22 research outputs found

    Genome-Wide Identification of Allele-Specific Expression (ASE) in Response to Marek’s Disease Virus Infection Using Next Generation Sequencing.

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    Background Marek’s disease (MD), a T cell lymphoma induced by the highly oncogenic α-herpesvirus Marek’s disease virus (MDV), is the main chronic infectious disease concern threatening the poultry industry. Enhancing genetic resistance to MD in commercial poultry is an attractive method to augment MD vaccines, which is currently the control method of choice. In order to optimally implement this control strategy through marker-assisted selection (MAS) and to gain biological information, it is necessary to identify specific genes that influence MD incidence. Methods A genome-wide screen for allele-specific expression (ASE) in response to MDV infection was conducted. The highly inbred ADOL chicken lines 6 (MD resistant) and 7 (MD susceptible) were inter-mated in reciprocal crosses and half of the progeny challenged with MDV. Splenic RNA pools at a single time after infection for each treatment group point were generated, sequenced using a next generation sequencer, then analyzed for allele-specific expression (ASE). To validate and extend the results, Illumina GoldenGate assays for selected cSNPs were developed and used on all RNA samples from all 6 time points following MDV challenge. Results RNA sequencing resulted in 11-13+ million mappable reads per treatment group, 1.7+ Gb total sequence, and 22,655 high-confidence cSNPs. Analysis of these cSNPs revealed that 5360 cSNPs in 3773 genes exhibited statistically significant allelic imbalance. Of the 1536 GoldenGate assays, 1465 were successfully scored with all but 19 exhibiting evidence for allelic imbalance. Conclusions ASE is an efficient method to identify potentially all or most of the genes influencing this complex trait. The identified cSNPs can be further evaluated in resource populations to determine their allelic direction and size of effect on genetic resistance to MD as well as being directly implemented in genomic selection programs. The described method, although demonstrated in inbred chicken lines, is applicable to all traits in any diploid species, and should prove to be a simple method to identify the majority of genes controlling any complex trait

    Genome-Wide Identification and Quantification of cis- and trans-Regulated Genes Responding to Marek’s Disease Virus Infection via Analysis of Allele-Specific Expression

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    Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by Marek’s disease virus (MDV), a naturally occurring oncogenic alphaherpesvirus. Selecting for increased genetic resistance to MD is a control strategy that can augment vaccinal control measures. To identify high-confidence candidate MD resistance genes, we conducted a genome-wide screen for allele-specific expression (ASE) amongst F1 progeny of two inbred chicken lines that differ substantially in MD resistance. High throughput sequencing was initially used to profile transcriptomes from pools of uninfected and infected individuals at 4 days post-infection to identify any genes showing ASE in response to MDV infection. RNA sequencing identified 22,655 single nucleotide polymorphisms (SNPs) of which 5,360 in 3,773 genes exhibited significant allelic imbalance. Illumina GoldenGate assays were subsequently used to quantify regulatory variation controlled at the gene (cis) and elsewhere in the genome (trans) by examining differences in expression between F1 individuals and artificial F1 RNA pools over six time periods in 1,536 of the most significant SNPs identified by RNA sequencing. Allelic imbalance as a result of cis-regulatory changes was confirmed in 861 of the 1,233 GoldenGate assays successfully examined. Furthermore we have identified seven genes that display trans-regulation only in infected animals and ∼500 SNP that show a complex interaction between cis- and trans-regulatory changes. Our results indicate ASE analyses are a powerful approach to identify regulatory variation responsible for differences in transcript abundance in genes underlying complex traits. And the genes with SNPs exhibiting ASE provide a strong foundation to further investigate the causative polymorphisms and genetic mechanisms for MD resistance. Finally, the methods used here for identifying specific genes and SNPs have practical implications for applying marker-assisted selection to complex traits that are difficult to measure in agricultural species, when expression differences are expected to control a portion of the phenotypic variance

    Molecular evolution of the Bovini tribe (Bovidae, Bovinae): Is there evidence of rapid evolution or reduced selective constraint in Domestic cattle?

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    BACKGROUND: If mutation within the coding region of the genome is largely not adaptive, the ratio of nonsynonymous (dN) to synonymous substitutions (dS) per site (dN/dS) should be approximately equal among closely related species. Furthermore, dN/dS in divergence between species should be equivalent to dN/dS in polymorphisms. This hypothesis is of particular interest in closely related members of the Bovini tribe, because domestication has promoted rapid phenotypic divergence through strong artificial selection of some species while others remain undomesticated. We examined a number of genes that may be involved in milk production in Domestic cattle and a number of their wild relatives for evidence that domestication had affected molecular evolution. Elevated rates of dN/dS were further queried to determine if they were the result of positive selection, low effective population size (N(e)) or reduced selective constraint. RESULTS: We have found that the domestication process has contributed to higher dN/dS ratios in cattle, especially in the lineages leading to the Domestic cow (Bos taurus) and Mithan (Bos frontalis) and within some breeds of Domestic cow. However, the high rates of dN/dS polymorphism within B. taurus when compared to species divergence suggest that positive selection has not elevated evolutionary rates in these genes. Likewise, the low rate of dN/dS in Bison, which has undergone a recent population bottleneck, indicates a reduction in population size alone is not responsible for these observations. CONCLUSION: The effect of selection depends on effective population size and the selection coefficient (N(e)s). Typically under domestication both selection pressure for traits important in fitness in the wild and Ne are reduced. Therefore, reduced selective constraint could be responsible for the observed elevated evolutionary ratios in domesticated species, especially in B. taurus and B. frontalis, which have the highest dN/dS in the Bovini. This may have important implications for tests of selection such as the McDonald-Kreitman test. Surprisingly we have also detected a significant difference in the supposed neutral substitution rate between synonymous and noncoding sites in the Bovine genome, with a 30% higher rate of substitution at synonymous sites. This is due, at least in part, to an excess of the highly mutable CpG dinucleotides at synonymous sites, which will have implications for time of divergence estimates from molecular data

    Testing the neutral theory of molecular evolution using genomic data: a comparison of the human and bovine transcriptome

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    Despite growing evidence of rapid evolution in protein coding genes, the contribution of positive selection to intra- and interspecific differences in protein coding regions of the genome is unclear. We attempted to see if genes coding for secreted proteins and genes with narrow expression, specifically those preferentially expressed in the mammary gland, have diverged at a faster rate between domestic cattle (Bos taurus) and humans (Homo sapiens) than other genes and whether positive selection is responsible. Using a large data set, we identified groups of genes based on secretion and expression patterns and compared them for the rate of nonsynonymous (dN) and synonymous (dS) substitutions per site and the number of radical (Dr) and conservative (Dc) amino acid substitutions. We found evidence of rapid evolution in genes with narrow expression, especially for those expressed in the liver and mammary gland and for genes coding for secreted proteins. We compared common human polymorphism data with human-cattle divergence and found that genes with high evolutionary rates in human-cattle divergence also had a large number of common human polymorphisms. This argues against positive selection causing rapid divergence in these groups of genes. In most cases dN/dS ratios were lower in human-cattle divergence than in common human polymorphism presumably due to differences in the effectiveness of purifying selection between long-term divergence and short-term polymorphism

    Phylogenetic reconstruction and the identification of ancient polymorphism in the Bovini tribe (Bovidae, Bovinae)

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    <p>Abstract</p> <p>Background</p> <p>The Bovinae subfamily incorporates an array of antelope, buffalo and cattle species. All of the members of this subfamily have diverged recently. Not surprisingly, a number of phylogenetic studies from molecular and morphological data have resulted in ambiguous trees and relationships amongst species, especially for Yak and Bison species. A partial phylogenetic reconstruction of 13 extant members of the Bovini tribe (Bovidae, Bovinae) from 15 complete or partially sequenced autosomal genes is presented.</p> <p>Results</p> <p>We identified 3 distinct lineages after the Bovini split from the Boselaphini and Tragelaphini tribes, which has lead to the (1) Buffalo clade (<it>Bubalus </it>and <it>Syncerus </it>species) and a more recent divergence leading to the (2) Banteng, Gaur and Mithan and (3) Domestic cattle clades. A fourth lineage may also exist that leads to Bison and Yak. However, there was some ambiguity as to whether this was a divergence from the Banteng/Gaur/Mithan or the Domestic cattle clade. From an analysis of approximately 30,000 sites that were amplified in all species 133 sites were identified with ambiguous inheritance, in that all trees implied more than one mutation at the same site. Closer examination of these sites has identified that they are the result of ancient polymorphisms that have subsequently undergone lineage sorting in the Bovini tribe, of which 53 have remained polymorphic since <it>Bos </it>and <it>Bison </it>species last shared a common ancestor with <it>Bubalus </it>between 5–8 million years ago (MYA).</p> <p>Conclusion</p> <p>Uncertainty arises in our phylogenetic reconstructions because many species in the Bovini diverged over a short period of time. It appears that a number of sites with ambiguous inheritance have been maintained in subsequent populations by chance (lineage sorting) and that they have contributed to an association between Yak and Domestic cattle and an unreliable phylogenetic reconstruction for the Bison/Yak clade. Interestingly, a number of these aberrant sites are in coding sections of the genome and their identification may have important implications for studying the neutral rate of mutation at nonsynonymous sites. The presence of these sites could help account for the apparent contradiction between levels of polymorphism and effective population size in domesticated cattle.</p

    An examination of positive selection and changing effective population size in Angus and Holstein cattle populations (Bos taurus) using a high density SNP genotyping platform and the contribution of ancient polymorphism to genomic diversity in Domestic ca

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    Background: Identifying recent positive selection signatures in domesticated animals could provide information on genome response to strong directional selection from domestication and artificial selection. With the completion of the cattle genome, private companies are now providing large numbers of polymorphic markers for probing variation in domestic cattle (Bos taurus). We analysed over 7,500 polymorphic single nucleotide polymorphisms (SNP) in beef (Angus) and dairy (Holstein) cattle and outgroup species Bison, Yak and Banteng in an indirect test of inbreeding and positive selection in Domestic cattle. Results: Outgroup species: Bison, Yak and Banteng, were genotyped with high levels of success (90%) and used to determine ancestral and derived allele states in domestic cattle. Frequency spectrums of the derived alleles in Angus and Holstein were examined using Fay and Wu's H test. Significant divergences from the predicted frequency spectrums expected under neutrality were identified. This appeared to be the result of combined influences of positive selection, inbreeding and ascertainment bias for moderately frequent SNP. Approximately 10% of all polymorphisms identified as segregating in B. taurus were also segregating in Bison, Yak or Banteng; highlighting a large number of polymorphisms that are ancient in origin. Conclusion: These results suggest that a large effective population size (Ne) of approximately 90,000 or more existed in B. taurus since they shared a common ancestor with Bison, Yak and Banteng ∼1-2 million years ago (MYA). More recently Ne decreased sharply probably associated with domestication. This may partially explain the paradox of high levels of polymorphism in Domestic cattle and the relatively small recent N in this species. The period of inbreeding caused Fay and Wu's H statistic to depart from its expectation under neutrality mimicking the effect of selection. However, there was also evidence for selection, because high frequency derived alleles tended to cluster near each other on the genome
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