Polymorphisms in MHC-DRA and -DRB alleles of water buffalo (Bubalus bubalis) reveal different features from cattle DR alleles

Seventy-five individuals of Bubalus bubalis belonging to four different breeds, three of river buffalo and one of swamp buffalo, were studied for polymorphism in MHC DRB (Bubu-DRB) and DRA (Bubu-DRA) loci. Eight alleles of Bubu-DRB were found, and all alleles in the swamp type were shared with the three river breeds. All alleles sampled from the breed of European origin (Mediterranean) were present in breeds sampled in Brazil, thus variability of this locus may have been preserved to a great extent in the more recently founded Brazilian population. Bubu-DRB alleles contained higher proportions of synonymous vs. non-synonymous substitutions in the non-peptide-binding sites (PBS) region, in contrast to the pattern of variation found in BoLA-DRB3, the orthologous locus in cattle. This indicated that either the first domain exon (exon 2) of Bubu-DRB has not undergone as much recombination and/or gene conversion as in cattle alleles, or Bubu-DRB may be more ancient than BoLA-DRB3 alleles. Phylogenetic analysis of DRB alleles from Bubalus, Syncerus c. caffer, the Cape buffalo, and domestic cattle demonstrated transspecies polymorphism. Water buffalo contained two alleles of DRA that differed from each other in two amino acid positions, including one in the PBS (alpha22) that was also shared with Anoa depressicornis, the anoa. Discovery of variation in DRA was surprising as the first domain of DRA is a highly conserved polypeptide in mammals in general and especially in ruminants, where no other substitution in PBS was seen.     

Evidence for an additional cattle DQB locus

 Studying the genetic polymorphism of the major histocompatibility complex class II genes in cattle, we identified a sequence (KUH1) which resembles those encoding class II β chains. The gene was shown to be transcribed in peripheral blood leukocytes. Sequence comparisons, Southern blot, and phylogenetic analyses indicate that (1) KUH1 represents a distinct DQB locus, which we propose to designate BoLA-DQB5, (2) DQB5 constitutes an ancient DQB locus which diverged from a common ancestor gene prior to the duplication resulting in DQB1 and DQB2, (3) DQB5 is associated with haplotypes which contain DQA5 and a duplicated DQ region. Received: 26 January 1999 / Revised: 20 April 1999     

Sequence polymorphism in the bovine major histocompatibility complex DQB loci

Polymorphism in DQB sequences of the bovine major histocompatibility complex was investigated in 22 British Friesian cattle. The first domain exon was amplified, cloned and sequenced. Eight different sequences were identified, six of which had not been identified previously. The high proportion of novel sequences suggests that additional polymorphisms within the DQB loci remain to be discovered in this breed. One sequence was present in at least 21 of the 22 cattle. This sequence, or a closely related sequence, has also been found in American Holstein Friesian, Swedish Red and White and Japanese Black cattle. The remarkably high sequence conservation suggests that the bovine DQB region may contain a locus with a low level of polymorphism and be more similar to the human DQB region than previously supposed. One sequence with three widely spaced frameshift insertions appeared to be a pseudogene.     

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.     

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.     

Gene duplications and sequence polymorphism of bovine class II DQB genes

The genetic diversity of bovine class II DQB genes was investigated by polymerase chain reaction amplification and DNA sequencing. The first domain exon was amplified from genomic DNA samples representing 14 class II haplotypes, defined by restriction fragment length polymorphism (RFLP) analysis. The presence of a polymorphism in the copy number of DQB genes was confirmed since two DQB sequences were isolated from certain haplotypes. Four subtypes of bovine DQB genes were found. DQB1 is the major type and was found in almost all haplotypes. DQB2 is very similar to DQB1 but was found only in the duplicated haplotypes DQ9 to 12. DQB3 and DQB4 are two quite divergent genes only present in certain duplicated haplotypes. The bovine DQB complexity thus resembles that in the human DRB region. Bovine DQB genes were found to be highly polymorphic as ten DQB1 alleles and four DQB2 alleles were identified. The observed sequence polymorphism correlated well with previously defined DQB RFLPs. Bovine and human DQB alleles show striking similarities at the amino acid level. In contrast, the frequency of silent substitutions is much higher in comparisons of DQB alleles between species than within species ruling out the possibility that any of the contemporary DQB alleles have been maintained since the divergence of humans and cattle. The frequency of silent substitutions between DQB alleles was markedly lower in cattle than in humans, in agreement with a previous comparison of human and bovine DRB alleles.     

Comparative FISH mapping of bovid X chromosomes reveals homologies and divergences between the subfamilies bovinae and caprinae

Comparative FISH mapping of river buffalo (Bubalus bubalis, BBU), sheep (Ovis aries, OAR), and cattle (Bos taurus, BTA) X chromosomes revealed homologies and divergences between the X chromosomes in the subfamilies Bovinae and Caprinae. Twenty-four and 17 loci were assigned for the first time to BBU X and OAR X, respectively, noticeably extending the physical map in these two species. Seventeen loci (four of which for the first time) were also FISH mapped to BTA X and used for comparative mapping studies on the three species, which show three morphologically different X chromosomes: an acrocentric (BBU X), an acrocentric with distinct short arms (OAR X), and a submetacentric (BTA X). The same order of loci were found on BTA X and BBU X, suggesting that a centromere transposition, with loss (cattle) or acquisition (river buffalo) of constitutive heterochromatin, differentiated the X chromosomes of these two bovids. Comparison of bovine (cattle and river buffalo) and caprine (sheep) X chromosomes revealed at least five common chromosome segments, suggesting that multiple transpositions, with retention or loss of constitutive heterochromatin, had occurred during their karyotypic evolution.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Sequence analysis of UTR and coding region of kappa-casein gene of Indian riverine buffalo (Bubalus bubalis).

In this study, complete nucleotide as well as derived amino acid sequence characterization of water buffalo (Bubalus bubalis) kappa-casein gene has been presented. Kappa-casein cDNA clones were identified and isolated from a buffalo lactating mammary gland cDNA library. Sequence analysis of kappa-casein cDNA revealed 850 nucleotides with an open reading frame (ORF) of 573 nucleotides, encoding mature peptide of 169 amino acids. The 5' untranslated region (UTR) comprised 71 nucleotides, while 3' UTR was of 206 nucleotides. A total of 11 nucleotide and seven amino acid changes were observed in, buffalo (Bubalus bubalis) as compared to cattle (Bos taurus), sheep (Ovis aries) and goat (Capra hircus). Among these nucleotide changes, eight were unique in buffalo as they were fully conserved in cattle, sheep and goat. Majority of the nucleotide changes and all the amino acid changes; 14 (Asp-Glu), 19(Asp/Ser-Asn), 96(Ala-Thr), 126(Ala-Val), 128(Ala/Gly-Val), 156(Ala/Pro-Val) and 168(Ala/Glu-Val) were limited to exon IV. Three glycosylation sites, Thr 131, Thr 133 and Thr 142 reported in cattle and goat kappa-casein gene were also conserved in buffalo, however, in sheep Thr 142 was replaced by Ala. Chymosin hydrolysis site, between amino acids Phe 105 and Met 106, important for rennet coagulation process, were found to be conserved across four bovid species. Buffalo kappa-casein with the presence of amino acids Thr 136 and Ala 148 seems to be an intermediate of "A" and "B" variants of cattle. Comparison with other livestock species revealed buffalo kappa-casein sharing maximum nucleotide (95.5%) and amino acid (92.6%) similarity with cattle, whereas with pig it showed least sequence similarity of 76.0% and 53.2%, respectively. Phylogenetic analysis based on both nucleotide and amino acid sequence indicated buffalo kappa-casein grouping with cattle, while sheep and goat forming a separate cluster close to them. The non-ruminant species viz. camel, horse and pig were distantly placed, in separate lineages.     

Multilocus phylogeographical analysis of Trypanosoma (Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants

Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host-parasite co-evolution, indicating that host switches have occurred across ruminants followed by divergences, giving rise to new trypanosome genotypes adapted exclusively to one host species.     

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.     

The red deer (Cervus elaphus) contains two expressed major histocompatibility complex class II DQB genes

The highly variable second exons of the red deer ( Cervus elaphu s) major histocompatibility complex (MHC) DQB genes were cloned and sequenced. Eight different expressed DQB sequences were isolated from four unrelated red deer. Either two or three different DQB sequences were isolated from each individual, demonstrating that more than one DQB gene is expressed in red deer. This is consistent with other ruminants, which also have multiple expressed copies of the DQB gene. The sequences ranged in similarity from 81·3% to 97·6% with an average similarity of 90·2%; this indicates that at least one of the DQB genes is highly polymorphic.     

黄牛、水牛体内人肉孢子虫18S rRNA基因研究及虫种鉴定

本文对自然感染的水牛源的人肉孢子虫以及黄牛源人孢子虫ＤＮＡ的１８Ｓ ｒＲＮＡ基因的ＰＣＲ扩增产物进行了测序。对年获的８６３ｂｐ的１８Ｓ ｒＲＮＡ基因分析比较表明,二者有较高的同源性,因此认为二者可能同是一种肉孢子虫－－人肉孢子虫（Ｓａｒｃｏｃｙｓｔｉｓ ｈｏｍｉｎｉｓ Ｒａｉｌｌｉｅｔ ａｎｄ Ｌｕｃｅｔ,１８９１）。由此推断,不仅黄牛可作为人肉孢子虫的中间宿主,水牛也可作为人肉孢子虫的中间宿主。     

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.     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.     

Partial purification and isoelectric focusing patterns of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) glutathione transferases.

1. Glutathione transferases from the liver, lung and kidney tissues of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) were partially purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration. 2. Liver tissue contains the highest enzyme activity when compared to the lung and kidney tissues. 3. The activity in cattle is higher than that in the buffalo. 4. Isoelectric focusing separates the activities into the acidic, near neutral and basic fractions. 5. The focused patterns are different for each of the tissues and in each of the species investigated.     

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.