On the search for genetic variations associated with tick resistance in cattle by means of molecular genetics

Laercio Ribeiro Porto Neto (2011). On the search for genetic variations associated with tick resistance in cattle by means of molecular genetics PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

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Author Laercio Ribeiro Porto Neto
Thesis Title On the search for genetic variations associated with tick resistance in cattle by means of molecular genetics
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-05
Thesis type PhD Thesis
Supervisor Prof Michael D'Occhio
Prof Nicholas N Jonsson
Dr William Barendse
Total pages 180
Total colour pages 25
Total black and white pages 155
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary In recent years there has been renewed interest in the tropical adaptation of cattle, including increased resistance to parasites. The interest in tick resistance is understandable given the major production and welfare implications of tick infestation in tropical and subtropical zones where around 70% of beef cattle are located. In Australia, it is estimated that up to AU$200 million are lost per year due to tick infestation and tick borne diseases. There have been a relatively small number of studies aimed at dissecting the genetic components involved in the tropical adaptation process. This thesis reports on the application of molecular genetic technologies to understand the adaptive trait of host resistance to tick infestation measured as the tick burden of cattle, contributing in two broad areas. The first is On the concepts of molecular markers - the identification of SNP markers in cattle and the improved understanding of the intrinsic characteristics of SNP that are now being routinely explored. The second is On the identification of molecular markers associated with tick resistance in cattle - a practical application of SNP markers towards the identification of genomic regions and genes associated with tick burden in cattle. Three quantitative trait loci (QTL) were fine-mapped and one positional candidate gene was tested in functional experiments. This contributed to the understanding of biological factors involved in host-parasite interactions between cattle and the cattle tick and to the development of indicators for tick-resistant cattle, to select cattle with reduced tick burdens. The identification of SNP markers for adaptive and production traits has recently become a relatively routine process. However, before applying the latest technologies on gene mapping studies it is important to better understand some inherent characteristics of SNP markers. Two publicly available SNP databases (Bovine Assembly and Bovine Interbreed) were found to have a high false-positive rate, mainly due to sequence artefacts that were miscalled as SNP. The identification of SNP listed in the bovine assembly and bovine interbreed SNP databases can be improved to increase the chance of successful genotyping by the use of filters applied to the sequence data. Three filters commonly applied to the sequence data are based on (1) establishment of a minimum quality score to call a base correctly (2) the observed depth of sequencing – the minimum number of copies observed of each allele and (3) the evaluated quality score of the SNP point and bases around it, creating a neighbourhood quality standard. Similar filters were first described and successfully applied for human SNP identification. In this thesis such filters were implemented in the software SNPValidator (Chapter 1.1), which is freely available for evaluation of SNP from the Bovine Assembly and Bovine Interbreed databases. SNPValidator assists the identification of false SNP in those databases, improving the efficiency with which this resource can be used. Another important characteristic of SNP markers and a concern for researchers, beyond the identification of the marker per se, is the potential ascertainment bias of SNP sets. Several SNP-discovery strategies imply an ascertainment bias in the markers identified, mainly due to the use of a small number of individuals from a small number of populations for SNP discovery. In cattle, a number of recent publications considered the potential effect of ascertainment bias on analyses but none undertook a systematic evaluation of its effects on routinely-used population-structure analyses. Chapter 1.2 addresses the evaluation of the ascertainment bias determined by the SNP discovery methods in the Bovine Hapmap experiment. It was confirmed that the allelic frequencies are biased between different SNP sets, while principal component and population structure analyses and analyses of molecular variance were less affected or even unaffected by ascertainment bias. Following the characterisation and better understanding of SNP markers, an analysis of association of validated SNP previously associated with milk traits to host resistance to tick infestation was performed. As a result, no significant effect of validated milk-related SNP on tick burden was found (Appendix A). Then, the question as to whether selection for high milk production would impact on the level of tick burden and vice-versa was addressed. The correlation of the allele effects, estimated using genome-wide SNP, for milk components and tick burden was relatively small, such that it could be concluded that selection for one trait may not have an undesirable effect on the other trait (Appendix A). These analyses, together with the detection of genome-wide associations for tick burden, supported the continuation of fine-mapping experiments for tick burden. Three QTL for tick burden in cattle previously identified in genome-wide association studies (GWAS) were further evaluated in this thesis. GWAS fine-mapping studies confirmed and expanded on the associations of SNP to a QTL on BTA10 (Chapter 2.1), BTA3 (Chapter 2.2) and BTA14 (Chapter 2.3), using two independent samples (n ~ 1,000 dairy cattle and n ~ 725 beef cattle). The BTA3 QTL region includes the ELTD1 gene and in this region more markers were added and analysed, reducing the confidence interval and confirming the association of the region with tick burden (Chapter 2.2). Similarly, for the BTA10 region more SNP were added to the region, in this case including newly-identified SNP from partial sequencing of the candidate gene ITGA11; the confidence interval was reduced and the region was confirmed as harbouring a QTL for tick burden (Chapter 2.1). For the BTA14 region, more markers were added to the analyses, including SNP newly identified by sequencing the entire positional candidate gene RIPK2. Association analyses were also performed, reducing the confidence interval and confirming the QTL for tick burden. The RIPK2 gene was previously shown to be a component of the innate and adaptive immune systems and is down-regulated in cattle skin under a tick infestation, providing support for further functional studies for this candidate gene. The influence of RIPK2 in antibody production against tick salivary-gland extract was assessed using a mouse gene-knockout model. The absence of the RIPK2 gene was associated with lower antibody IgG response. Western blotting analyses demonstrated that antibodies of the different mice strains did not all target the same proteins (Chapter 2.3). Following these analyses, it could be speculated that the effect of the RIPK2 on tick burden is related to the magnitude of the antibody response, and also to the differentially targeted antigens. Finally, host resistance to ticks in cattle is a complex trait influenced by many different factors potentially many interacting genes and biological pathways. This complexity of resistance to ticks in cattle is reflected in the number of genomic regions that contain associations with tick burden, all of which have a relatively small effect (SNP/haplotype effects, R2 ~ 1% of the phenotypic variance). The complexity of the tick-resistance process is investigated in a meta-analysis that combined gene-mapping and gene-expression data associated with tick infestation (Chapter 2.4). The identification of causal mutations contributes to the better understanding of the biological process underpinning the observed trait. This step is a time-consuming and costly procedure that is not always supported by the livestock industry. In livestock, a large proportion of molecular genetics research aims to identify markers to be used in genetic selection programs for improvement of animal production. For this purpose, the identification of causal variants may not be necessary and, in this case, the identification of a molecular marker validated as associated to the functional variant might be sufficient. Because each SNP accounts for only a small proportion of the variance, what is required is the development of marker panels that together explain a reasonable proportion of the variance available for genetic improvement. The number of genes or functional mutations identified as associated with quantitative traits is expected to increase as a consequence of the development of molecular genetics technologies (e.g. advances in SNP genotyping platforms, development of algorithms that robustly impute SNP genotypes and next-generation sequencing applied to whole genomes). The work presented here will assist with the identification of causal mutations associated with tick burden and allow a more focussed approach in future research.
Keyword QTL, Bovine, SNP, Parasite, molecular genetics, knockout mouse
Additional Notes 1, 10, 14, 15, 18, 38, 46, 55, 57, 58, 59, 62, 65, 69, 70, 72, 86, 87, 102, 103, 106, 121, 123, 132, 142

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Created: Wed, 15 Jun 2011, 10:22:53 EST by Mr Laercio Porto Neto on behalf of Library - Information Access Service