186 research outputs found
FISH mapping of bovine U21, U1 and U7 molecular markers to river buffalo chromosomes 3p, 5q and 5p.
No full textThree bovine cosmid-derived microsatellites (IDVGA49, IDVGA7 and IDVGA47), previously assigned to cattle syntenic groups U1, U7 and U21, respectively, were fluorescence in situ hybridization (FISH) mapped to river buffalo (Bubalus bubalis, L., 2n = 50) chromosomes (BBU) 3p22 (IDVGA47, U21), BBU 5q21 (IDVGA49, U1) and BBU 5p19 (IDVGA7, U7) using sequential FISH and R-banding techniques. These localizations allowed the assignment, for the first time, of the bovine syntenic groups U21, U1 and U7 to specific river buffalo chromosomes. FISH mapping of IDVGA7 (U7) to cattle rob(1;29) p-arms confirms the banding homologies between BTA 29 and BBU 5p and further supports the idea that cattle standard karyotypes need adjustments
Cytogenetic elaboration of a novel reciprocal translocation in sheep
No full textReciprocal translocations represent one of the most common structural chromosomal rearrangements observed in both humans and domestic animals. In these translocations, the balanced forms are most frequent but may remain undetected because the carriers show a normal phenotype. For this reason, routine cytogenetic analysis of domestic animals should necessarily rely on banded karyotypes. In fact, during a screening analysis, carried out on phenotypically normal young sheep (Ovis aries, OAR, 2n = 54) from Laticauda-Comisana hybrids, a new structural rearrangement was detected. Two abnormal acrocentric chromosomes (the smallest and the largest one) were found in all metaphases of this carrier animal, suggesting the presence of a reciprocal translocation (rcp). CBA and RBA banding were performed in order to characterize the translocation, and FISH with chromosome-specific BAC probes and telomere probes was applied to confirm the cytogenetic data. The translocation was classified as rcp(4q;12q)(q13;q25)
Cytogenetic Elaboration of a Novel Reciprocal Translocation in Sheep
No full textReciprocal translocations represent one of the most common structural chromosomal rearrangements observed in both humans and domestic animals. In these translocations, the balanced forms are most frequent but may remain undetected because the carriers show a normal phenotype. For this reason, routine cytogenetic analysis of domestic animals should necessarily rely on banded karyotypes. In fact, during a screening analysis, carried out on phenotypically normal young sheep (Ovis aries, OAR, 2n = 54) from Laticauda-Comisana hybrids, a new structural rearrangement was detected. Two abnormal acrocentric chromosomes (the smallest and the largest one) were found in all metaphases of this carrier animal, suggesting the presence of a reciprocal translocation (rcp). CBA and RBA banding were performed in order to characterize the translocation, and FISH with chromosome-specific BAC probes and telomere probes was applied to confirm the cytogenetic data. The translocation was classified as rcp(4q;12q)(q13;q25)
Advanced comparative cytogenetic analysis of X chromosomes in river buffalo, cattle, sheep, and human
No full textBased on a recently generated comprehensive
gene map for Ovis aries chromosome X (OARX)
with an approximately even locus distribution, we
assigned selected bacterial artificial chromosome
(BAC) probes corresponding to these OARX loci to
Bubalus bubalis (BBU) and Bos taurus (BTA) by
comparative fluorescence in-situ hybridization (FISH)
to improve cytogenetically the X chromosome maps in these species. Twenty-five added loci in BBUX and
BTAX, respectively, contribute to a more detailed
description of the cytogenetic organization of these
chromosomes. Further seven loci were identified in
OARX and two DNA probes were assigned to X and
Y chromosomes in river buffalo, cattle, and sheep,
respectively, and thus identified loci in the pseudoautosomal
region. The additional assignments double the
number of cytogenetic loci in BBUX and increase
their number in BTAX and OARX. The larger quantity
of cytogenetic anchors allows a more precise morphological
comparison of bovid X chromosomes among
each other and with the Homo sapiens (HSA) X chromosome.
The anchor loci confirm and refine syntenic
fragments in HSAX and identify several evolutionary
breakpoints between the compared chromosomes. The
cytogenetic assignments in BBUX, BTAX, and
OARX represent useable anchors for the ongoing genome
sequence assembly in Bovidae
The utility of chromosome microdissection in clinical cytogenetics: a new reciprocal translocation in sheep.
No full textLocal sheep breeders and scientists in Italy cooperate and conduct research on the genetic improvement of autochthonous genetic types (AGTs) by various approaches, including a cytogenetic breeding selection since 2011. The Laticauda sheep (Ovis aries, 2n = 54) breed is one of the AGTs reared in the Campania region (southern Italy). Performing cytogenetic analyses, we have detected and described a novel reciprocal translocation in a Laticauda sheep identified as 54,XX t(18;23)(q14;q26). Our data support recurring appeals that suggest the regular performance of cytogenetic analyses for monitoring genetic health of livestock species. In total, 5 cases of reciprocal translocations in sheep are known, including the new case. None of them has any phenotypic effect on the living offspring. However, affected animals are characterized by sterility or have a low fertility which can have an effect on breeding success and on economical balance. Presence and kind of the described novel chromosomal aberration were detected by performing CBA-banding and FISH mapping with telomeric probes. RBA-banding allowed the karyotyping of sheep chromosomes and the identification of aberrant chromosomes and regions involved in the new reciprocal translocation. Whole chromosome painting (WCP) probes received from equivalent chromosomes in cattle and the derivative sheep chromosome 18 confirmed the cytogenetic data. This way, our study underlined both the importance of WCP probes by chromosome microdissection and a new way to use WCP probes directly generated from derivative chromosome
Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes.
No full textThe development of new molecular techniques (array CGH, M-FISH, SKY-FISH, etc.) has led to great advancements in the entire field of molecular cytogenetics. However, the application of these methods is still very limited in farm animals. In the present study, we report, for the first time, the production of 13 river buffalo (Bubalus bubalis, 2n = 50) chromosome-specific painting probes, generated via chromosome microdissection and the DOP-PCR procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of river buffalo chromosome/arms, namely, 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X, and Y, using both conventional and late-replicating banded chromosome preparations counterstained by DAPI. The provided 'bank' of chromosome-specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for other species of the family Bovidae, such as cattle, sheep, and goats, for chromosome abnormality diagnosis, and, more generally, for evolutionary studies
High-Throughput De Novo Sequencing of Laser Microdissected Y Chromosome in the Mediterranean River Buffalo (2n = 50,XY)
No full textThe sequencing and correct assembly of the Y chromosome sequences is still a challenge in mammals. In fact, apart from the pseudoautosomal regions (PARs), the Y chromosome lacks a homologous counterpart in the X chromosome. Therefore, in males, the use of genomic DNA as a template for NGS allows isolating the specific Y sequences only as a difference from the X sequences, with many potential gaps and assembly errors for the contemporary presence of X and Y. To overcome this problem, we present a high-throughput sequencing approach based on the direct isolation of Y-chromosomes by laser microdissection in the Mediterranean river buffalo.
Peripheral blood lymphocytes from 10 buffalo bulls were cultured in vitro for normal cultures. Fixed lymphocytes were spread on a polyethylene naphthalate membrane (PEN), which was attached to a 24 × 60 glass slide and treated for GTG-banding. Ten copies of the Y-chromosome from each bull were laser microdissected and collected in individual PCR tubes for DOP-PCR amplification and labeling. FISH confirmed the specific hybridization of each Y-probe on lymphocyte metaphases before NGS. Library preparation was performed with the NEB Next Ultra II DNA Library Prep Kit for Illumina. High-throughput sequencing was performed by Illumina technology with the NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles). Raw data were processed by TrimGalore (v0.6.7), and de novo assembly was accomplished by SPAdes genome assembler v3.15.5. Gene sequences were predicted by AUGUSTUS (v3.5.0).
We generated about 40 Gb (90×) of Illumina short reads. Total assembly length was 2,260,027 bp, with an average GC% of 48.11%. A total of 861 genes were predicted, and 807 of them have a hit-to-reference, 210 are uncharacterized, and around 30 are without description. The total number of microsatellites identified was 552. Variant calling was conducted using the GATK4 pipeline, specifically employing the HaplotypeCaller tool for each sample separately and also in a multi-sample version. The total number of variants across all samples was 25,100.
Our approach yielded valuable insights into the genomic characteristics of the Y chromosome, and our results represent a milestone for the river buffalo
50,XY gonadal dysgenesis (Swyer's syndrome) in a female river buffalo (Bubalus bubalis)
No full textSex determination in mammals occurs during fertilisation. Generally, the presence of the Y chromosome gives rise to male sex (or male offspring), even when more than one X is present. In human beings, the term '2n=46,XY gonadal dysgenesis (Swyer's syndrome)' is characterised by a 46,XY karyotvpe and incomplete testicular determinations. The lack of a medullary zone in the non-differentiated gonads affects the formation of testicles even when the Y chromosome is present. Many studies have revealed that this sex reversal may be the result of mutations in some Y-specific genes, in particular those of the sex determining region (SRY). In cattle, the most frequent cases of intersex are found in females which are co-twins with males (freemartin syndrome) because of placental anastomoses between the two co-twins. While male co-twins are generally normal, female co-twins are sterile but with normal external features. A few cases of XY intersexes have been reported as gonadal digenesis, true hermaphrodites and pseudohermaphrodites . Sex reversal seems to be much more common in horses, and several cases have been reported. In river buffalo, only two cases of sex chromosome abnormalities have been reported: a female which was trisomic, and a female which was monosomic for X chromosomes. The female which was trisomic was 10 years old and had two births, whereas the monosomic female was four-years-old and sterile. This short communication describes the first case of a female river buffalo 2n=50,XY with
gonadal dysgenesis, otherwise known as Swyer's syndrome. A normally developed five-year-old female river buffalo with reproductive problems, had prominent withers, as in males, a horn base with a circumference of 38 0 cm (26 to 28 cm in normal females), a pubic bone shorter than normal, a normal vagina, a normal vestibule, and a normal clitoris. The buffalo was slaughtered due to its infertility. Subsequent anatomical observation of the reproductive organs revealed slight hypoplasia of derivative Muller's ducts, small cervix uteri, very small gonads with ovary structure, a slight hydrosalpingitis, and a well-developed uteri interhorn ligament. Peripheral blood cultures were performed using two experiments. Cells were left untreated or treated with 5-bromo-2'-deoxyuridine (B3rdU) and bisbenzimize dye (33258; Hoechst) (15 pg/ml each) six hours before harvesting to obtain normal chromosome preparations and chromosomes with R-banded patterns, respectively. Slides obtained from norm3al cultures were treated for CBA-banding as
described by Sumner (1972), and acridine orange staining. Slides treated with BrdU were stained with Giemsa to obtain R13(;-banding. River buLtffalo chromosome identification followed the standard karyotype. Two hundred cells were examined from the tvo cultures, and all showed a male constitution (2n=50,XY), as demonstrated by both CBA-banding and RBG-banding techniques.
Despite external female sex structures (normal vagina, vestibule and clitoris), both the horns (larger than in normal females) and withers (prominent as in males) suggested the action of Y-specific genes affecting the body conformation. However, the absence of testicles caused the lack of Mullerian regression factor production and this justifies the development of Mullerian duct derivatives, even when the Y chromosome was present. This study demonstrates that both breeders and veterinary practitioners should be alert during animal breeding to avoid such problems as retaining females which never produce calves (or milk) on a farm. The female river buffalo in this
study showed some clear male features (horns and withers) which should have suggested earlier cytogenetic investigation
Physical Mapping of 20 Unmapped Fragments of the Btau_4.0 Genome Assembly in Cattle, Sheep and River Buffalo
No full textThe recent advances in sequencing technology and bioinformatics have revolutionized genomic research, making the decoding of the genome an easier task. Genome sequences are currently available for many species, including cattle, sheep and river buffalo. The available reference genomes are very accurate, and they represent the best possible order of loci at this time. In cattle, despite the great accuracy achieved, a part of the genome has been sequenced but not yet assembled: these genome fragments are called unmapped fragments. In the present study, 20 unmapped fragments belonging to the Btau_4.0 reference genome have been mapped by FISH in cattle (Bos taurus, 2n = 60), sheep (Ovis aries, 2n = 54) and river buffalo (Bubalus bubalis, 2n = 50). Our results confirm the accuracy of the available reference genome, though there are some discrepancies between the expected localization and the observed localization. Moreover, the available data in the literature regarding genomic homologies between cattle, sheep and river buffalo are confirmed. Finally, the results presented here suggest that FISH was, and still is, a useful technology to validate the data produced by genome sequencing programs
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