Description of bovine major histocompatibility complex class IIa haplotypes using parthenogenetic embryo-derived cells.

In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.

A high-resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA.

The major histocompatibility complex (MHC) in mammals codes for antigen-presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene-dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle-derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH5000 ), we generated a high-resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR5000 ) was aligned with the cattle MHC RH map (cR12000 ) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two-point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single- and double-color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library.

River buffalo genome analyses have advanced significantly in the last decade, and the genome sequence of Bubalus bubalis will be available shortly. Nonetheless, large-insert DNA library resources such as bacterial artificial chromosomes (BAC) are still required for validation and accurate assembly of the genome sequence. We constructed a river buffalo BAC library containing 52,224 clones with an average insert size of 97 kb, representing 1.7 × coverage of the genome. This genomic resource for river buffalo will facilitate further studies in this economically important species allowing for instance, whole genome physical mapping and isolation of genes and gene clusters, contributing to the elucidation of gene organization and identification of regulatory elements.

Polymorphism and gene organization of water buffalo MHC-DQB genes show homology to the BoLA DQB region.

In cattle (Bos taurus), there is evidence of more than 50 alleles of BoLA-DQB (bovine lymphocyte antigen DQB) that are distributed across at least five DQB loci, making this region one of the most complex in the BoLA gene family. In this study, DQB alleles were analysed for the water buffalo (Bubalus bubalis), another economically important bovine species. Twelve alleles for Bubu-DQB (Bubalis bubalis DQB) were determined by nucleotide sequence analysis. A phylogenetic analysis revealed numerous trans-species polymorphisms, with alleles from water buffalo assigned to at least three different loci (BoLA-DQB1, BoLA-DQB3 and BoLA-DQB4) that are also found in cattle. These presumptive loci were analysed for patterns of synonymous (d(S)) and non-synonymous (d(N)) substitution. Like BoLA-DQB1, Bubu-DQB1 was observed to be under strong positive selection for polymorphism. We conclude that water buffalo and cattle share the current arrangement of their DQB region because of their common ancestry.

Comparative RH maps of the river buffalo and bovine Y chromosomes.

Radiation hybrid maps were constructed for river buffalo and cattle Y chromosomes. A total of 41 cattle-derived Y-chromosome molecular markers were selected and tested with 2 previously described 5,000-rad whole-genome radiation hybrid (RH) panels (river buffalo - BBURH(5000) and cattle - BTARH(5000)) for generation of maps. Among the initial 41 selected markers, a subset of 26 markers generated PCR products suitable for scoring with the BBURH(5000) panel. Of these, 19 markers (73%) were distributed in 1 linkage group spanning 341.3 cR. Retention frequencies (RF) for individual markers ranged from 17.8% for SMCY to 56.7% for BTY1, with an average RF of 37.6%. From the selected markers, 37 generated reliable scores using the BTARH(5000) panel. The newly constructed BTAY RH map contains 28 markers distributed within 1 linkage group. Twenty-four of these markers had been previously mapped on BTAY using a 7,000-rad cattle-hamster WG-RH panel and 4 markers were mapped for the first time (ZFY, SeqRep, RepSeqS4 and BTY1). The length of the BTAY RH map was estimated to be 602.4 cR. Retention frequencies for individual mapped markers ranged from 10% (INRA126) to 63.3% (SeqRep), with an average RF of 35.3%. RH marker positions along the Y chromosome were compared between BBUY and BTAY, which revealed differences in the order of some of the markers. The BBUY pseudoautosomal region (PAR) is delineated by 3 BTAY PAR markers (MAF45, TGLA325 and UMN2008). These markers are telomeric in both species but are not found in the same order. Here we have demonstrated the effective use of bovine Y chromosome markers for the development of the first BBUY RH map. Likewise, these set of markers can be used for comparative assessment of Y chromosomes in other members of the Bovidae family.

First radiation hybrid map of the river buffalo X chromosome (BBUX) and comparison with BTAX.

We report the first radiation hybrid map of the river buffalo X chromosome generated from a recently constructed river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel (BBURH(5000)). This map contains a total of 33 cattle-derived markers, including 10 genes, four ESTs and 19 microsatellites. The markers are distributed in two linkage groups: LG1 contains eight markers spanning 125.6 cR, and LG2 contains 25 markers spanning 366.3 cR. LG1 contains six markers in common with bovine sequence assembly build 3.1. With the exception of BMS2152, the order of these markers on our BBUX map is shuffled when compared to the cow X chromosome (Bos taurus; BTAX). From LG2, two markers (AMELX and BL22) map to a more distal portion of BTAX compared to BBUX. In addition, two pairs of LG2 markers exhibit inversions compared to BTAX (ILSTS017 and ATRX; XBM38 and PPEF1). Alternatively, when compared to the most recent bovine RH map (Bov-Gen 3000rads), BL1098 and BMS2227 from LG1 as well as PLS3 and BMS1820 from LG2 showed inverted positions on the BBUX map. These discrepancies in buffalo and cattle maps may reflect evolutionary divergence of the chromosomes or mapping errors in one of the two species. Although the set of mapped markers does not cover the entire X chromosome, this map is a starting point for the construction of a high-resolution map, which is necessary for characterization of small rearrangements that might have occurred between the Bubalus bubalis and Bos taurus X chromosomes.

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.

Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3.

We present the first radiation hybrid (RH) map of river buffalo (Bubalus bubalis) chromosome 6 (BBU6) developed with a recently constructed river buffalo whole-genome RH panel (BBURH(5000)). The preliminary map contains 33 cattle-derived markers, including 12 microsatellites, 19 coding genes and two ESTs, distributed across two linkage groups. Retention frequencies for markers ranged from 14.4% to 40.0%. Most of the marker orders within the linkage groups on BBU6 were consistent with the cattle genome sequence and RH maps. This preliminary RH map is the starting point for comparing gene order between river buffalo and cattle, presenting an opportunity for the examination of micro-rearrangements of these chromosomes. Also, resources for positional candidate cloning in river buffalo are enhanced.

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.

A methodological approach for the construction of a radiation hybrid map of bovine chromosome 5

A bovine 5,000 rad WG-RH panel was used to construct an RH map of bovine chromosome 5 (BTA5). Twenty-one microsatellites and thirteen genes were scored in the panel using PAGE and radioactive labeling. Marker retention ranged from 8.9 percent-25.8 percent and averaged 17.8 percent. Pairwise locus analysis placed all markers in a single syntenic group with a LOD support of 4.0. At a LOD support of 8.0, a centromeric group of 23 syntenic markers was formed. Telomeric groups of 11 and 9 markers were assembled with a LOD support of 6.0 and 8.0, respectively. All markers were ordered by maximum likelihood methods using the program RHMAP. Only 13 markers were ordered with a LOD support of at least 3.0, while 25 and 29 markers were ordered with a support of at least 2.0 and 1.0, respectively. Total length of the comprehensive RH map was 435.9 cR5,000, with an average marker separation of 12.8 cR5,000. The largest gaps in the map were 55.0 and 30.4 cR5,000 in length. The locus orders of markers common to both the RH map and the USDA-MARC linkage map were identical. The relationship between the RH and linkage maps was calculated to be 3.74 cR5,000/cM.

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.

RH maps of bovine chromosomes 15 and 29: conservation of human chromosomes 11 and 5.

Comparative mapping data on evolutionary conserved coding sequences and synteny maps between human and cattle are insufficient to define the extent and distribution of conserved segments between these two species, because the order of loci is often rearranged. A 5000-rad cattle whole-genome radiation hybrid (WG-RH) panel was constructed to provide high-resolution comparative maps and also to integrate linkage maps of microsatellites with evolutionary conserved genes and transcripts in a single ordered map. We used the WG-RH panel to construct radiation hybrid maps of bovine Chromosomes (Chrs) 15 and 29 (BTA15 and BTA29), integrating microsatellites from published linkage maps with selected genes. The comprehensive map of BTA15 consists of 24 markers, 13 of which were placed in the framework map. Eleven molecular markers compose the comprehensive map of BTA29, seven of which were placed in the framework map. We identified the homologous regions between bovine Chr 15 (BTA15) and human Chrs 5 and 11 (HSA5 and HSA11), as well as between BTA29 and HSA11. The present study demonstrates that WG-RH mapping is an efficient method for integrating multiple genetic maps into one map and for incorporating monomorphic Type I loci into ordered maps for comparison between species.