Advances in genomic sequencing using Bovine SNP BeadChip in Deer

© Slovak University of Agriculture in Nitra Faculty of Agriobiology and Food Resources, 2014 http://www.fapz.uniag.sk/ DOI: 10.15414/afz.2014.17.02.65–71 Organisms traditionally considered of „minor“ importance by national and international funding agencies generally suffer from a paucity of genomewide sequence and polymorphism data which severely limits the implementation of genomic approaches for addressing biological question in these species (Seabury et al., 2011). One of such underserved species is deer, a highly successful and widely distributed ruminant mammal species of the order Artiodactyla and family Cervidae (Wilson and Reeder, 2005).


Characterization of deer species based on mitochondrial DNA variations
The family Cervidae includes 40 species of deer distributed throughout the northern hemisphere, as well as in South America and Southeast Asia, and an additional five species which has been assigned to the Moschidae family (Gilbert et al., 2006;Grubb and Groves, 2003).The subfamily Cervinae are found in Eurasia, with the exception of the North American wapiti (Cervus elaphus), and are referred to as the Old World deer species.The Odocoileinae, or New World deer species, can be found in both North America, such as the whitetailed deer (Odocoileus viginianus), in South America such as the brocket deer (Mazama), and in Eurasia, such as the roe deer (Capreolus capreolus).Tribal status within the Cervidae has been assigned to the holarctic deer species of reindeer (caribou; Rangifer sp.) and moose (Alces alces).More difficult to place have been the muntjac deer, which now have subfamily status within the Cervidae (Muntiacinae), and even more so the Chinese water deer (Hydropotes inermis) which is the only extant species in the subfamily of Hydropotinae.
Complication of the taxonomic designations were the various hybridizations that occured between deer species, such as the well-described hybridizations between members of the genera Cervus, particularly for Sika (C.nippon), red deer and North American wapiti (C.elaphus).Further complications have arisen due to the various morphotypes of C. elaphus, where the subspecies status has been even more controversal (Ludt et al., 2004).The red deer (Cervus elaphus) is one of the biggest free-ranging mammals of Central Europe and, although not endangered in terms of population numbers, is the perfect model for study the population genetic effects of a multitude of deliberate and unintentional anthropogenic influences on natural populations over a long period of time.Red deer gene pools were affected by habitat fragmentation, keeping of populations in enclosures, translocations, (re) introductions, and trophy hunting.Various schedules of population regulation by hunting were applied throughout Europe.Many autochthonous stocks have been hybridized with the introduced animals, thus blurring the historical boundaries between formerly natural populations (Hartl et al., 2003;Zachos et al., 2003).
Complete mitochondrial DNA sequence represents data for determination of the population structure and also identification of subspecies and species of deer (Wan et al., 2004).In recent years has been recorded a rapid expansion in the sequencing of mitochondrial genomes of different species in family Cervidae.The complete mitochondrial genome sequence of deer for determining phylogenetic positions, providing also important information for molecular-based animal breeding and genetic improvement was determined on the semidomestic red deer (Cervus elaphus) of New Zealand by Wada et al. (2010).Database homology searches showed that the mitochondrial DNA (mtDNA) sequence from the New Zealand semidomestic deer was similar to partial mtDNA sequences from the European, Norwegian (C.e. atlanticus) and Spanish red deer (C.e. hispanicus).Saebury et al. (2011) generated complete mitochondrial genome sequence and identified putative SNPs distributed throughout the white-tailed deer (Odocoileus virginianus) nuclear and mitochondrial genomes.A SNP validation study designed to test specific classes of putative SNPs provided evidence of 10,476 genome-wide SNPs in the dataset.Based on cytogenetic evidence for homology between cattle (Bos taurus) and white-tailed deer chromosomes, they demonstrated that a divergent genome may be used for estimation of the relative distribution and density of de novo contigs as well as putative SNPs for species without draft genome assemblies.Yang et al. (2012a) and Pan et al. (2013) determined complete mitochondrial genome of the Chinese Sika deer -Sichuan Sika deer (Cervus nippon sichuanicus) and the South China Sika deer (Cervus nippon kopschi).Kim et al. (2013) completed mitochondrial genome of a water deer subspecies (Hydropotes inermis argyropus).The close relatedness between Siberian red deer (C.e. sibirica) and North American wapiti (C.e. canadensis) based on mitochondrial DNA variation of cytochrome B gene confirmed Kuznetsova et al. (2012).Zhang and Zhang (2012) used mitochondrial DNA sequences to estimated phylogenetic relationships among animal taxa and for molecular phylogenetic evolution analysis.They prepared phylogenetic analyses of 19 species of Cervidae, with Bos taurus as the outgroup and used neighbour joining, maximum likelihood, maximum parsimony, and Bayesian inference methods on whole mitochondrial genome sequences.
Current studies prepared to examination in order to understand the origin, phylogeny, and phylogeography of the family Cervidae are mainly based on DNA sequence variation of mitochondrial protein -coding genes, analysed in species of deer represent most of the taxonomic diversity of the family.

Utilization of SNPs in deer as non-model species
The development of genetic markers in deer has followed the development of biochemistry and molecular biology, from the initial studies that utilized allozymes to studies that incorporate molecular markers derived from both mitochondrial DNA and the nuclear genome.Much of the work in the development of nuclear DNA markers has relied upon comparative work already completed for other species, in particular other ruminants (Hall, 2009).Single nucleotide polymorphisms (SNPs) are growing in popularity as a genetic markers for investigating evolutionary processes.A panel of SNPs is often developed by comparing large quantities of DNA sequence data across multiple individuals to identify polymorphic sites (Haynes and Latch, 2012).Genomic sequencing programmes in livestock species such as cattle and sheep have enabled the building and application of large SNP chip containing more than 50,000 markers.
Other livestock species (non-model), including deer do not have a large scale genomic sequence or they do have other adequate supporting tools to enable trait to marker associations to be established (Bixley et al., 2009).Single nucleotide polymorphisms are the most abundant type of genetic polymorphism in most, if not all, genomes (Slate et al., 2009).Genetic markers that were applied in past studies fall into two general categories: markers that identify anonymous genetic variation and markers that identify genetic variation in specific segments of genome.SNPs fall into the second category, but are broadly distributed and can represent variation in all of the different genomic regions (coding, noncoding, microsatellite, mitochondrial and chloroplast DNA) (Garvin et al., 2010).SNPs are attractive markers for many reasons, including the availability of high numbers of annotated markers, low-scoring error rates, relative ease of calibration among laboratories compared to length-based markers and the associated ability to assemble combined temporal and spatial data sets from multiple laboratories (Helyar et al., 2011).Additionally, the potential for high-throughput genotyping improved genotyping results for poor quality samples, a simple mutation model, and the ability to examine both neutral variation and regions under selection offers unparalleled scope for expansive screening of genomes and large sample sizes from natural populations (Smith et al., 2011).SNP discovery in many non-model organisms is still primarily performed with chain-termination (Sanger) sequencing (Seeb et al., 2011).Most of the studies attempted to address ascertainment bias including representative samples in the discovery panel, and some were able to use large sample sizes, but most included only a handful of samples.The number of potential SNPs discovered and reported ranged from 2 to 1700, which demonstrated that large numbers of SNPs can be discovered with Sanger sequencing, although coast associated with larger studies were not available (Garvin et al., 2010).In model organisms, which are species with fully sequenced genomes, can be panels of SNPs devised to allow marker-trait association studies of high statistical power and accuracy (Brooks et al., 2010).Utilization of SNPs in study of non-model organisms is different, in connection with microsatellite markers can be used to detection of kinship and parentage (Hauser et al., 2011), individuals (Wiliams et al., 2010) and population structure (Morin et al., 2009).The newly mature next-generation sequencing technologies provide access to a wealth of sequence information on non-model organisms (Tautz et al., 2010).
Even though the next generation sequencing should reduce the cost of DNA sequencing, it is still outside the resources of many projects.One means of SNP discovery that does not require extensive sequencing is to use commercially available SNP chips developed for a related, well-studied model species (Haynes and Latch, 2012).In the last period have been used SNP chips from livestock to identify SNPs in closely related species to non-model species (Miller et al., 2011;Pertoldi et al., 2010), including deer.Bixley et al. ( 2009) have produced a reduced representational sequence of >160 million base pairs (Mbp), of which they mapped 44 Mbp to unique positions on the bovine genome.From this was selected 768 SNPs to be included in a Golden Gate (IlluminaTM) SNP chip.Subsequently have been assembled a mapping pedigree in order to quality control check these and other SNPs and to produce a genetic map.This mapping population was also used to assess recombination rates and to reorder the deer sequence from bovine physical order to deer order.Other immediate outputs from this SNP chip were new parentage assignment and breed composition panels.Haynes and Latch (2012) used the Bovine SNP50 BeadChip developed in cattle (Bos taurus) for identifying polymorphic SNPs in Cervids Odocoileus hemionus (mule deer and black-tailed deer) and O. virginianus (white-tailed deer) in the Pacific Northwest.They found that 38.7 % of loci could be genotyped, of which 5 % (n = 1068) were polymorphic.Of these 1068 polymorphic SNPs, a mixture of putatively neutral loci (n = 878) and loci under selection (n = 190) were identified with the F ST -outlier method.A range of population genetic analyses were implemented using these SNPs and a panel of 10 microsatellite loci.The three types of deer could readily be distinguished with both the SNP and microsatellite datasets.This study demonstrates that commercially developed SNP chips are a viable means of SNP discovery for non-model organisms, even when used between very distantly related species (the Bovidae and Cervidae families diverged some 2 512 301 million years before present).

Significance of SNPs in genes important for different deer traits
Until now has been described in family Cervidae a number of genetic markers (or candidate genes) affecting fitnessrelated traits.At the present are identified through sequencing mainly SNPs of individual genes such as the major histocompatibility complex, reproductive and growth hormones and the prion protein genes (Hall, 2009;Slate et al., 2002).

Genes encoding major histocompatibility complex
The major histocompatibility complex (MHC) is highly polymorphic genomic region that is divided into three tightly linked regions, termed class I, class II, and class III (Wan et al., 2011).Genes encoding MHC molecules are the most polymorphic genes described in vertebrates with polymorphism occurring predominantly at peptide binding site (Winter et al., 1995).DR and DQ genes have been identified as two-principal class II molecule in ruminants.They are tightly linked with class III and class I genes (Van Eijk et al., 1995).The primarily role of the MHC is recognize foreign proteins, present them to specialist immune cells and initiate an immune response.The MHC can reflect the fitness and adaptive potential of a given species due to its association with the immune system (Liu et al., 2013).MHC genes encode cell surface glycoproteins that bind and present antigenic peptides to T cells.Individuals from natural populations deal constantly with diverse range of pathogens and the polymorphism at MHC loci determines the diversity of foreign antigens that the host immune system can recognize (Fernández de Mera et al., 2009).The MHC has also been implicated in mate selection and antler development may be associated with pathogen resistance in deer and thus could be a signal of genetic quality (Kennedy et al., 2010).The MHC class II genes encode polymorphic cell-surface glycoproteins comprising non-covalently linked α and β subunits.These play a pivotal role in the initiation of the immune response to pathogen-derived peptide antigens (Hughes and Yeager, 1998).The second exon has been shown to be highly polymorphic and under positive selection, and the class II DQA gene has recently attracted more attention (Goüy et al., 2009).
In artiodactyls, research on the DQ gene structure and polymorphisms of economically important animals such as cattle and sheep is remarkably extensive (Zhou and Hickford, 2004).In contrast, studies on MHC class II genes in deer are largely restricted to the DRB locus (Fernández de Mera et al., 2009;Kennedy et al., 2010;Li et al., 2012;Li et al., 2013), only rarely considering the DQ gene (Wan et al., 2011;Wu et al., 2012;Liu et al., 2013).

Growth hormone gene
Many studies in this case evaluated genes, which affect antler growth, because antler belongs to the most important products of deer farming with great economic value.Antler is one of the fastest growing parts of deer body and can be considered as the secondary sex characteristics of male deer (Bartos et al., 2009).Antler formation and growth is mainly controlled by the concentration of sex hormones including progesterone, thyroid hormone, adrenocortical hormone and chorionic gonadotropin (Bubenik et al., 1997;Li et al., 2003;Bartos et al., 2009), but few molecular studies analysed and associated antler growing traits with candidate genes encoding growth hormone (GH) and major histocompatibility complex (Li, 2003;Du and Bai, 2004;Wallis et al., 2006).Pituitary growth hormone (GH) is a protein hormone from the pituitary gland which is found in most vertebrates, and which regulates overall somatic growth and various aspects of metabolism.In mammals, GH is generally a strongly conserved protein, but in primates and cetartiodactyls (Cetacea plus Artiodactyla) bursts of rapid evolution led to GHs that differ considerably from those of other mammals (Wallis et al., 2006).
Growth hormone secretion from anterior pituitary is regulated by the hypothalamus and the principal physiological regulation mechanisms of GH secretion are neural endogenous rhythm, sleep, stress, exercise, and nutritional and metabolic signals (Møller and Jørgensen, 2009).Growth hormone is essential for normal linear growth and the attainment of an adult mature height, and it also has roles during cartilage growth and the attainment of normal bone mass (Bennett, 2005).The genetic polymorphism and potential effects of GH gene have been intensively studied in domestic animals such as cattle and pigs and wild animals like red deer (Wallis et al., 2006;Zhang et al., 2011;Yang et al., 2012b).
Another analyzed gene as candidate for antler growth was gene encoding androgen receptor (AR) (Xiong et al., 2012).Androgen is important for male reproductive organ development and function maintenance.Androgen receptor is one of type I steroid receptors and is a ligand-development transregulator protein.Bubenik et al. (2002) showed that the antler cycle is very sensitive to manipulation of androgen levels.These studies showed that antler growing is process affected with several genes and therefore is screening more candidate very important.Potential molecular markers related to antler growth could be utilized for marked assisted selection programs in deer farming.

Prion protein gene
The genetic of the prion protein (PRNP) play a crucial role in determining the relatively susceptibility to transmissible spongioform encephalopathies (TSEs) in several mammalian species (Peletto et al., 2009).Transmissible spongiform encephalopathy (TSE), or prion diseases, are a group of infectious neurodegenerative diseases, which leads the hosts to death.Without available healing and prevention programs, TSE becomes more important as new researches have shown its comprehensive global spread and the large number of animal groups susceptible to developing TSEs.The most popular TSEs are Creutzfeldt-Jacob disease in humans, Bovine Spongiform Encephalopathy (BSE), and scrapie in ovine.Another prion disease less known, but not less important, is the Chronic Wasting Disease (CWD) which attacks animals from Cervidae family, and is the only form of TSE that attacks wild animals (Falcão and Garcia, 2012).CWD is a prion disease of North American cervids, currently affecting both captive and wild elk (Cervus elaphus), mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and moose (Alces alces) (Matsumoto et al., 2012).Disease management in wild cervids populations has been severaly impeded by efficient horizontal transmission of the disease agent, which has resulted in substantial economic losses to farming, gaming, and tourism industries (Bishop, 2004).Direct and indirect transmission is result from prion infectivity in various tissues fluids, and carcasses of infected animals (Mathiason et al., 2006).The degree of similarity of amino acid sequence of the Prnp gene among different species will have consequences on the transmission of TSEs among these species.Often, the same polymorphisms in two species result in similar effects with respect to susceptibility to prion disease (Goldmann, 2008).Associations between variation in the primary sequence of the prion protein gene and disease modulation have been shown for CWD and a number of studies have analysed the genetic variability of the PRNP gene in cervids species (Peletto et al., 2009;Jeong et al., 2009;Ernest et al., 2010;Cullingham et al., 2011).

Conclusions
Finally, single nucleotide polymorphisms are used for number of different applications in molecular genetics research of deer species, which are important for conservation and utilization of their genetic diversity.In addition SNPs can be used also as genetic markers for fitness-related or economic important traits of deer.The application of SNPs to genetic studies will continue to expand because SNPs are abundant, co-dominant markers that are broadly distributes in genomes and amenable to high-throuhput screening.Advances in DNA sequencing and genotyping technologies are now