The river buffalo (Bubalus bubalis, 2n = 50) cytogenetic map: assignment of 64 loci by fluorescence in situ hybridization and R-banding

Sixty-four genomic BAC-clones mapping five type I (ADCYAP1, HRH1, IL3, RBP3B and SRY) and 59 type II loci, previously FISH-mapped to goat (63 loci) and cattle (SRY) chromosomes, were fluorescence in situ mapped to river buffalo R-banded chromosomes, noticeably extending the physical map of this species. All mapped loci from 26 bovine syntenic groups were located on homeologous chromosomes and chromosome regions of river buffalo and goat (cattle) chromosomes, confirming the high degree of chromosome homeologies among bovids. Furthermore, an improved cytogenetic map of the river buffalo with 293 loci from all 31 bovine syntenic groups is reported.     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

An advanced sheep (Ovis aries, 2n = 54) cytogenetic map and assignment of 88 new autosomal loci by fluorescence in situ hybridization and R-banding

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Comparative FISH mapping in river buffalo and sheep chromosomes: assignment of forty autosomal type I loci from sixteen human chromosomes.

Forty autosomal type I loci earlier mapped in goat were comparatively FISH mapped on river buffalo (BBU) and sheep (OAR) chromosomes, noticeably extending the physical map in these two economically important bovids. All loci map on homoeologous chromosomes and chromosome bands, with the exception of COL9A1 mapping on BBU10 (homoeologous to cattle/goat chromosome 9) and OAR9 (homoeologous to cattle/goat chromosome 14). A FISH mapping control with COL9A1 on both cattle and goat chromosomes gave the same results as those obtained in river buffalo and sheep, respectively. Direct G- and R-banding comparisons between Bovinae (cattle and river buffalo) and Caprinae (sheep and goat) chromosomes 9 and 14 confirmed that a simple translocation of a small pericentromeric region occurred between the two chromosomes. Comparisons between physical maps obtained in river buffalo and sheep with those reported in sixteen human chromosomes revealed complex chromosome rearrangements (mainly translocations and inversions) differentiating bovids (Artiodactyls) from humans (Primates).     

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

The 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.     

Quantitative trait loci (QTL) mapping of resistance to strongyles and coccidia in the free-living Soay sheep (Ovis aries)

A genome-wide scan was performed to detect quantitative trait loci (QTL) for resistance to gastrointestinal parasites and ectoparasitic keds segregating in the free-living Soay sheep population on St. Kilda (UK). The mapping panel consisted of a single pedigree of 882 individuals of which 588 were genotyped. The Soay linkage map used for the scans comprised 251 markers covering the whole genome at average spacing of 15cM. The traits here investigated were the strongyle faecal egg count (FEC), the coccidia faecal oocyst count (FOC) and a count of keds (Melophagus ovinus). QTL mapping was performed by means of variance component analysis so that the genetic parameters of the study traits were also estimated and compared with previous studies in Soay and domestic sheep. Strongyle FEC and coccidia FOC showed moderate heritability (h(2)=0.26 and 0.22, respectively) in lambs but low heritability in adults (h(2)<0.10). Ked count appeared to have very low h(2) in both lambs and adults. Genome scans were performed for the traits with moderate heritability and two genomic regions reached the level of suggestive linkage for coccidia FOC in lambs (logarithm of the odds=2.68 and 2.21 on chromosomes 3 and X, respectively). We believe this is the first study to report a QTL search for parasite resistance in a free-living animal population and therefore may represent a useful reference for similar studies aimed at understanding the genetics of host-parasite co-evolution in the wild.     

A set of polymorphic DNA microsatellites useful in swamp and river buffalo (Bubalus bubalis)

DNA microsatellites have found widespread application in gene mapping, pedigree determination and population genetics. In closely related species such as bovids, heterologous polymerase chain reaction (PCR) primers may in some cases be used, bypassing the need to isolate and characterize microsatellite-containing sequences and design PCR primers. We report on the ability of a set of eighty bovine derived DNA microsatellite primers to amplify sequences in the two types (swamp and river) of water buffalo ( Bubalus bubalis ). Number of alleles and per cent heterozygosities in a large number of animals were determined on a subset of microsatellite loci selected on the robustness of the primers. These loci will form the basis of a set of polymorphic DNA markers for use in water buffalo.     

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

Total lipids and phospholipids in buffalo semen (Bubalus bubalis)

Spermatozoal and seminal plasma concentrations of total lipids from 50 ejaculates and phospholipids and their fractions from 30 ejaculates were quantified in the semen of five Murrah buffalo bulls. Sperm lipid content ranged from 0.93 to 1.72 mg/10(9) cells with an overall average 1.32 +/- 0.03 mg/10(9) cells. Its concentration in seminal plasma varied from 1.39 to 2.22 mg/ml with overall average of 1.75 +/- 0.03 mg/ml. Spermatozoal total phospholipid content ranged from 0.44 to 0.94 mg/10(9) cells with overall mean being 0.64 +/- 0.02 mg/10(9) cells. The corresponding values for seminal plasma were 0.53 and 0.88 mg/ml with an overall mean of 0.69 +/- 0.02 mg/ml. Phosphatidyl choline constituted the major fraction both in the spermatozoa and and seminal plasma.     

ZFX and ZFY loci in water buffalo (Bubalus bubalis):: Potential for sex identification

In the present study a small region of ZFX and ZFY loci in buffalo have been amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) was also uncovered that can distinguish between male and female buffalo DNA. The study also found a second copy of the ZFX loci (ZFXR) present in both male and female. Sequence analysis showed that ZFXR has a single base deletion that results in a redundant putative protein     

Microisolation and microcloning of bovine X-chromosomes for identification of sorted buffalo (Bubalus bubalis) spermatozoa

Flow-cytometry sorting technology has been successfully used to separate the X- and Y-chromosome bearing spermatozoa for production of sex-preselected buffalo. However, an independent technique should be employed to validate the sorting accuracy. In the present study, X-chromosomes of bovine were micro-dissected from the metaphase spreads by using glass needles. Then X-chromosomes were then amplified by PCR and labelled with Cy3-dUTP for use as a probe in hybridization of the unsorted and sorted buffalo spermatozoa -chromosome. The results revealed that 47.7% (594/1246) of the unsorted buffalo spermatozoa were positive for X- chromosome probe, which was conformed to the sex ratio in buffalo (X:Y spermatozoa=1:1); 9.6% (275/2869) of the Y-sorted buffalo spermatozoa and 86.1% (1529/1776) of the X-sorted buffalo spermatozoa showed strong X-chromosome FISH signals. Flow cytometer re-analysis revealed that the proportions of X- and Y-bearing spermatozoa in the sorted X and Y semen was 89.6% and 86.7%, respectively. There were no significant differences between results assayed by flow-cytometry re-analysis and by FISH in this study. In conclusion, FISH probe derived from bovine X- chromosomes could be used to verify the purity of X and Y sorted spermatozoa in buffalo.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.     

Comparative FISH mapping of mucin 1, transmembrane (MUC1) among cattle, river buffalo, sheep and goat chromosomes: comparison between bovine chromosome 3 and human chromosome 1

Four bovine BAC clones (0494F01, 0069D07, 0060B06, and 0306A12) containing MUC1, as confirmed by mapping MUC1 on a RH3000 radiation hybrid panel, were hybridised on R-banded chromosomes of cattle (BTA), river buffalo (BBU), sheep (OAR) and goat (CHI). MUC1 was FISH-mapped on BTA3q13, BBU6q13, OAR1p13 and CHI3q13 and both chromosomes and chromosome bands were homoeologous confirming the high degree of chromosome homoeologies among bovids and adding more information on the pericentromeric regions of these species' chromosomes. Indeed, MUC1 was more precisely assigned to BTA3 and assigned for the first time to BBU6, OAR1p and CHI3. Moreover, detailed and improved cytogenetic maps of BTA3, CHI3, OAR1p and BBU6 are shown and compared with HSA1.