes cytochrome c oxidase subunit 1 (COI), mitochondrial ATP synthase 6 and 8 (ATPase 6/8), and NADH dehydrogenase subunit 2 (ND2) were used in conjunction with chromosomal data to characterize molecularly and cytogenetically populations of Astyanax and Psalidodon from rivers and streams of the Ivaí River Basin (Paraná Basin). The results demonstrated the effectiveness of the integrative use of molecular and cytogenetic techniques, with the confirmation of at least three species for the sampled sites: A. lacustris, P. paranae, and P. fasciatus, which showed inter- and intrapopulation karyotype variations. In addition, extensive haplotypic variation can be observed for these species within the Ivaí River Basin and throughout the Paraná River Basin. The data demonstrate a hidden diversity among the species analyzed, enrich the ichthyofaunistic knowledge of small rivers and streams, and contribute to future conservation projects in these areas.

Understanding microchromosomal organization and evolution in four representative woodpeckers (Picidae, Piciformes) through BAC-FISH analysis.

The genome organization of woodpeckers has several distinctive features e.g., an uncommon accumulation of repetitive sequences, enlarged Z chromosomes, and atypical diploid numbers. Despite the large diversity of species, there is a paucity of detailed cytogenomic studies for this group and we thus aimed to rectify this. Genome organization patterns and hence evolutionary change in the microchromosome formation of four species (Colaptes campestris, Veniliornis spilogaster, Melanerpes candidus, and Picumnus nebulosus) was established through fluorescence in situ hybridization using bacterial artificial chromosomes originally derived from Gallus gallus and Taeniopygia guttata. Findings suggest that P. nebulosus (2n = 110), which was described for the first time, had the most basal karyotype among species of Picidae studied here, and probably arose as a result of fissions of avian ancestral macrochromosomes. We defined a new chromosomal number for V. spilogaster (2n = 88) and demonstrated microchromosomal rearrangements involving C. campestris plus a single, unique hitherto undescribed rearrangement in V. spilogaster. This comprised an inversion after a fusion involving the ancestral microchromosome 12 (homologous to chicken microchromosome 12). We also determined that the low diploid number of M. candidus is related to microchromosome fusions. Woodpeckers thus exhibit significantly rearranged karyotypes compared to the putative ancestral karyotype.

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Evolution of bird sex chromosomes: a cytogenomic approach in Palaeognathae species.

BACKGROUND: Different patterns of sex chromosome differentiation are seen in Palaeognathae birds, a lineage that includes the ratites (Struthioniformes, Rheiformes, Apterygiformes, Casuariiformes, and the sister group Tinamiformes). While some Tinamiform species have well-differentiated W chromosomes, both Z and W of all the flightless ratites are still morphologically undifferentiated. Here, we conducted a comprehensive analysis of the ZW differentiation in birds using a combination of cytogenetic, genomic, and bioinformatic approaches. The whole set of satDNAs from the emu (Dromaius novaehollandiae) was described and characterized. Furthermore, we examined the in situ locations of these satDNAs alongside several microsatellite repeats and carried out Comparative Genomic Hybridizations in two related species: the greater rhea (Rhea americana) and the tataupa tinamou (Crypturellus tataupa). RESULTS: From the 24 satDNA families identified (which represent the greatest diversity of satDNAs ever uncovered in any bird species), only three of them were found to accumulate on the emu's sex chromosomes, with no discernible accumulation observed on the W chromosome. The W chromosomes of both the greater rhea and the emu did not exhibit a significant buildup of either C-positive heterochromatin or repetitive DNAs, indicating their large undifferentiation both at morphological and molecular levels. In contrast, the tataupa tinamou has a highly differentiated W chromosome that accumulates several DNA repeats. CONCLUSION: The findings provide new information on the architecture of the avian genome and an inside look at the starting points of sex chromosome differentiation in birds.

Interspecific cytogenomic comparison reveals a potential chromosomal centromeric marker in Proceratophrys frog species.

Among the repetitive elements, satellite DNA (SatDNA) emerges as extensive arrays of highly similar tandemly repeated units, spanning megabases in length. Given that the satDNA PboSat01-176, previously characterized in P. boiei, prompted our interest for having a high abundance in P. boiei and potential for centromeric satellite, here, we employed various approaches, including low coverage genome sequencing, followed by computational analysis and chromosomal localization techniques in four Proceratophrys species and, investigating the genomic presence and sharing, as well as its potential for chromosomal centromere marker in Proceratophrys frog species. Our findings demonstrate that PboSat01-176 exhibits high abundance across all four Proceratophrys species, displaying distinct characteristics that establish it as the predominant repetitive DNA element in these species. The satellite DNA is prominently clustered in the peri/centromeric region of the chromosomes, particularly in the heterochromatic regions. The widespread presence of PboSat01-176 in closely related Proceratophrys species reinforces the validity of the library hypothesis for repetitive sequences. Thus, this study highlighted the utility of the satDNA family PboSat01-176 as a reliable centromeric marker in Proceratophrys species, with potential to be applied in other species of anuran amphibians. The observed sharing and maintenance of this sequence within the genus suggest possibilities for future research, particularly through expanded sampling to elucidate parameters that underlie the library hypothesis and the evolutionary dynamics of satDNA sequences.

Comparative Cytogenetics in Tyrannidae (Aves, Passeriformes): High Genetic Diversity despite Conserved Karyotype Organization.

INTRODUCTION: Passeriformes has the greatest species diversity among Neoaves, and the Tyrannidae is the richest in this order with about 600 valid species. The diploid number of this family remains constant, ranging from 2n = 76 to 84, but the chromosomal morphology varies, indicating the occurrence of different chromosomal rearrangements. Cytogenetic studies of the Tyrannidae remain limited, with approximately 20 species having been karyotyped thus far. This study aimed to describe the karyotypes of two species from this family, Myiopagis viridicata and Sirystes sibilator. METHODS: Skin biopsies were taken from each individual to establish fibroblast cell cultures and to obtain chromosomal preparations using the standard methodology. The chromosomal distribution of constitutive heterochromatin was investigated by C-banding, while the location of simple repetitive sequences (SSRs), 18S rDNA, and telomeric sequences was found through fluorescence in situ hybridization. RESULTS: The karyotypes of both species are composed of 2n = 80. The 18S rDNA probes hybridized into two pairs of microchromosomes in M. viridicata, but only a single pair in S. sibilator. Only the telomeric portions of each chromosome in both species were hybridized by the telomere sequence probes. Most of the SSRs were found accumulated in the centromeric and telomeric regions of several macro- and microchromosomes in both species, which likely correspond to the heterochromatin-rich regions. CONCLUSION: Although both species analyzed showed a conserved karyotype organization (2n = 80), our study revealed significant differences in their chromosomal architecture, rDNA distribution, and SSR accumulation. These findings were discussed in the context of the evolution of Tyrannidae karyotypes.

Cytomolecular diversity among Vigna Savi (Leguminosae) subgenera.

The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.

Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service.

OBJECTIVES: Male infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience. RESULTS: We evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

Satellite DNAs, heterochromatin, and sex chromosomes of the wattled jacana (Charadriiformes; Jacanidae): a species with highly rearranged karyotype.

Charadriiformes, which comprises shorebirds and their relatives, is one of the most diverse avian orders, with over 390 species showing a wide range of karyotypes. Here, we isolated and characterized the whole collection of satellite DNAs (satDNAs) at both molecular and cytogenetic levels of one of its representative species, named the wattled jacana (Jacana jacana), a species that contains a typical ZZ/ZW sex chromosome system and a highly rearranged karyotype. In addition, we also investigate the in situ location of telomeric and microsatellite repeats. A small catalog of 11 satDNAs was identified that typically accumulated on microchromosomes and on the W chromosome. The latter also showed a significant accumulation of telomeric signals, being (GA)10 the only microsatellite with positive hybridization signals among all the 16 tested ones. These current findings contribute to our understanding of the genomic organization of repetitive DNAs in a bird species with high degree of chromosomal reorganization contrary to the majority of bird species that have stable karyotypes.

Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting.

INTRODUCTION: Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGS to detect copy number variants (CNVs) in clinical cytogenetics. MATERIALS AND METHODS: DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75 kb to 90.3 Mb, including aneuploidies and two cases of mosaicism. RESULTS: All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. DISCUSSION: Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

A new insight into the pathway behind spontaneous recurrent pregnancy loss: decreased CYR61 gene expression

ABSTRACT OBJECTIVE: Investigating the potential role of CYR61 in recurrent pregnancy loss is critical for developing diagnostic approaches and treatments for recurrent pregnancy loss. METHODS: In this prospective case-control study, we have investigated the expression patterns of CYR61 in blood samples from participants with recurrent pregnancy loss in their medical history and control group (n=20 vs n=10). Peripheral blood mononuclear cells from study and control groups were isolated and the expression patterns of the CYR61 gene were determined by real-time semi-quantitative reverse transcriptase PCR. RESULTS: A significant decrease in CYR61 gene expression was demonstrated in patients with two or more clinically recognized miscarriages compared with patients without miscarriages or with a history of miscarriage (p<0.01), which may make the CYR61 gene a potential candidate for predicting the risk of recurrent pregnancy loss. DISCUSSION: This study provides a basis for a detailed investigation of candidate biomarkers and molecular players involved in the development of recurrent pregnancy loss and for the development of potential treatment approaches to prevent recurrent pregnancy loss.

U2 and U4 snDNA Comparative Chromosomal Mapping in the Neotropical Fish Genera Apareiodon and Parodon (Characiformes: Parodontidae).

Small nuclear DNA (snDNA) are valuable cytogenetic markers for comparative studies in chromosome evolution because different distribution patterns were found among species. Parodontidae, a Neotropical fish family, is known to have female heterogametic sex chromosome systems in some species. The U2 and U4 snDNA sites have been found to be involved in Z and W chromosome differentiation in Apareiodon sp., Apareiodon affinis, and Parodon hilarii. However, few studies have evaluated snDNA sites as propulsors of chromosome diversification among closely related fish species. In this study, we investigated the distribution of U2 and U4 snDNA clusters in the chromosomes of 10 populations/species belonging to Apareiodon and Parodon, aiming to identify chromosomal homeologies or diversification. In situ localization data revealed a submetacentric pair carrying the U2 snDNA site among the populations/species analyzed. Furthermore, all studied species demonstrated homeology in the location of U4 snDNA cluster in the proximal region of metacentric pair 1, besides an additional signal showing up with a divergence in Apareiodon. Comparative chromosomal mapping of U4 snDNA also helped to reinforce the proposal of the ZZ/ZW1W2 sex chromosome system origin in an A. affinis population. According to cytogenetic data, the study corroborates the diversification in Parodontidae paired species with uncertain taxonomy.

Cross-species chromosome painting and repetitive DNA mapping illuminate the karyotype evolution in true crocodiles (Crocodylidae).

Crocodilians have maintained very similar karyotype structures and diploid chromosome numbers for around 100 million years, with only minor variations in collinearity. Why this karyotype structure has largely stayed unaltered for so long is unclear. In this study, we analyzed the karyotypes of six species belonging to the genera Crocodylus and Osteolaemus (Crocodylidae, true crocodiles), among which the Congolian endemic O. osborni was included and investigated. We utilized various techniques (differential staining, fluorescence in situ hybridization with repetitive DNA and rDNA probes, whole chromosome painting, and comparative genomic hybridization) to better understand how crocodile chromosomes evolved. We studied representatives of three of the four main diploid chromosome numbers found in crocodiles (2n = 30/32/38). Our data provided new information about the species studied, including the identification of four major chromosomal rearrangements that occurred during the karyotype diversification process in crocodiles. These changes led to the current diploid chromosome numbers of 2n = 30 (fusion) and 2n = 38 (fissions), derived from the ancestral state of 2n = 32. The conserved cytogenetic tendency in crocodilians, where extant species keep near-ancestral state, contrasts with the more dynamic karyotype evolution seen in other major reptile groups.

Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

Cytogenetics Meets Genomics: Cytotaxonomy and Genomic Relationships among Color Variants of the Asian Arowana Scleropages formosus.

Scleropages formosus (Osteoglossiformes, Teleostei) represents one of the most valued ornamental fishes, yet it is critically endangered due to overexploitation and habitat destruction. This species encompasses three major color groups that naturally occur in allopatric populations, but the evolutionary and taxonomic relationships of S. formosus color varieties remain uncertain. Here, we utilized a range of molecular cytogenetic techniques to characterize the karyotypes of five S. formosus color phenotypes, which correspond to naturally occurring variants: the red ones (Super Red); the golden ones (Golden Crossback and Highback Golden); the green ones (Asian Green and Yellow Tail Silver). Additionally, we describe the satellitome of S. formosus (Highback Golden) by applying a high-throughput sequencing technology. All color phenotypes possessed the same karyotype structure 2n = 50 (8m/sm + 42st/a) and distribution of SatDNAs, but different chromosomal locations of rDNAs, which were involved in a chromosome size polymorphism. Our results show indications of population genetic structure and microstructure differences in karyotypes of the color phenotypes. However, the findings do not clearly back up the hypothesis that there are discrete lineages or evolutionary units among the color phenotypes of S. formosus, but another case of interspecific chromosome stasis cannot be excluded.

Analysis in Proceratophrys boiei genome illuminates the satellite DNA content in a frog from the Brazilian Atlantic forest.

Satellite DNAs (satDNAs) are one of the most abundant elements in genomes. Characterized as tandemly organized sequences that can be amplified into multiple copies, mainly in heterochromatic regions. The frog P. boiei (2n = 22, ZZ♂/ZW♀) is found in the Brazilian Atlantic forest and has an atypical pattern of heterochromatin distribution when compared to other anuran amphibians, with large pericentromeric blocks on all chromosomes. In addition, females of Proceratophrys boiei have a metacentric sex chromosome W showing heterochromatin in all chromosomal extension. In this work, we performed high-throughput genomic, bioinformatic, and cytogenetic analyses to characterize the satellite DNA content (satellitome) in P. boiei, mainly due to high amount of C-positive heterochromatin and the highly heterochromatic W sex chromosome. After all the analyses, it is remarkable that the satellitome of P. boiei is composed of a high number of satDNA families (226), making P. boiei the frog species with the highest number of satellites described so far. Consistent with the observation of large centromeric C-positive heterochromatin blocks, the genome of P. boiei is enriched with high copy number of repetitive DNAs, with total satDNA abundance comprising 16.87% of the genome. We successfully mapped via Fluorescence in situ hybridization the two most abundant repeats in the genome, PboSat01-176 and PboSat02-192, highlighting the presence of certain satDNAs sequences in strategic chromosomal regions (e.g., centromere and pericentromeric region), which leads to their participation in crucial processes for genomic organization and maintenance. Our study reveals a great diversity of satellite repeats that are driving genomic organization in this frog species. The characterization and approaches regarding satDNAs in this species of frog allowed the confirmation of some insights from satellite biology and a possible relationship with the evolution of sex chromosomes, especially in anuran amphibians, including P. boiei, for which data were not available.

Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses.

Chromosomal abnormalities are largely associated with fertility impairments in the domestic horse. To date, over 600 cases of individuals carrying abnormal chromosome complements have been reported, making the domestic horse the species with the highest prevalence. However, studies analyzing the prevalence of chromosomal diseases in whole populations are scarce. We, therefore, employed a two-step molecular tool to screen and diagnose chromosomal abnormalities in a large population of 25,237 Pura Raza Español horses. Individuals were first screened using short tandem repeats parentage testing results and phenotypic evaluations. Those animals showing results suggesting chromosomal abnormalities were re-tested using a single nucleotide polymorphism (SNP)-based diagnostic methodology to accurately determine the chromosomal complements. Thirteen individuals showed a positive screening, all of which were diagnosed as chromosomally abnormal, including five 64,XY mares with sex development disorders (DSD) and four cases of blood chimerism (two male/female and two female/female cases). In addition, we detected one Turner and one Klinefelter syndrome and two individuals carrying complex karyotypes. The overall prevalence in the entire population was ~0.05%, with the prevalence of 64,XY DSD and blood chimerism ~0.02% and ~0.016%, respectively. However, the overall results should be taken with caution since the individuals carrying Turner syndrome (in full (63,X) or mosaic (mos 63,X/64,XX) forms) cannot be detected due to limitations in the methodology employed. Finally, the lack of agreement between populational studies performed using karyotyping or molecular methods is discussed. To our knowledge, this is the largest populational study performed evaluating the prevalence of the most common chromosomal abnormalities in the domestic horse.