Sub-populations and evolutionary forces

62 Utilisation and conservation of farm animal genetic resources John Woolliams and Miguel Toro Box 3.3. he attributes and advantages of types of DNA markers. Marker Attributes: he following desirable attributes for a marker type can be identiied. Widely distributed. Wide distribution of markers throughout the genome allows the mapping of the whole genome and tracking of gene low in populations, although maybe with only low resolution. Locally dense. Ability to ind many markers within small genomic regions for the purpose of ine mapping. Ability to localise. he marker can be placed at a physical location in the genome. Highly polymorphic. he utility of a marker depends on its ability to distinguish between segments of homologous chromosomes. his will depend on the frequency of heterozygosity, which increases with the number of alleles at the marker locus and the more equal they are in frequency. he amount of information contained by a marker is oten deined by its information content Lynch and Walsh, 1998. Co-dominant. Ideally, both alleles at a marker locus can be distinguished. For some types of marker the individuals carrying 1 or 2 copies of an allele cannot be distinguished. Low Mutation Rate. For many uses of marker information the long-term stability of the marker over generations is important for inferences about identity by descent IBD. he higher the mutation rate, the less certain the inferences become. It is oten desirable for the marker locus to have a mutation activity that is representative of coding or regulatory sequences. High hroughput. Determined by a number of elements, including the amenability to PCR, which allows more genotypes to be obtained from the same quantity of DNA, ability to automate and multiplex assay procedures, and speed of assay. Low Technical Cost. Expressed per genotype. Repeatable. Assays should be highly repeatable, both between assays within laboratories and between laboratories. Types of Marker Mini-satellites MiniS. A sequence of DNA base pairs, typically of containing 10’s of base pairs, repeated a variable number of times. DNA Fingerprints. Classically a multiple array of mini-satellites. Restriction Fragment Length Polymorphism RFLP. An early bi-allelic marker type based on recognition sites for restriction enzymes. Randomly Ampliied Polymorphic DNA RAPD. Markers formed from an arbitrary set of PCR primers, resulting in a random set of ampliied segments. Micro-satellite MicroS. Based upon sites in which the same short sequence is repeated multiple times. AFLP. AFLPs are a multiple array of RFLPs displayed in a single gel. Single Nucleotide Polymorphism SNP. Point mutation in the genome sequence, pre- dominantly bi-allelic, but feasible to have 4 alleles, with each of the 4 nucleotide bases appearing in the same location. ▷▷▷ • • • • • • • • • • • • • • • • Utilisation and conservation of farm animal genetic resources 63 Chapter 3. What is genetic diversity? is provided by the pedigree Box 3.4. hese options are simply doing what we could do before, but removing some of the limitations. However, the availability of DNA allows us to measure diversity in diferent ways, since we can obtain the nucleotide sequence of individuals in speciic areas of the genome and identify the alleles that are segregating in a population at each position and the genotypes of each individual. Options for addressing diversity with this information include the following: Table 3.1. A general guide to the attributes of diferent marker types. Attribute MiniS Fingerprint RFLP RAPD MicroS AFLP SNP Widely distributed Moderate Moderate Very good Very good Very good Very good Very good Locally dense Poor Poor Moderate Moderate Good Moderate Very good Ability to localise Poor Very poor Very good Poor Very good Moderate Very good Polymorphic Very good Very good Poor Moderate Very good Poor Poor co-dominant Yes No Yes No Yes No 1 Yes Mutation rate Rapid Rapid Reasonable Reasonable Rapid Reasonable Reasonable hroughput Very low Very low Moderate Moderate Moderate Moderate Very high 2 Technical costs Very high Very high Very high Very high Moderate Moderate Very low Repeatable Poor Poor Good Very poor Good Good Very good 1 Inferences on genotypes for AFLPs can be improved using densitometry. 2 As an example, DNA chips now ofer 50,000 SNPs in a single reaction. Current trends in marker choice: SNPs are becoming the marker of choice. heir disadvantage of being pre-dominantly bi-allelic, therefore with lower information content, is being overcome by the number and density of markers available coupled to their high throughput and low cost. Microsatellites retain a use since they can provide considerable information within a few genotypes e.g. as required for pedigree assignment and in some applications this will ofset the relative disadvantage of throughput in comparison to SNPs. 64 Utilisation and conservation of farm animal genetic resources John Woolliams and Miguel Toro 1. Examining the diversity in allele frequency, by deining an allele frequency for an individual as 0, ½ or 1 depending on whether it carries 0, 1 or 2 copies of the allele. his trait can be treated as it was a continuous trait, and diversity measured, both between and within breeds, as described above. he idea of individual allele frequency as a trait is an important one, and the trait has a useful property that all Box 3.4. Pedigree expectations and DNA estimation of shared alleles. When we examine genetic variation using the pedigree we are using expectations, but the use of the DNA genotypes helps to estimate the true situation. Consider two full-sibs, each will receive half their autosomal DNA from their sire, and half from their dam, but they will not receive the same half as their sib, and which allele is passed to each ofspring is an entirely random process; therefore on average the two sibs may be expected to have only half of the genes passed by the sire and half the genes passed by the dam in common, and it is this average that is assumed when using the pedigree. In reality this proportion of genes shared may be much larger or much less. his proportion can be estimated directly when markers are available that are distributed throughout the genomes of the parents, and are informative for each parent, so that they are capable of distinguishing which of the sire’s two homologous alleles were passed to the ofspring and which of the dam’s. More dense and more informative markers make the estimate of true proportions of shared alleles more precise. However a caution: simple estimates of proportion using low density markers may introduce substantial distortion outweighing any potential beneit, and the available observed pedigree remains of primary importance unless very dense informative markers are used. For example, Toro et al. 2002 compared inbreeding of 62 Iberian pigs from two related strains either calculated from a pedigree going back 20 generations or with molecular coancestries estimated from 49 microsatellites. he correlation was negative for Guadyerbas –0.32 and low for Torbiscal 0.19 but substantial for all animals together 0.69. Furthermore, the attempt to infer coancestries from molecular markers gave results severely biased because the inference requires information on the true allelic frequencies of markers in the true base population – and these are usually not known. Slate et al. 2004 examined 590 sheep of the Coopworth breed with known pedigree for seven generations and genotyped 101 microsatellites: again the correlation was remarkably low 0.17 concluding that, for the correlation between the genealogical and the molecular inbreeding to be substantial, a considerable number of loci and, more important, a high variance of the genealogical inbreeding values is required. his does not demonstrate a lack of relationship but demonstrates the noise attached to molecular estimates, for example Daetwyler et al. 2006 shows that in Canadian Holstein the observed log heterozygosity based on 10,000 SNP markers has the expected regression of 1 on log 1-F where F was calculated from pedigree. In conclusion it should be preferable to use pedigree information whenever available, and limiting the use of markers to verify, correct, complete or even implement pedigree recording Fernández et al., 2005.