Identification of Some Errors in the Genome Assembly of Bovidae by FISH.

From an economic point of view, Bovidae represent the most important family of the Ruminantia suborder. Thus, the mitochondrial and nuclear genomes of Bos taurus were among the first genomes to be sequenced after the sequencing of the human genomes. Over the millennia, the evolution of the genomes of the 3 main species belonging to the Bovidae family - B. taurus (BTA), Ovis aries (OAR), and Capra hircus (CHI) - has led to few chromosome rearrangements. Certainly, the availability and free access to the animal genomes significantly contributed to the improvement of animal genetics; however, some errors may exist due to the high automation in the genomic assembly construction process. In this work, some differences between the genomes of cattle, goat, and sheep highlighted by bioinformatics analysis have been verified by FISH, confirming that some errors persist even in the most recent genome assemblies. This type of approach has allowed us to detect a misassembly of a region belonging to BTA16 and to the homologues OAR12 and CHI16, a misassembly of a short tract in BTA22, OAR19, and CHI22, an incorrect mapping of a region of BTA21 and of CHI27 and OAR26, a discrepancy in the BTA26, OAR22, and CHI26 assemblies, a missed inversion in CHI1 compared to BTA1 and OAR1, and the exact assembly of a region of about 7 Mb in OAR10 and CHI12. Incorrect positioning of genomic tracts can cause unintended consequences in genetic analyses, especially when the data represent a starting point for the construction of genetic tools. In the new genomic assemblies published after the conclusion of our experiments, however, the accuracy in the construction of animal assemblies has been much improved, even if the new assemblies present more extended unmapped portions than the previous versions. The gap could be filled by comparative analyses between similar species or FISH.

Cytogenetic Characterization of a Small Evolutionary Rearrangement Involving Chromosomes BTA21 and OAR18.

Both cattle (Bos taurus) and sheep (Ovis aries) belong to the Bovidae family but to different subfamilies, Bovinae and Caprinae, respectively. From a chromosomal point of view, apart from the already known centric fusions (that occurred during the evolutionary process in the Bovidae family) and the small differences in the chromosome classification, the 2 karyotypes are very similar in banding patterns. In this study, the combination of bioinformatics techniques and physical mapping of DNA markers enabled the identification of a micro-rearrangement, a small inversion involving bovine chromosome 21 (BTA21) and the corresponding sheep chromosome 18 (OAR18). The aim of this study was the cytogenetic characterization of this difference in genomic assemblies between cattle and sheep in this single chromosome region. To verify the inversion in FISH experiments, we used the BACs 442H08 and 222H03 from the INRA library and BACs 134H22 and 436P08 from the sheep-specific CHORI library. The results confirmed the presence of the inverted fragment in sheep compared to the cattle genome. Genomic rearrangements may have consequences depending on their influence on gene activity, but in this case no gene or transcribed DNA portion seemed to be involved. In conclusion, we showed for the first time, concerning autosomes, that besides the already known centric fusions also other differences exist between the bovine and sheep karyotypes. Furthermore, we demonstrated that the combination of a bioinformatics approach and physical mapping is a valid tool for the identification of currently unknown rearrangements between related species.

Centromere Repositioning in Cattle (Bos taurus) Chromosome 17.

Eukaryotic organisms have developed a structure, called centromere, able to preserve the integrity of the genome during cell division. A young bull from the Marchigiana breed, with a normal external phenotype, underwent routine cytogenetic analysis to enter the reproduction center. All metaphases analyzed showed an unusual biarmed chromosome of medium size despite a diploid set of chromosomes (2n = 60,XY). FISH analysis excluded a pericentric inversion or a reciprocal translocation, but highlighted a repositioning of the centromere in BTA17. The satellite DNA was still in an acrocentric position. The telomeres were normally present. The primary constriction on the abnormal chromosome was C-band negative. Finally, the absence of a large genomic deletion in the BTA17 pericentromeric region was demonstrated by both array-CGH analysis and SNP array. To our knowledge, this is the first case of centromere repositioning reported in cattle.

New cryptic karyotypic differences between cattle (Bos taurus) and goat (Capra hircus).

Cattle (Bos taurus) and goat (Capra hircus) belong to the Bovidae family, and they share a common ancestor 19.7-21.5 Ma ago (MYA). The Bovidae family apparently experienced a rapid species radiation in the middle Miocene. The present day cattle and goat possess the same diploid chromosome number (2n = 60) and structurally similar autosomes, except that a small subcentromeric portion of cattle chromosome nine has been translocated to goat chromosome 14. In this study, we adopted a new strategy that involves the use of bioinformatics approach to detect unknown cryptic chromosome divergences between cattle and goat using and subsequent validation using the fluorescence in situ hybridization (FISH) of bacterial artificial chromosome clones. We identified two hypothetical discrepancies between the cattle and goat genome assemblies: an inversion in the goat chromosome 13 and a transposition in the goat chromosome 6. The FISH technique allowed clear validation of the existence of a new 7.4 Mb chromosomal inversion in the goat chromosome 13. Regarding the transposition in the goat chromosome six, FISH analyses revealed that the cattle and goat genomes shared the same organization, with the assembly of the goat genome being the correct one. Moreover, we defined, for the first time, the size and orientation of the translocated fragment involved in the evolutionary translocation between cattle chromosomes 9 and goat chromosome 14. Our results suggest that bioinformatics represents an efficient method for detecting cryptic chromosome divergences among species.

Genomic analysis of cattle rob(1;29).

Robertsonian translocation (rob) involving chromosomes 1 and 29 represents the most frequent chromosome abnormality observed in cattle breeds intended for meat production. The negative effects of this anomaly on fertility are widely demonstrated, and in many countries, screening programs are being carried out to eliminate carriers from reproduction. Although rob(1;29) was first observed in 1964, the genomic structure of this anomaly is partially unclear. In this work, we demonstrate that, during the fusion process, around 5.4 Mb of the pericentromeric region of BTA29 moves to the q arm, close to the centromere, of rob(1;29). We also clearly show that this fragment is inverted. We find that no deletion/duplication involving sequences reported in the BosTau6 genome assembly occurred during the fusion process which originates this translocation.

Clinical, genetic, and pathological features of male pseudohermaphroditism in dog.

Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition.

Integration of bovine herpesvirus 4 genome into cultured persistently infected host cell genome.

Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play a secondary causal role, along with bacterial infection, in bovine post-partum metritis. Mechanisms of maintenance of BoHV-4 persistent infection are not understood. We previously generated in vitro models of BoHV-4 persistent infection in human rhadomyosarcoma and bovine macrophage cell lines by drug selection of cells infected with BoHV-4 carrying a drug-resistance marker, and demonstrated circular episomal BoHV-4 genomes. In the present study, we used fluorescent in situ hybridization (FISH) to demonstrate BoHV-4 genomes also integrated into the genomes of these persistently infected cells.

Persistent Müllerian Duct Syndrome in a German Shepherd Dog.

In mammals, the regression of the müllerian ducts is regulated by the action of the AMH hormone which is produced by testes during embryonic development. The action of this hormone is mediated by the only known receptor AMHR2. Mutations occurring in the AHM hormone and/or in the AMHR2 receptor gene cause the lack of regression of müllerian ducts, which may therefore persist even in male embryos carrying a XY chromosomal arrangement. This is known as the persistent müllerian duct syndrome (PMDS). A female German Shepherd dog was referred to the veterinary clinic because of urinary incontinence. She also showed an anatomical structure that protruded from and enlarged the vulvar labia. From the morphological appearance, one gonad resembled an ovary and the other a testicle. The histological examination instead showed that the gonads were both testes with an underdeveloped parenchyma and without signs of spermatogenetic activity. No alterations were found with regard to the uterus which showed a correctly developed body, cervix, and horns. Genetic analysis, performed on DNA extracted from blood, showed (i) the presence of both X and Y chromosomes, (ii) the absence of chromosome XX/XY chimerism, (iii) a normal SRY gene coding sequence, (iv) a normal AMHR2 gene coding sequence, and (v) a normal AMH gene coding sequence. In this study, we report and characterize a new case of PMDS in a dog excluding that the only mutation hitherto found in the AMHR2 gene is responsible for the observed phenotype.

XY (SRY-positive) Ovarian Disorder of Sex Development in Cattle.

In mammals, the sex of the embryo depends on the SRY gene. In the presence of at least one intact and functional copy of this genetic factor (XY embryo) undifferentiated gonads will develop as testicles that subsequently determine the male phenotype. When this factor is not present, i.e., in subjects with 2 X chromosomes, an alternative pathway induces the development of ovaries, hence a female phenotype. In this case study, we describe a female cattle affected by a disorder of sex development (DSD). The subject, despite having a chromosomal XY constitution, did not develop testicles but ovaries, although they were underdeveloped. Moreover, genetic analysis highlighted the presence of the SRY gene with a normal coding region in both blood- and tissue-derived DNA. A chimeric condition was excluded in blood by sexing more than 350 cells and by allele profile investigation of 18 microsatellite markers. Array CGH analysis showed the presence of a not yet described 99-kb duplication (BTA18), but its relationship with the phenotype remains to be demonstrated. Gonadal histology demonstrated paired ovaries: the left one containing a large corpus luteum and the right one showing an underdeveloped aspect and very few early follicles. To our knowledge, we describe the first case of XY (SRY+) DSD in cattle with a normal SRY gene coding sequence.

Testicular XX (SRY-Negative) Disorder of Sex Development in Cat.

In most mammals, the sex of an individual is genetically determined by the Y chromosome-specific SRY gene. The presence of at least one functional copy of this gene determines the development of the primordial gonads into testes. However, testicular tissue does develop in the absence of SRY, albeit rarely, which is the case in testicular XX (SRY-negative) disorder of sex development (DSD). This condition is very important for studying the process of sexual determination because it allows the identification of genetic factors that are able to promote the male developmental pathway in the absence of SRY and thereby enables a better understanding of this process. Until now, this condition has been identified in various animal species but has never been reported in cat. In this study, we describe the first case of an XX (SRY-negative) DSD cat. The cat possesses a tortoiseshell coat associated with male-like external genitalia, including normal scrotum with 2 palpably normal testicles. Histological analysis confirmed the presence of the testes, and cytogenetic and genetic analyses showed a female karyotype associated with the absence of the SRY gene. Finally, sequencing of the RSPO1 gene revealed no mutation, and FISH analysis of the SOX9 locus did not reveal any large abnormalities.

A very rare clinical case of a holstein heifer with two vulvae and a scrotum.

The physical and gynecological examination of a Holstein heifer single-born with a disorder of sexual development showed anatomical abnormalities such as the presence of a scrotum and 2 vulvae and an anal sphincter that was positioned on the right side of the body. Also, an early pregnancy was diagnosed. Cytogenetic and hormone analysis was requested, and the animal showed normal female metaphases (60,XX) and hormonal profiles. However, in gross anatomy and histological examinations, a structure compatible with a penis, the absence of a uterine body, 2 exophytic structures, and a septum in the vagina were detected.

A Revised Genome Assembly of the Region 5' to Canine SOX9 Includes the RevSex Orthologous Region.

The SOX gene family includes many genes that play a determinant role in several developmental pathways. The SOX9 gene has been identified as a major factor in testis development in mammals after it is activated by the SRY gene. However, duplication of the gene itself in some mammalian species, or of a well-delimited upstream 'RevSex' region in humans, has been shown to result in testis development in the absence of the SRY gene. In the current study, we present an accurate analysis of the genomic organization of the SOX9 locus in dogs by both in silico and FISH approaches. Contrary to what is observed in the current dog genome assembly, we found that the genomic organization is quite similar to that reported in humans and other mammalian species, including the position of the RevSex region in respect to SOX9. The analysis of the conserved sequences within this region in 7 mammalian species facilitated the highlighting of a consensus sequence for SRY binding. This new information could help in the identification of evolutionarily conserved elements relevant for SOX9 gene regulation, and could provide valid targets for mutation analysis in XY DSD patients.

A Clinical Case of an SRY-Positive Intersex/Hermaphrodite Holstein Cattle.

A single-born, 15-month-old Holstein cattle, diagnosed as hermaphrodite, was investigated for estrous cycle, hormonal profiles, karyotype, presence of SRY, as well as anatomopathological and histological aspects. Normal continuous estrous cycles and basal testosterone levels were reported. Necropsy showed the presence of a female genital tract that mismatched a vulvar opening and a male pelvic urethra continued within a penis. Moreover, we observed islands of seminiferous tubules with the presence of germline cells, 2 pampiniform plexi, the corpus cavernosum, the penile urethra, the corpus spongiosum and the glans. Cytogenetic analyses of the blood cells showed an XX karyotype, while the molecular analyses revealed the presence of the SRY gene in several tissues, including blood. This is the first report in the scientific literature of an SRY-positive hermaphrodite Holstein cattle with continuous ovarian cycles.

Sox9 duplications are a relevant cause of Sry-negative XX sex reversal dogs.

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.