The microsatellite markers are assayed by polymerase chain reaction (PCR) amplification using fluorescent-labeled primers. Primers are short (20 base pairs), single-strand lengths of DNA that are complementary to a specific region of the genome. PCR is the amplification of a section of DNA contained between two primers designed to complement the unique sequence flanking the STR. The PCR products are then resolved by electrophoresis based on their length. Figure 52-1 shows a single microsatellite marker in three different animals. This marker is polymorphic and would be a useful marker for individual identification or parentage. Because the markers show differences between individuals, a collection of these markers can be used as a form of identification of an animal. High statistical significance can be obtained with as few as 10 markers, depending on the species and breed. The DNA type of an animal will not change over its lifetime and can therefore be used as a form of permanent identification.

Many purebred registries require parentage verification for registration purposes. To accomplish parentage verification, a DNA sample must be available from both parents as well as the offspring. DNA samples are taken in the form of hair, blood, or buccal swabs (depending on the species and registry) and submitted at the time registration is requested. Each animal inherits one copy of each marker from its sire and one copy from its dam, so the markers can also be used to verify parentage. The most useful marker has a high polymorphism rate because that type of marker will be most likely to show differences not only between the sire and the dam, but also between the two copies (alleles) of the marker. A set of polymorphic markers (10 to 20) is used to verify parentage to ensure a high probability that the parentage is correct. Table 52-1 shows the allele sizes for a set of markers in a parentage case. For marker A the offspring inherited a 122 and a 126 allele. The 122 came from its dam, so the 126 came from the sire. Because sire 1 does not have a 126 allele, it has been excluded. In this example, sire 1 is excluded as the sire of the offspring, and sire 2 is verified on the basis of the results for all three markers.

Early attempts to construct whole-genome maps of large animal species were based on two technologies that had been used to construct the first human genome maps: somatic cell genetics and in situ hybridization. This technology involved using labeled complementary DNA or RNA strands (i.e., probes) to localize a specific DNA or RNA sequence in somatic cells. Initial mapped markers were generally genes, or gene products, highly conserved across mammalian genomes. These markers, defined as syntenic (genes on the same chromosome but not necessarily linked) and cytogenic locations, were determined through sequences hybridizing specific DNA probes. Therefore, the first “genome maps” were defined as synteny groups: genes on the same chromosome defined by gene products that segregated in hybrid somatic cell lines. Synteny groups were developed for cattle, horses, and sheep. The next stage of mapping required the use of highly polymorphic markers, including microsatellites, within families of animals to create linkage maps. Collaborative efforts between researchers interested in large animal genomics were required to create these linkage maps for cattle, horses, sheep, and goats (Table 52-2). Radiation hybrid (RH) maps, which used x-ray breakage of chromosomes to determine the distances between DNA markers, followed. These RH maps allowed for the incorporation of genetic (i.e., linkage) and physical maps. The first RH maps involved only specific chromosomes, but later generations included whole genome RH and comparative maps. The isolation of bacterial artificial chromosome (BAC) clones for a large assortment of loci in the various large animal species was instrumental in facilitating chromosomal locations for these loci. In the horse, a complete physical BAC contig map of the entire genome was developed.

Whole genome sequencing and genome assembly of large animal species was completed in the early 2000s (horse, September 2007; cow, October 2011; sheep, August 2012; goat, December 2012). In the horse, a Thoroughbred mare (Twilight) was selected for sequencing because of her low heterozygosity rate (1/1380 base pairs). In 2010, a Quarter Horse mare underwent whole genome sequencing and was aligned to Twilight.¹ In cattle, a single partially inbred Hereford cow was selected to contribute 6x whole-genome shotgun (WGS) reads and another 1.5x came from individual animals of the Holstein, Angus, Jersey, Limousin, Brahman, and Norwegian Red breeds for detection of single nucleotide polymorphisms (SNPs). Following sequencing of each species, over 20,000 protein-coding genes were annotated on the sequences by virtue of previously sequenced cDNAs as well as by prediction software that compare known genome sequences in other species with newly sequenced genomes. These annotated genome databases are publicly available (see Table 52-2).

With the advent of whole genome sequencing and assemblies, there has been a rapid expansion in the number of SNPs discovered in the genomes of large animal species. SNPs are now considered the next generation of markers to conduct breed diversity and to map disease-causing traits. The availability of such a tool as an SNP-chip facilitates rapid mapping of diseases to specific chromosomal regions and analysis of candidate genes. SNP arrays are currently available on many large animal species (see Table 52-2) and are currently being used in mapping studies of various complex disorders. Haplotype structures are being identified across species with targeted resequencing and, most recently, whole genome next-generation sequencing, of animals from different breeds.

In addition to genomic mapping, functional genomic tools have become more readily available and affordable to researchers. Expression arrays have been designed in the horse, cow, and sheep (see Table 52-2). Most recently, sequencing of the transcriptome, through technologies such as RNA sequencing (RNA-seq), has allowed researchers to evaluate gene expression differences among tissues and between animals of different disease states.

Identifying Genetic Mutations

Initial genetic mutations in large animal species were discovered through the use of comparative genomics. Genes involved in a specific disease were targeted because of equivalent diseases in other species, namely humans. In the horse (Table 52-3), the genetic mutations for many diseases that have genetic tests currently available, including hyperkalemic periodic paralysis (HYPP)² and severe combined immunodeficiency (SCID),³ were uncovered by evaluating candidate genes that had been associated with similar diseases in humans. With the sequencing and annotation of whole genome maps, other diseases were discovered through whole genome linkage mapping (hereditary equine regional dermal asthenia [HERDA]⁴), genome-wide association studies with microsatellites (type I polysaccharide storage myopathy [PSSM]⁵) and genome-wide association studies using SNP array technology (Lavender foal syndrome⁶).

A similar theme is evident in cattle. Initial genetic mutations were discovered based on sequencing of candidate genes known to cause similar disease in humans (bovine leukocyte adhesion deficiency [BLAD]⁷). Later studies used microsatellite markers and performed linkage analysis (complex vertebral malformation⁸) and, most recently, the use of SNP-based genome-wide association studies has identified recessive defects (congenital muscular dystony types 1 and 2⁹). At the time of publication, there are 90 genetic tests available in cattle (Table 52-4). It is worth noting that the majority of genetic tests currently available are for diseases/traits that are inherited as autosomal recessive traits. With the current technologies available through SNP-association mapping and next-generation sequencing, we should expect to further our understanding of polygenic traits and diseases.

TABLE 52-4

Genetic Tests for Cattle

Disease/Trait	Gene	Mode of Inheritance	Reference(s)
Disease
Abortion/Stillbirth	MIMT1	Maternally imprinted	32
Abortion	APAF1	Autosomal recessive lethal	33
	GART	Autosomal recessive lethal	34
	CWC15	Autosomal recessive lethal	35
	SHBG	Autosomal recessive lethal	34
	SLC37A2	Autosomal recessive lethal	34
Acrodermatitis enteropathica (bovine hereditary zinc deficiency or lethal trait A46)	SLC39A4	Autosomal recessive	36
Anhidrotic ectodermal dysplasia	EDA	X-linked recessive	37
Arachnomelia (spider limbs)	MOCS1	Autosomal recessive	38
	SUOX	Autosomal recessive	39
Axonopathy (Demetz syndrome)	MFN2	Autosomal recessive	40
Beta-lactoglobulin, aberrant low expression	PAEP	Autosomal	41
Brachyspina	FANCI	Autosomal recessive	42
Cardiomyopathy and woolly haircoat syndrome	PPP1R13L	Autosomal recessive	43
Cardiomyopathy, dilated	OPA3	Autosomal recessive	44
Chediak-Higashi syndrome	LYST	Autosomal recessive	45
Chondrodysplasia	EVC2	Autosomal	46
Citrullinemia	ASS1	Recessive	47
Complex vertebral malformation	SLC35A3	Autosomal recessive	8
Congenital muscular dystonia 1	ATP2A1	Autosomal recessive	9
Congenital muscular dystonia 2	SCL6A5	Autosomal recessive	9
Deficiency of uridine monophosphate synthase (DUMPS)	UMPS	Autosomal recessive	48
Dominant white with bilateral deafness	MITF	Autosomal dominant	49
Dwarfism, Angus	PRKG2	Autosomal recessive	50
Dwarfism, Dexter	ACAN	Autosomal recessive lethal	51
Dwarfism, growth-hormone deficiency	GH1	Autosomal recessive	52
Ehlers-Danlos syndrome, Holstein variant	EPYC	Autosomal recessive	53
Ehlers-Danlos syndrome, type VII (Dermatosparaxis)	ADAMTS2	Autosomal	54
Epidermolysis bullosa	KRT5	Autosomal dominant	55
Epidermolysis bullosa, dystrophic	COL7A1	Autosomal recessive	56
Factor XI deficiency	F11	Autosomal recessive	57
Forelimb-girdle muscle anomaly	GFRA1	Autosomal recessive	58
Glycogen storage disease II (Pompe disease)	GAA	Recessive	59
Glycogen storage disease V	PYGM	Autosomal recessive	60
Goiter, familial	TG	Autosomal	61
Hemophilia A	F8	X-linked	62
Hypotrichosis	HEPHL1	Autosomal recessive	63
Ichthyosis congenita	ABCA12	Autosomal recessive	9
Lethal multi-organ developmental dysplasia	KDM2B	Autosomal recessive	64
Leukocyte adhesion deficiency, type I	ITGB2	Autosomal recessive	7
Mannosidosis
Alpha	MAN2B1	Autosomal recessive	65
Beta	MANBA	Autosomal recessive	66
Maple syrup urine disease	BCKDHA	Autosomal recessive	67
Marfan syndrome	FBN1	Autosomal dominant	68
Mucopolysaccharidosis IIIB	NAGLU	Autosomal recessive	69
Multiple ocular defects	WFDC1	Autosomal recessive	70
Muscular hypertrophy (double muscling)	MSTN	Autosomal recessive	13–15
Myasthenic syndrome, congenital	CHRNE	Autosomal	71
Myoclonus	GLRA1	Autosomal recessive	72
Myopathy of the diaphragmatic muscles	HSPA1A	Autosomal recessive	73
Neuronal ceroid lipofuscinosis 5	CLN5	Autosomal recessive	74
Osteopetrosis	SLC4A2	Autosomal recessive	75
Ovotesticular disorder of sexual development	SRY	Y-linked	76
Polled and multisystemic syndrome	ZEB2	Autosomal dominant	77
Protoporphyria	FECH	Autosomal	78
Pseudomyotonia, congenital	ATP2A1	Autosomal recessive	79
Renal dysplasia	CLDN16	Autosomal recessive	80
Scurs, type 2	TWIST1	Autosomal dominant	81
Spherocytosis	SLC4A1	Autosomal incompletely dominant	82
Spinal dysmyelination	SPAST	Autosomal recessive	83
Spinal muscular atrophy	KDSR	Autosomal recessive	84
Syndactyly	LRP4	Autosomal recessive	85, 86
Tail, crooked	MRC2	Autosomal recessive	87
Thrombopathia	RASGRP2	Unknown	88
Trimethylaminuria	FMO3	Autosomal recessive	89
Yellow fat	BCO2	Unknown	90
Trait
Milk yield and composition	GHR	Polygenic	91
	DGAT1	Polygenic	92
	Leptin	Polygenic	93, 94
Meat tenderness	Calpastatin	Polygenic	95, 96
	µ-Calpain	Polygenic	97, 98

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