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1.  A second generation radiation hybrid map to aid the assembly of the bovine genome sequence 
BMC Genomics  2006;7:283.
Background
Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6× coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process.
Results
An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6× bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map.
Conclusion
Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6× sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.
doi:10.1186/1471-2164-7-283
PMCID: PMC1636650  PMID: 17087818
2.  A physical map of the bovine genome 
Genome Biology  2007;8(8):R165.
A new physical map of the bovine genome has been constructed by integrating data from genetic and radiation hybrid maps, and a new bovine BAC map, with the bovine genome draft assembly.
Background
Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.
Results
A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.
Conclusion
Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.
doi:10.1186/gb-2007-8-8-r165
PMCID: PMC2374996  PMID: 17697342
3.  Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array 
BMC Genomics  2012;13:376.
Background
Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases.
Results
In this study using the high density BovineHD SNP array, we performed high resolution CNV analyses on both Btau_4.0 and UMD3.1 with 674 animals of 27 cattle breeds. We first compared CNV results derived from these two different SNP array platforms on Btau_4.0. With two thirds of the animals shared between studies, on Btau_4.0 we identified 3,346 candidate CNV regions representing 142.7 megabases (~4.70%) of the genome. With a similar total length but 5 times more event counts, the average CNVR length of current Btau_4.0 dataset is significantly shorter than the previous one (42.7 kb vs. 205 kb). Although subsets of these two results overlapped, 64% (91.6 megabases) of current dataset was not present in the previous study. We also performed similar analyses on UMD3.1 using these BovineHD SNP array results. Approximately 50% more and 20% longer CNVs were called on UMD3.1 as compared to those on Btau_4.0. However, a comparable result of CNVRs (3,438 regions with a total length 146.9 megabases) was obtained. We suspect that these results are due to the UMD3.1 assembly's efforts of placing unplaced contigs and removing unmerged alleles. Selected CNVs were further experimentally validated, achieving a 73% PCR validation rate, which is considerably higher than the previous validation rate. About 20-45% of CNV regions overlapped with cattle RefSeq genes and Ensembl genes. Panther and IPA analyses indicated that these genes provide a wide spectrum of biological processes involving immune system, lipid metabolism, cell, organism and system development.
Conclusion
We present a comprehensive result of cattle CNVs at a higher resolution and sensitivity. We identified over 3,000 candidate CNV regions on both Btau_4.0 and UMD3.1, further compared current datasets with previous results, and examined the impacts of genome assemblies on CNV calling.
doi:10.1186/1471-2164-13-376
PMCID: PMC3583728  PMID: 22866901
Cattle genome; Breed; Copy number variation (CNV); Single nucleotide polymorphism (SNP)
4.  A first generation whole genome RH map of the river buffalo with comparison to domestic cattle 
BMC Genomics  2008;9:631.
Background
The recently constructed river buffalo whole-genome radiation hybrid panel (BBURH5000) has already been used to generate preliminary radiation hybrid (RH) maps for several chromosomes, and buffalo-bovine comparative chromosome maps have been constructed. Here, we present the first-generation whole genome RH map (WG-RH) of the river buffalo generated from cattle-derived markers. The RH maps aligned to bovine genome sequence assembly Btau_4.0, providing valuable comparative mapping information for both species.
Results
A total of 3990 markers were typed on the BBURH5000 panel, of which 3072 were cattle derived SNPs. The remaining 918 were classified as cattle sequence tagged site (STS), including coding genes, ESTs, and microsatellites. Average retention frequency per chromosome was 27.3% calculated with 3093 scorable markers distributed in 43 linkage groups covering all autosomes (24) and the X chromosomes at a LOD ≥ 8. The estimated total length of the WG-RH map is 36,933 cR5000. Fewer than 15% of the markers (472) could not be placed within any linkage group at a LOD score ≥ 8. Linkage group order for each chromosome was determined by incorporation of markers previously assigned by FISH and by alignment with the bovine genome sequence assembly (Btau_4.0).
Conclusion
We obtained radiation hybrid chromosome maps for the entire river buffalo genome based on cattle-derived markers. The alignments of our RH maps to the current bovine genome sequence assembly (Btau_4.0) indicate regions of possible rearrangements between the chromosomes of both species. The river buffalo represents an important agricultural species whose genetic improvement has lagged behind other species due to limited prior genomic characterization. We present the first-generation RH map which provides a more extensive resource for positional candidate cloning of genes associated with complex traits and also for large-scale physical mapping of the river buffalo genome.
doi:10.1186/1471-2164-9-631
PMCID: PMC2625372  PMID: 19108729
5.  Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping 
Animal Genetics  2007;38(2):120-125.
High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina® BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine–hamster whole-genome radiation hybrid panel (WGRH3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine whole-genome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.
doi:10.1111/j.1365-2052.2006.01564.x
PMCID: PMC2063635  PMID: 17302794
bovine; illumina; map; single nucleotide polymorphism
6.  A high resolution radiation hybrid map of bovine chromosome 14 identifies scaffold rearrangement in the latest bovine assembly 
BMC Genomics  2007;8:254.
Background
Radiation hybrid (RH) maps are considered to be a tool of choice for fine mapping closely linked loci, considering that the resolution of linkage maps is determined by the number of informative meiosis and recombination events which may require very large mapping populations. Accurately defining the marker order on chromosomes is crucial for correct identification of quantitative trait loci (QTL), haplotype map construction and refinement of candidate gene searches.
Results
A 12 k Radiation hybrid map of bovine chromosome 14 was constructed using 843 single nucleotide polymorphism markers. The resulting map was aligned with the latest version of the bovine assembly (Btau_3.1) as well as other previously published RH maps. The resulting map identified distinct regions on Bovine chromosome 14 where discrepancies between this RH map and the bovine assembly occur. A major region of discrepancy was found near the centromere involving the arrangement and order of the scaffolds from the assembly. The map further confirms previously published conserved synteny blocks with human chromosome 8. As well, it identifies an extra breakpoint and conserved synteny block previously undetected due to lower marker density. This conserved synteny block is in a region where markers between the RH map presented here and the latest sequence assembly are in very good agreement.
Conclusion
The increase of publicly available markers shifts the rate limiting step from marker discovery to the correct identification of their order for further use by the research community. This high resolution map of bovine chromosome 14 will facilitate identification of regions in the sequence assembly where additional information is required to resolve marker ordering.
doi:10.1186/1471-2164-8-254
PMCID: PMC1959194  PMID: 17655763
7.  Use of “one-pot, mix-and-read” peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle 
Veterinary Research  2014;45(1):50.
Peptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine β2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype.
doi:10.1186/1297-9716-45-50
PMCID: PMC4018993  PMID: 24775445
8.  Recent and historical recombination in the admixed Norwegian Red cattle breed 
BMC Genomics  2011;12:33.
Background
Comparison of recent patterns of recombination derived from linkage maps to historical patterns of recombination from linkage disequilibrium (LD) could help identify genomic regions affected by strong artificial selection, appearing as reduced recent recombination. Norwegian Red cattle (NRF) make an interesting case study for investigating these patterns as it is an admixed breed with an extensively recorded pedigree. NRF have been under strong artificial selection for traits such as milk and meat production, fertility and health.
While measures of LD is also crucial for determining the number of markers required for association mapping studies, estimates of recombination rate can be used to assess quality of genomic assemblies.
Results
A dataset containing more than 17,000 genome-wide distributed SNPs and 2600 animals was used to assess recombination rates and LD in NRF. Although low LD measured by r2 was observed in NRF relative to some of the breeds from which this breed originates, reports from breeds other than those assessed in this study have described more rapid decline in r2 at short distances than what was found in NRF. Rate of decline in r2 for NRF suggested that to obtain an expected r2 between markers and a causal polymorphism of at least 0.5 for genome-wide association studies, approximately one SNP every 15 kb or a total of 200,000 SNPs would be required. For well known quantitative trait loci (QTLs) for milk production traits on Bos Taurus chromosomes 1, 6 and 20, map length based on historic recombination was greater than map length based on recent recombination in NRF.
Further, positions for 130 previously unpositioned contigs from assembly of the bovine genome sequence (Btau_4.0) found using comparative sequence analysis were validated by linkage analysis, and 28% of these positions corresponded to extreme values of population recombination rate.
Conclusion
While LD is reduced in NRF compared to some of the breeds from which this admixed breed originated, it is elevated over short distances compared to some other cattle breeds. Genomic regions in NRF where map length based on historic recombination was greater than map length based on recent recombination coincided with some well known QTL regions for milk production traits.
Linkage analysis in combination with comparative sequence analysis and detection of regions with extreme values of population recombination rate proved to be valuable for detecting problematic regions in the Btau_4.0 genome assembly.
doi:10.1186/1471-2164-12-33
PMCID: PMC3030550  PMID: 21232164
9.  The great diversity of major histocompatibility complex class II genes in Philippine native cattle 
Meta Gene  2014;2:176-190.
Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle.
doi:10.1016/j.mgene.2013.12.005
PMCID: PMC4287811  PMID: 25606401
MHC, major histocompatibility complex; SBT, sequence-based typing; BoLA, bovine MHC; PCR, polymerase chain reaction; HLA, human leukocyte antigen.; BoLA-DRB3 allele; Philippine; Sequence-based typing; Allele frequency; Population tree; Cattle breed; Major histocompatibility complex
10.  High resolution radiation hybrid maps of bovine chromosomes 19 and 29: comparison with the bovine genome sequence assembly 
BMC Genomics  2007;8:310.
Background
High resolution radiation hybrid (RH) maps can facilitate genome sequence assembly by correctly ordering genes and genetic markers along chromosomes. The objective of the present study was to generate high resolution RH maps of bovine chromosomes 19 (BTA19) and 29 (BTA29), and compare them with the current 7.1X bovine genome sequence assembly (bovine build 3.1). We have chosen BTA19 and 29 as candidate chromosomes for mapping, since many Quantitative Trait Loci (QTL) for the traits of carcass merit and residual feed intake have been identified on these chromosomes.
Results
We have constructed high resolution maps of BTA19 and BTA29 consisting of 555 and 253 Single Nucleotide Polymorphism (SNP) markers respectively using a 12,000 rad whole genome RH panel. With these markers, the RH map of BTA19 and BTA29 extended to 4591.4 cR and 2884.1 cR in length respectively. When aligned with the current bovine build 3.1, the order of markers on the RH map for BTA19 and 29 showed inconsistencies with respect to the genome assembly. Maps of both the chromosomes show that there is a significant internal rearrangement of the markers involving displacement, inversion and flips within the scaffolds with some scaffolds being misplaced in the genome assembly. We also constructed cattle-human comparative maps of these chromosomes which showed an overall agreement with the comparative maps published previously. However, minor discrepancies in the orientation of few homologous synteny blocks were observed.
Conclusion
The high resolution maps of BTA19 (average 1 locus/139 kb) and BTA29 (average 1 locus/208 kb) presented in this study suggest that by the incorporation of RH mapping information, the current bovine genome sequence assembly can be significantly improved. Furthermore, these maps can serve as a potential resource for fine mapping QTL and identification of causative mutations underlying QTL for economically important traits.
doi:10.1186/1471-2164-8-310
PMCID: PMC2064936  PMID: 17784962
11.  A genome-wide association study of immune response traits in Canadian Holstein cattle 
BMC Genomics  2014;15(1):559.
Background
Breeding for enhanced immune response (IR) has been suggested as a tool to improve inherent animal health. Dairy cows with superior antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) have been demonstrated to have a lower occurrence of many diseases including mastitis. Adaptive immune response traits are heritable, and it is, therefore, possible to breed for improved IR, decreasing the occurrence of disease. The objective of this study was to perform genome-wide association studies to determine differences in genetic profiles among Holstein cows classified as High or Low for AMIR and CMIR. From a total of 680 cows with immune response phenotypes, 163 cows for AMIR (81 High and 82 Low) and 140 for CMIR (75 High and 65 Low) were selectively genotyped using the Illumina Bovine SNP50 BeadChip. Results were validated using an unrelated population of 164 Holstein bulls IR phenotyped for AMIR and 146 for CMIR.
Results
A generalized quasi likelihood score method was used to determine single nucleotide polymorphisms (SNP) and chromosomal regions associated with immune response. After applying a 5% chromosomal false discovery rate, 186 SNPs were significantly associated with AMIR. The majority (93%) of significant markers were on chromosome 23, with a similar peak found in the bull population. For CMIR, 21 SNP markers remained significant. Candidate genes within 250,000 base pairs of significant SNPs were identified to determine biological pathways associated with AMIR and CMIR. Various pathways were identified, including the antigen processing and presentation pathway, important in host defense. Candidate genes included those within the bovine Major Histocompatability Complex such as BoLA-DQ, BoLA-DR and the non-classical BoLA-NC1 for AMIR and BoLA-DQ for CMIR, the complement system including C2 and C4 for AMIR and C1q for CMIR, and cytokines including IL-17A, IL17F for AMIR and IL-17RA for CMIR and tumor necrosis factor for both AMIR and CMIR. Additional genes associated with CMIR included galectins 1, 2 and 3, BCL2 and β-defensin.
Conclusions
The significant genetic variation associated with AMIR and CMIR in this study may imply feasibility to include immune response in genomic breeding indices as an approach to improve inherent animal health.
doi:10.1186/1471-2164-15-559
PMCID: PMC4099479  PMID: 24996426
Immune response; Dairy cattle; Health; Genome-wide association study; Antibody; Mastitis; Major histocompatability complex; Cytokine
12.  Assignment of chromosomal locations for unassigned SNPs/scaffolds based on pair-wise linkage disequilibrium estimates 
BMC Bioinformatics  2010;11:171.
Background
Recent developments of high-density SNP chips across a number of species require accurate genetic maps. Despite rapid advances in genome sequence assembly and availability of a number of tools for creating genetic maps, the exact genome location for a number of SNPs from these SNP chips still remains unknown. We have developed a locus ordering procedure based on linkage disequilibrium (LODE) which provides estimation of the chromosomal positions of unaligned SNPs and scaffolds. It also provides an alternative means for verification of genetic maps. We exemplified LODE in cattle.
Results
The utility of the LODE procedure was demonstrated using data from 1,943 bulls genotyped for 73,569 SNPs across three different SNP chips. First, the utility of the procedure was tested by analysing the masked positions of 1,500 randomly-chosen SNPs with known locations (50 from each chromosome), representing three classes of minor allele frequencies (MAF), namely >0.05, 0.01
Conclusion
The LODE procedure described in this study is an efficient and accurate method for positioning SNPs (MAF>0.05), for validating and checking the quality of a genome assembly, and offers a means for positioning of unordered scaffolds containing SNPs. The LODE procedure will be helpful in refining genome sequence assemblies, especially those being created from next-generation sequencing where high-throughput SNP discovery and genotyping platforms are integrated components of genome analysis.
doi:10.1186/1471-2105-11-171
PMCID: PMC2859757  PMID: 20370931
BMC Genetics  2009;10:18.
Background
Recent technological advances have made it possible to efficiently genotype large numbers of single nucleotide polymorphisms (SNPs) in livestock species, allowing the production of high-density linkage maps. Such maps can be used for quality control of other SNPs and for fine mapping of quantitative trait loci (QTL) via linkage disequilibrium (LD).
Results
A high-density bovine linkage map was constructed using three types of markers. The genotypic information was obtained from 294 microsatellites, three milk protein haplotypes and 6769 SNPs. The map was constructed by combining genetic (linkage) and physical information in an iterative mapping process. Markers were mapped to 3,155 unique positions; the 6,924 autosomal markers were mapped to 3,078 unique positions and the 123 non-pseudoautosomal and 19 pseudoautosomal sex chromosome markers were mapped to 62 and 15 unique positions, respectively. The linkage map had a total length of 3,249 cM. For the autosomes the average genetic distance between adjacent markers was 0.449 cM, the genetic distance between unique map positions was 1.01 cM and the average genetic distance (cM) per Mb was 1.25.
Conclusion
There is a high concordance between the order of the SNPs in our linkage map and their physical positions on the most recent bovine genome sequence assembly (Btau 4.0). The linkage maps provide support for fine mapping projects and LD studies in bovine populations. Additionally, the linkage map may help to resolve positions of unassigned portions of the bovine genome.
doi:10.1186/1471-2156-10-18
PMCID: PMC2680908  PMID: 19393043
BMC Genomics  2013;14:43.
Background
Though India has sequenced water buffalo genome but its draft assembly is based on cattle genome BTau 4.0, thus de novo chromosome wise assembly is a major pending issue for global community. The existing radiation hybrid of buffalo and these reported STR can be used further in final gap plugging and “finishing” expected in de novo genome assembly. QTL and gene mapping needs mining of putative STR from buffalo genome at equal interval on each and every chromosome. Such markers have potential role in improvement of desirable characteristics, such as high milk yields, resistance to diseases, high growth rate. The STR mining from whole genome and development of user friendly database is yet to be done to reap the benefit of whole genome sequence.
Description
By in silico microsatellite mining of whole genome, we have developed first STR database of water buffalo, BuffSatDb (Buffalo MicroSatellite Database (http://cabindb.iasri.res.in/buffsatdb/) which is a web based relational database of 910529 microsatellite markers, developed using PHP and MySQL database. Microsatellite markers have been generated using MIcroSAtellite tool. It is simple and systematic web based search for customised retrieval of chromosome wise and genome-wide microsatellites. Search has been enabled based on chromosomes, motif type (mono-hexa), repeat motif and repeat kind (simple and composite). The search may be customised by limiting location of STR on chromosome as well as number of markers in that range. This is a novel approach and not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of the selected markers enabling researcher to select markers of choice at desired interval over the chromosome. The unique add-on of degenerate bases further helps in resolving presence of degenerate bases in current buffalo assembly.
Conclusion
Being first buffalo STR database in the world , this would not only pave the way in resolving current assembly problem but shall be of immense use for global community in QTL/gene mapping critically required to increase knowledge in the endeavour to increase buffalo productivity, especially for third world country where rural economy is significantly dependent on buffalo productivity.
doi:10.1186/1471-2164-14-43
PMCID: PMC3563513  PMID: 23336431
de novo; Microsatellites; Primers; Radiation hybrid; Water buffalo
Mammalian Genome  2010;21(11-12):592-598.
High-throughput sequencing of RNA (RNA-Seq) was developed primarily to analyze global gene expression in different tissues. However, it also is an efficient way to discover coding SNPs. The objective of this study was to perform a SNP discovery analysis in the milk transcriptome using RNA-Seq. Seven milk samples from Holstein cows were analyzed by sequencing cDNAs using the Illumina Genome Analyzer system. We detected 19,175 genes expressed in milk samples corresponding to approximately 70% of the total number of genes analyzed. The SNP detection analysis revealed 100,734 SNPs in Holstein samples, and a large number of those corresponded to differences between the Holstein breed and the Hereford bovine genome assembly Btau4.0. The number of polymorphic SNPs within Holstein cows was 33,045. The accuracy of RNA-Seq SNP discovery was tested by comparing SNPs detected in a set of 42 candidate genes expressed in milk that had been resequenced earlier using Sanger sequencing technology. Seventy of 86 SNPs were detected using both RNA-Seq and Sanger sequencing technologies. The KASPar Genotyping System was used to validate unique SNPs found by RNA-Seq but not observed by Sanger technology. Our results confirm that analyzing the transcriptome using RNA-Seq technology is an efficient and cost-effective method to identify SNPs in transcribed regions. This study creates guidelines to maximize the accuracy of SNP discovery and prevention of false-positive SNP detection, and provides more than 33,000 SNPs located in coding regions of genes expressed during lactation that can be used to develop genotyping platforms to perform marker-trait association studies in Holstein cattle.
doi:10.1007/s00335-010-9297-z
PMCID: PMC3002166  PMID: 21057797
BMC Genomics  2009;10:180.
Background
We present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque.
Results
The assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information.
Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly.
Conclusion
The biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.
doi:10.1186/1471-2164-10-180
PMCID: PMC2686734  PMID: 19393050
BMC Genetics  2012;13:86.
Background
WC1 co-receptors belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1+ γδ T cells. We have previously identified partial or complete genomic sequences for thirteen different WC1 genes through annotation of the bovine genome Btau_3.1 build. We also identified two WC1 cDNA sequences from other cattle that did not correspond to sequences in the Btau_3.1 build. Their absence in the Btau_3.1 build may have reflected gaps in the genome assembly or polymorphisms among animals. Since the response of γδ T cells to bacterial challenge is determined by WC1 gene expression, it was critical to understand whether individual cattle or breeds differ in the number of WC1 genes or display polymorphisms.
Results
Real-time quantitative PCR using DNA from the animal whose genome was sequenced (“Dominette”) and sixteen other animals representing ten breeds of cattle, showed that the number of genes coding for WC1 co-receptors is thirteen. The complete coding sequences of those thirteen WC1 genes is presented, including the correction of an error in the WC1-2 gene due to mis-assembly in the Btau_3.1 build. All other cDNA sequences were found to agree with the previous annotation of complete or partial WC1 genes. PCR amplification and sequencing of the most variable N-terminal SRCR domain (domain 1 which has the SRCR “a” pattern) of each of the thirteen WC1 genes showed that the sequences are highly conserved among individuals and breeds. Of 160 sequences of domain 1 from three breeds of cattle, no additional sequences beyond the thirteen described WC1 genes were found. Analysis of the complete WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 molecular forms.
Conclusion
The bovine WC1 multi-gene family is composed of thirteen genes coding for three structural forms whose sequences are highly conserved among individual cattle and breeds. The sequence diversity necessary for WC1 genes to function as a multi-genic pattern recognition receptor array is encoded in the genome, rather than generated by recombinatorial diversity or hypermutation.
doi:10.1186/1471-2156-13-86
PMCID: PMC3511184  PMID: 23072335
Bovine; WC1; γδ T cells
BMC Genomics  2009;10:211.
Background
Whole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig (Sus scrofa) chromosome 10 (SSC10) using the IMNpRH212,000-rad porcine WG-RH panel, integrated with the IMpRH7000-rad WG-RH, genetic and BAC fingerprinted contig (FPC) maps.
Results
Map vectors from the IMNpRH212,000-rad and IMpRH7,000-rad panels were merged to construct parallel framework (FW) maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR7,000 and 1814.5 cR12,000, respectively. The second group covers SSC10q at map distances of 1336.9 cR7,000 and 3353.6 cR12,000, yielding an overall average map resolution of 16.4 kb/cR12,000 or 393.5 kb per marker on SSC10. This represents a ~2.5-fold increase in map resolution over the IMpRH7,000-rad panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built.
Conclusion
This initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH212,000-rad and IMpRH7,000-rad, genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10.
doi:10.1186/1471-2164-10-211
PMCID: PMC2689272  PMID: 19426492
BMC Genomics  2012;13:45.
Background
Cow milk is a complex bioactive fluid consumed by humans beyond infancy. Even though the chemical and physical properties of cow milk are well characterized, very limited research has been done on characterizing the milk transcriptome. This study performs a comprehensive expression profiling of genes expressed in milk somatic cells of transition (day 15), peak (day 90) and late (day 250) lactation Holstein cows by RNA sequencing. Milk samples were collected from Holstein cows at 15, 90 and 250 days of lactation, and RNA was extracted from the pelleted milk cells. Gene expression analysis was conducted by Illumina RNA sequencing. Sequence reads were assembled and analyzed in CLC Genomics Workbench. Gene Ontology (GO) and pathway analysis were performed using the Blast2GO program and GeneGo application of MetaCore program.
Results
A total of 16,892 genes were expressed in transition lactation, 19,094 genes were expressed in peak lactation and 18,070 genes were expressed in late lactation. Regardless of the lactation stage approximately 9,000 genes showed ubiquitous expression. Genes encoding caseins, whey proteins and enzymes in lactose synthesis pathway showed higher expression in early lactation. The majority of genes in the fat metabolism pathway had high expression in transition and peak lactation milk. Most of the genes encoding for endogenous proteases and enzymes in ubiquitin-proteasome pathway showed higher expression along the course of lactation.
Conclusions
This is the first study to describe the comprehensive bovine milk transcriptome in Holstein cows. The results revealed that 69% of NCBI Btau 4.0 annotated genes are expressed in bovine milk somatic cells. Most of the genes were ubiquitously expressed in all three stages of lactation. However, a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage. This indicates the ability of milk somatic cells to adapt to different molecular functions according to the biological need of the animal. This study provides a valuable insight into the biology of lactation in the cow, as well as many avenues for future research on the bovine lactome.
doi:10.1186/1471-2164-13-45
PMCID: PMC3285075  PMID: 22276848
BMC Genomics  2011;12:639.
Background
The sequencing of the cow genome was recently published (Btau_4.0 assembly). A second, alternate cow genome assembly (UMD2), based on the same raw sequence data, was also published. The two assemblies have been subsequently updated to Btau_4.2 and UMD3.1, respectively.
Results
We compared the Btau_4.2 and UMD3.1 alternate assemblies. Inconsistencies were grouped into three main categories: (i) DNA segments showing almost coincidental chromosomal mapping but discordant orientation (inversions); (ii) DNA segments showing a discordant map position along the same chromosome; and (iii) sequences present in one chromosomal assembly but absent in the corresponding chromosome of the other assembly. The latter category mainly consisted of large amounts of scaffolds that were unassigned in Btau_4.2 but successfully mapped in UMD3.1. We sampled 70 inconsistencies and identified appropriate cow BACs for each of them. These clones were then utilized in FISH experiments on cow metaphase or interphase nuclei in order to disambiguate the discrepancies. In almost all instances the FISH results agreed with the UMD3.1 assembly. Occasionally, however, the mapping data of both assemblies were discordant with the FISH results.
Conclusions
Our work demonstrates how FISH, which is assembly independent, can be efficiently used to solve assembly problems frequently encountered using the shotgun approach.
doi:10.1186/1471-2164-12-639
PMCID: PMC3268123  PMID: 22208360
Cow genome; alternate assemblies of cow genomes; genomic comparison; unassigned scaffolds; BAC-FISH mapping
Pulmonary Circulation  2011;1(4):462-469.
High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12-18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TG-interacting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10-14), cell development and cell signaling as the top cellular functions (P<1.20×10-08), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10-09) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH.
doi:10.4103/2045-8932.93545
PMCID: PMC3329076  PMID: 22530101
brisket disease; microarray analysis; hypoxia
Farm animals remain at risk of endemic, exotic and newly emerging viruses. Vaccination is often promoted as the best possible solution, and yet for many pathogens, either there are no appropriate vaccines or those that are available are far from ideal. A complementary approach to disease control may be to identify genes and chromosomal regions that underlie genetic variation in disease resistance and response to vaccination. However, identification of the causal polymorphisms is not straightforward as it generally requires large numbers of animals with linked phenotypes and genotypes. Investigation of genes underlying complex traits such as resistance or response to viral pathogens requires several genetic approaches including candidate genes deduced from knowledge about the cellular pathways leading to protection or pathology, or unbiased whole genome scans using markers spread across the genome.
Evidence for host genetic variation exists for a number of viral diseases in cattle including bovine respiratory disease and anecdotally, foot and mouth disease virus (FMDV). We immunised and vaccinated a cattle cross herd with a 40-mer peptide derived from FMDV and a vaccine against bovine respiratory syncytial virus (BRSV). Genetic variation has been quantified. A candidate gene approach has grouped high and low antibody and T cell responders by common motifs in the peptide binding pockets of the bovine major histocompatibility complex (BoLA) DRB3 gene. This suggests that vaccines with a minimal number of epitopes that are recognised by most cattle could be designed. Whole genome scans using microsatellite and single nucleotide polymorphism (SNP) markers has revealed many novel quantitative trait loci (QTL) and SNP markers controlling both humoral and cell-mediated immunity, some of which are in genes of known immunological relevance including the toll-like receptors (TLRs).
The sequencing, assembly and annotation of livestock genomes and is continuing apace. In addition, provision of high-density SNP chips should make it possible to link phenotypes with genotypes in field populations without the need for structured populations or pedigree information. This will hopefully enable fine mapping of QTL and ultimate identification of the causal gene(s). The research could lead to selection of animals that are more resistant to disease and new ways to improve vaccine efficacy.
doi:10.1016/j.vetimm.2011.05.009
PMCID: PMC3413884  PMID: 21621277
Cattle; Genetics; Vaccine response; Bovine respiratory syncytial virus; Foot and mouth disease virus; Quantitative trait loci; Whole genome scan; Polymorphism; Bovine major histocompatibility complex; BoLA; Toll like receptor
PLoS ONE  2012;7(8):e42680.
We analyzed the whole genome sequence coverage in two versions of the Bos taurus genome and identified all regions longer than five kilobases (Kbp) that are duplicated within chromosomes with >99% sequence fidelity in both copies. We call these regions High Fidelity Duplications (HFDs). The two assemblies were Btau 4.2, produced by the Human Genome Sequencing Center at Baylor College of Medicine, and UMD Bos taurus 3.1 (UMD 3.1), produced by our group at the University of Maryland. We found that Btau 4.2 has a far greater number of HFDs, 3111 versus only 69 in UMD 3.1. Read coverage analysis shows that 39 million base pairs (Mbp) of sequence in HFDs in Btau 4.2 appear to be a result of a mis-assembly and therefore cannot be qualified as segmental duplications. UMD 3.1 has only 0.41 Mbp of sequence in HFDs that are due to a mis-assembly.
doi:10.1371/journal.pone.0042680
PMCID: PMC3411808  PMID: 22880081
BMC Genomics  2011;12:559.
Background
One of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle.
Results
The genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs), 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel) between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs). Ten randomly selected CNVs, five genic and five non-genic, were successfully validated using quantitative real-time PCR. The CNVs are enriched for immune system genes and include genes that may contribute to lactation capacity. The majority of the CNVs (69%) were detected as regions with higher abundance in the Holstein bull.
Conclusions
Substantial genetic differences exist between the Black Angus and Holstein animals sequenced in this work and the Hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. The deeply annotated SNPs and CNVs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering DNA differences.
doi:10.1186/1471-2164-12-559
PMCID: PMC3229636  PMID: 22085807
PLoS ONE  2012;7(11):e48792.
The African buffalo, Syncerus caffer, is one of the most abundant and ecologically important species of megafauna in the savannah ecosystem. It is an important prey species, as well as a host for a vast array of nematodes, pathogens and infectious diseases, such as bovine tuberculosis and corridor disease. Large-scale SNP discovery in this species would greatly facilitate further research into the area of host genetics and disease susceptibility, as well as provide a wealth of sequence information for other conservation and genomics studies. We sequenced pools of Cape buffalo DNA from a total of 9 animals, on an ABI SOLiD4 sequencer. The resulting short reads were mapped to the UMD3.1 Bos taurus genome assembly using both BWA and Bowtie software packages. A mean depth of 2.7× coverage over the mapped regions was obtained. Btau4 gene annotation was added to all SNPs identified within gene regions. Bowtie and BWA identified a maximum of 2,222,665 and 276,847 SNPs within the buffalo respectively, depending on analysis method. A panel of 173 SNPs was validated by fluorescent genotyping in 87 individuals. 27 SNPs failed to amplify, and of the remaining 146 SNPs, 43–54% of the Bowtie SNPs and 57–58% of the BWA SNPs were confirmed as polymorphic. dN/dS ratios found no evidence of positive selection, and although there were genes that appeared to be under negative selection, these were more likely to be slowly evolving house-keeping genes.
doi:10.1371/journal.pone.0048792
PMCID: PMC3492240  PMID: 23144973

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