Stephen S. Moore

Development and Characterization of a High Density SNP Genotyping Assay for Cattle

The success of genome-wide association (GWA) studies for the detection of sequence variation affecting complex traits in human has spurred interest in the use of large-scale high-density single nucleotide polymorphism (SNP) genotyping for the identification of quantitative trait loci (QTL) and for marker-assisted selection in model and agricultural species. A cost-effective and efficient approach for the development of a custom genotyping assay interrogating 54,001 SNP loci to support GWA applications in cattle is described. A novel algorithm for achieving a compressed inter-marker interval distribution proved remarkably successful, with median interval of 37 kb and maximum predicted gap of <350 kb. The assay was tested on a panel of 576 animals from 21 cattle breeds and six outgroup species and revealed that from 39,765 to 46,492 SNP are polymorphic within individual breeds (average minor allele frequency (MAF) ranging from 0.24 to 0.27). The assay also identified 79 putative copy number variants in cattle. Utility for GWA was demonstrated by localizing known variation for coat color and the presence/absence of horns to their correct genomic locations. The combination of SNP selection and the novel spacing algorithm allows an efficient approach for the development of high-density genotyping platforms in species having full or even moderate quality draft sequence. Aspects of the approach can be exploited in species which lack an available genome sequence. The BovineSNP50 assay described here is commercially available from Illumina and provides a robust platform for mapping disease genes and QTL in cattle.

A genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 individuals in full sibling pedigrees. One hundred and seventy–one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.

SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries.

High-density single-nucleotide polymorphism (SNP) arrays have revolutionized the ability of genome-wide association studies to detect genomic regions harboring sequence variants that affect complex traits. Extensive numbers of validated SNPs with known allele frequencies are essential to construct genotyping assays with broad utility. We describe an economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations. Using nearly 50 million sequences generated on an Illumina Genome Analyzer from DNA of 66 cattle representing three populations, we identified 62,042 putative SNPs and predicted their allele frequencies. Genotype data for these 66 individuals validated 92% of 23,357 selected genome-wide SNPs, with a genotypic and sequence allele frequency correlation of r = 0.67. This approach for simultaneous de novo discovery of high-quality SNPs and population characterization of allele frequencies may be applied to any species with at least a partially sequenced genome.

The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species.

The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.

A medium-density genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.USDA-MARC family and data for validating this family. P. Creighton, C. Skidmore, T. Holm, and A. Georgoudis provided some validation data for the BOVMAP families. R. Fries, S. Johnson, S. Solinas Toldo, and A. Mezzelani kindly made some of their FISH assignments available before publication. We wish to thank all those researchers who kindly sent us probes and DNA primers.

Whole genome linkage disequilibrium maps in cattle

BackgroundBovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides background information concerning the extent of long range linkage disequilibrium in cattle.ResultsLinkage disequilibrium was assessed using r2 among all pairs of syntenic markers within eight breeds of cattle from the Bos taurus and Bos indicus subspecies. Bos taurus breeds included Angus, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black and Limousin while Bos indicus breeds included Brahman and Nelore. Approximately 2670 markers spanning the entire bovine autosomal genome were used to estimate pairwise r2 values. We found that the extent of linkage disequilibrium is no more than 0.5 Mb in these eight breeds of cattle.ConclusionLinkage disequilibrium in cattle has previously been reported to extend several tens of centimorgans. Our results, based on a much larger sample of marker loci and across eight breeds of cattle indicate that in cattle linkage disequilibrium persists over much more limited distances. Our findings suggest that 30,000–50,000 loci will be needed to conduct whole genome association studies in cattle.

Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism

Abstract We report construction of an initial genetic linkage map for the black tiger shrimp, Penaeus monodon. Mapping was carried out using polymorphic markers derived from 23 Amplified Fragment Length Polymorphism (AFLP) primer pairs. These were analysed on three reference families of known pedigree. A total of 673 polymorphic AFLP loci that conformed to expected Mendelian segregation ratios were scored in three families, and these were used to construct separate male and female linkage maps for each family. AFLP markers that consisted of a segregating fragment of the same size, amplified with the same primer pair in two or more of the reference families, were considered to be common markers. 116 such common AFLP markers were used to construct a common linkage map across the three families. This linkage map has 20 linkage groups covering a total genetic distance of 1412 cM. Future directions for genetic mapping in P. monodon are discussed in light of these initial data.

Characterization of 65 bovine microsatellites

Microsatellites or simple sequence repeat (SSR) polymorphisms are used widely in the construction of linkage maps in many species. High levels of polymorphism coupled with the ease of analysis of the polymerase chain reaction (PCR) have resulted in this type of maker being one of the most widely used for genetic analysis. In this paper we describe 58 polymorphic bovine microsatellites that were isolated from insert size selected bovine genomic libraries. Primer sequences, number of alleles, and heterozygosity levels in cattle reference families are reported. Chromosomal locations for 47 of these microsatellites as well as for 7 previously described systems derived from entries in the Genbank or EMBL databases have been determined. The markers map to 24 syntenic or chromosomal locations. Polymorphic bovine microsatellites were estimated to occur, on average, every 320 kb, and there is no evidence of clustering in the genome. Thirty of the bovine-derived microsatellite systems gave specific and polymorphic products in sheep, adding to the number of useful markers in that species.

Linkage of microbial ecology to phenotype: correlation of rumen microbial ecology to cattle's feed efficiency

Linkage of rumen microbial structure to host phenotypical traits may enhance the understanding of host-microbial interactions in livestock species. This study used culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the microbial profiles in the rumen of cattle differing in feed efficiency. The analysis of detectable bacterial PCR-DGGE profiles showed that the profiles generated from efficient steers clustered together and were clearly separated from those obtained from inefficient steers, indicating that specific bacterial groups may only inhabit in efficient steers. In addition, the bacterial profiles were more likely clustered within a certain breed, suggesting that host genetics may play an important role in rumen microbial structure. The correlations between the concentrations of volatile fatty acids and feed efficiency traits were also observed. Significantly higher concentrations of butyrate (P < 0.001) and valerate (P = 0.006) were detected in the efficient steers. Our results revealed potential associations between the detectable rumen microbiota and its fermentation parameters with the feed efficiency of cattle.

The development and application of genetic markers for the Kuruma prawn Penaeus japonicus

Microsatellite and Amplified Fragment Length Polymorphism (AFLP) DNA markers have been characterised for use in establishing pedigrees, linkage mapping and identifying Quantitative Trait Loci (QTL) influencing commercially important traits in P. japonicus. Low efficiency of Microsatellite characterisation from P. japonicus genomic DNA, due to the high frequency and extended length of simple sequence repeats, make these DNA markers unsuitable for linkage mapping studies. Unique sequence flanking repeats necessary for PCR primer design were difficult to obtain due the extended lengths of the repeats. Microsatellites that were characterised displayed between 4-24 alleles and heterozygosities between 47-91% in unrelated animals. No P. japonicus derived microsatellite successfully amplified sequences in P. monodon, P. esculentus or P. stylirostris. AFLPs were developed as an alternative to microsatellites. Over 570 polymorphic loci were defined using different primer combinations. AFLPs are robust with some polymorphisms conserved across families. PCR amplification and sequencing of excised bands allowed development of Sequence Tagged Sites from AFLPs. A primary linkage map based on a three generation pedigree, genotyped at 246 AFLP loci has been constructed. It incorporates 129 markers in 44 linkage groups with an estimated genome coverage of approximately 57%.