A high-resolution comparative RH map of the proximal part of bovine chromosome 1

Current comparative maps between human chromosome 21 and the proximal part of cattle chromosome 1 are insufficient to define chromosomal rearrangements because of the low density of mapped genes in the bovine genome. The recently completed sequence of human chromosome 21 facilitates the detailed comparative analysis of corresponding segments on BTA1. In this study eight bovine bacterial artificial chromosome (BAC) clones containing bovine orthologues of human chromosome 21 genes, i.e. GRIK1, CLDN8, TIAM1, HUNK, SYNJ1, OLIG2, IL10RB, and KCNE2 were physically assigned by fluorescence in situ hybridization (FISH) to BTA1q12.1-q12.2. Sequence tagged site (STS) markers derived from these clones were mapped on the 3000 rad Roslin/Cambridge bovine radiation hybrid (RH) panel. In addition to these eight novel markers, 17 known markers from previously published BTA1 linkage or RH maps were also mapped on the Roslin/Cambridge bovine RH panel resulting in an integrated map with 25 markers of 355.4 cR(3000) length. The human-cattle genome comparison revealed the existence of three chromosomal breakpoints and two probable inversions in this region.     

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA X.     

A comparative map of interstitial bovine chromosome 5 with human chromosomes 12 and 22

We have constructed a high-density comparative radiation hybrid map of the interstitial region of bovine chromosome 5 (BTA5) using a recently constructed 12,000-rad, whole-genome, cattle-hamster radiation hybrid (WGRH) panel. Sixty-two bovine EST markers were selected which have orthologous sequences on human chromosomes 12 and 22 (HSA12 and HSA22). Sixty markers were included in the multi-point framework map at LOD 3.0. Our comprehensive RH map contains more than twice as many markers (88) than previous generation maps. Because of a higher marker density and increased resolution of the RH(12,000) panel, all markers were placed into a single linkage group based on two-point analysis at a LOD score 6.0. As a result, this new comparative map reveals new blocks of synteny and extensive gene order alterations between species. Breakpoints of synteny are located with high accuracy. Overall, this work reveals widespread chromosomal rearrangements between bovine, human and mouse genomes.     

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 bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping

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.     

A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.     

A radiation hybrid map of bovine chromosome 24 and comparative mapping with human chromosome 18

We present herein a bovine chromosome 24 (BTA24) radiation hybrid (RH) map using 40 markers scored on a panel of 90 RHs. Of these markers, 29 loci were ordered with odds of at least 1000:1 in a framework map. An average retention frequency of 17.4% was observed, with relatively higher frequencies near the centromere. The length of the comprehensive map was 640 centiray5000 (cR5000) with an average marker interval of approximately 17.3 cR5000. The observed locus order is generally consistent with currently published bovine linkage and physical maps. Nineteen markers were either Type I loci or closely associated with expressed sequences and thus could be used to compare the BTA24 RH map with human mapping information. All genes located on BTA24 were located on human chromosome 18, and previously reported regions of conserved synteny were extended. The comparative data revealed the presence of at least six conserved regions between these chromosomes.     

A radiation hybrid map of chicken chromosome 15

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.     

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20-specific DNA library

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.     

Characterisation of a bovine/murine hybrid cell panel informative for all bovine autosomes

A bovine/murine hybrid cell panel consisting of 57 cell lines was typed with 124 markers by PCR, Southern hybridisation and isozyme analysis in order to establish its utility as a resource for genome mapping. All bovine chromosomes, including the sex chromosomes were represented in the panel. Computerised analysis of syntenies indicated that there are no cell lines containing only a single bovine chromosome. The panel was used to map 10 new bovine microsatellite markers, and the MYL6 and CPE genes. This panel is informative for all bovine chromosomes other than the sex-specific region of the X chromosome and can be used in synteny mapping studies. At present, due to the relatively small number of markers typed, the resolution of the panel does not go beyond the chromosomal level.     

A Radiation Hybrid Map of BTA23: Identification of a Chromosomal Rearrangement Leading to Separation of the Cattle MHC Class II Subregions

Bovine chromosome 23 (BTA23) contains the bovine major histocompatibility complex (MHC) and is thus of particular interest because of the role of MHC genes in immunity. Previous studies have shown cattle MHC class II genes to be subdivided into two distinct subregions separated by a variable genetic distance of 15–30 cM. To elucidate the genetic events that resulted in the present organization of the class II and other MHC genes, a framework radiation hybrid (RH) map of BTA23 was developed by testing DNA samples from a 5000 rad whole genome RH panel. Twenty-six markers were screened with an average retention frequency of 0.27, ranging from 0.14 to 0.42. Total length of the chromosome was 220 cR₅₀₀₀, with 4.1 cR₅₀₀₀/cM when compared to linkage data. Gene orders for the markers common to both the RH framework map and the consensus framework linkage map are identical. Large centiray intervals,D23S23–D23S7, DYA–D23S24andCYP21–D23S31,were observed compared to linkage distances. These data may indicate a much larger physical distance or suppression of recombination in the interval separating the class II subregions and also within the class I region than previously estimated. Comparison of 13 Type I genes conserved between BTA23 and the human homolog HSA6p suggests the occurrence of an inversion encompassing the centromeric half of the bovine chromosome, thus explaining the large distance between the bovine class IIa and IIb clusters. These results exemplify the power of RH mapping in solving problems in comparative genomics and evolution. Furthermore, noncongruence of the genetic and physical RH map distances indicates that caution must be observed in using either resource alone in searching for candidate genes controlling traits of economic importance.     

Genomic relationships based on X chromosome markers and accuracy of genomic predictions with and without X chromosome markers

Background

Although the X chromosome is the second largest bovine chromosome, markers on the X chromosome are not used for genomic prediction in some countries and populations. In this study, we presented a method for computing genomic relationships using X chromosome markers, investigated the accuracy of imputation from a low density (7K) to the 54K SNP (single nucleotide polymorphism) panel, and compared the accuracy of genomic prediction with and without using X chromosome markers.

Methods

The impact of considering X chromosome markers on prediction accuracy was assessed using data from Nordic Holstein bulls and different sets of SNPs: (a) the 54K SNPs for reference and test animals, (b) SNPs imputed from the 7K to the 54K SNP panel for test animals, (c) SNPs imputed from the 7K to the 54K panel for half of the reference animals, and (d) the 7K SNP panel for all animals. Beagle and Findhap were used for imputation. GBLUP (genomic best linear unbiased prediction) models with or without X chromosome markers and with or without a residual polygenic effect were used to predict genomic breeding values for 15 traits.

Results

Averaged over the two imputation datasets, correlation coefficients between imputed and true genotypes for autosomal markers, pseudo-autosomal markers, and X-specific markers were 0.971, 0.831 and 0.935 when using Findhap, and 0.983, 0.856 and 0.937 when using Beagle. Estimated reliabilities of genomic predictions based on the imputed datasets using Findhap or Beagle were very close to those using the real 54K data. Genomic prediction using all markers gave slightly higher reliabilities than predictions without X chromosome markers. Based on our data which included only bulls, using a G matrix that accounted for sex-linked relationships did not improve prediction, compared with a G matrix that did not account for sex-linked relationships. A model that included a polygenic effect did not recover the loss of prediction accuracy from exclusion of X chromosome markers.

Conclusions

The results from this study suggest that markers on the X chromosome contribute to accuracy of genomic predictions and should be used for routine genomic evaluation.     

A Mapping Method for SACCHAROMYCES CEREVISIAE Using rad52-Induced Chromosome Loss

Saccharomyces cerevisiae diploids homozygous for the rad52-1 mutation have previously been shown to lose chromosomes mitotically. Spontaneous events and events following low levels of X-ray or methyl methanesulfonate treatment result in monosomic diploids, whereas higher levels of treatment result in near haploidization. This rad52-1-dependent chromosome loss has been used to develop a new mapping method which can be used to assign a previously unmapped gene to a chromosome. Chromosome loss mapping can be done in either of two ways: if a diploid, homozygous for rad52-1 but heterozygous for a variety of other recessive markers, is constructed with an unmapped recessive mutation in coupling with known chromosomal markers, chromosome loss will result in the coordinate expression of the mutation and other recessive markers on the same chromosome; if, however, the diploid is constructed with the unmapped mutation in repulsion to chromosomal markers, then even haploidization will never result in the coordinate expression of the unmapped mutation and other markers on the same homologous chromosome pair--This mapping method and subsequent tetrad analyses have been used to locate hom6 on chromosome X, ade4 on chromosome XIII and cdc31 on chromosome XV and to demonstrate that met5, previously assigned to chromosome V, actually maps to chromosome X; the met- marker on chromosome V has been shown to be met6. GAL80 and SUP5, previously assigned to an unmapped fragment, have now been mapped to the right arm of chromosome XIII.     

A high-resolution comparative RH map of the telomeric end of bovine chromosome 2 with human chromosomes 1 and 2

High density livestock to human comparative maps are necessary for the implementation of comparative positional candidate gene cloning. We have constructed a high-density comparative radiation hybrid (RH) map of the telomeric end of bovine chromosome 2 (BTA2) using a 12,000-rad whole genome cattle-hamster radiation hybrid (WGRH) panel. Eighteen bovine EST markers with orthologues on human chromosomes 1 and 2 (HSA1 and HSA2), together with nine microsatellite markers, were typed against the 180 cell lines of the WGRH panel. Twenty-one markers were included in the multi-point framework map at LOD =3.0. The comparative analysis reveals a new segment of highly conserved synteny between HSA2 and BTA2.     

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6)

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.     

A radiation hybrid map of bovine chromosome one

Radiation hybrid (RH) mapping has proven to be an extremely powerful approach to constructing high density maps of human chromosomes and is experiencing increased use in other animals, including cattle. A 5000 rad bovine whole-genome radiation hybrid panel was recently constructed in order to integrate existing cattle linkage maps with evolutionarily conserved genes and provide high resolution comparative maps relative to humans and mice. We utilized this panel to construct a 19 marker framework map of bovine chromosome 1 (BTA1), which included 8 Type I loci and 11 Type II loci ordered with at least 1000:1 odds. A 35 marker comprehensive map including 15 Type I loci and 20 Type II loci was also produced. Of the 15 Type I loci ordered on the comprehensive map, three are ordered on HSA3 and five are ordered in three blocks on HSA21 on the human cytogenetic maps.     

Mutagenesis in cloned yeast genes. The effect of mutation rad2 on the frequency of gene mutation in plasmid and chromosome

The influence of rad2 mutation blocking incision of pyrimidine dimers on frequency of UV-light and 6-hydroxylaminopurine (6-GAP)-induced adenine-independent revertants was studied in the strains of Saccharomyces cerevisiae containing the same mutant allele of gene ADE2 in episomic plasmid and in chromosome. It was shown that the strains carrying the ade2 mutation in chromosome and in plasmid did not differ in sensitivity to lethal action of UV-light and 6-GAP. However, in the plasmid rad2 strain reversions were induced by UV-light more frequently (approximately 100 times), as compared to the chromosome strain. We observed no significant differences between reversion frequencies in plasmid and chromosome RAD strains. The tendency to enhanced 6-GAP-induced mutagenesis, less sharply expressed, was observed in the chromosome rad2 strain, as compared to the plasmid one. However, the plasmid RAD strain was characteristic of higher reversion frequency induced by 6-GAP, as compared to the chromosome strain. The possible mechanisms of these phenomena are discussed.     

Assignment of five polymorphic ovine microsatellites to bovine syntenic groups

A panel of bovine somatic cell hybrids was used to map ovine microsatellites. Five of seven microsatellites were assigned to five bovine syntenic groups. These microsatellites were designated D5S10 (MAF23), D1S4 (MAF46), D13S1 (MAF18), D4S3 (MAF50), and DXS2 (MAF45), mapped to syntenic groups U3 (chromosome 5), U10 (chromosome 1), U11, U13, and the X chromosome, respectively. Two remaining sheep microsatellites amplified rodent DNA in the hybrid somatic cell panel, and were not assigned to bovine syntenic groups. Assignment of ovine-derived microsatellites to bovine syntenic groups provides additional evidence of the usefulness of microsatellites for mapping closely related species. The use of ovine and bovine microsatellites will aid in development of comparative genomic maps for these two species.     

An integrated radiation hybrid map of bovine chromosome 18 that refines a critical region associated with multiple ocular defects in cattle

Congenital multiple ocular defects (MOD) of Japanese black cattle is a hereditary ocular disorder with an autosomal recessive mode of inheritance showing developmental defects of the lens, retina and iris, persistent embryonic eye vascularization and microphthalmia. The MOD locus has been mapped by linkage analysis to a 6.6-cM interval on the proximal end of bovine chromosome 18, which corresponds to human chromosome 16q and mouse chromosome 8. To refine the MOD region in cattle, we constructed an integrated radiation hybrid (RH) map of the proximal region of bovine chromosome 18, which consisted of 17 genes and 10 microsatellite markers, using the SUNbRH7000 panel. Strong conservation of gene order was found among the corresponding chromosomal regions in cattle, human and mouse. The MOD-critical region was fine mapped to a 59.5-cR region that corresponds to a 6.3-Mb segment of human chromosome 16 and a 4.8-Mb segment of mouse chromosome 8. Several positional candidate genes, including FOXC2 and USP10, were identified in this region.