MICROSATELLITE LOCI BASED ON THE CHICKEN GENOME
CHAPTER 5 CHARACTERISATION OF GRUS MICROSATELLITE LOCI
5.1 INTRODUCTION
5.1.2 General locus characteristics
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conditionsThese conditions refer to the optimised MgCh concentrations, annealing temperature (Ta) used toamplify each pair of primers, number of annealing cycles,and the duration time for extension. Optimising these conditions to will allow for easy replication of PCR reactions throughout this study. Optimisation is essential to obtain high quality PCR products for accurate allele scoring during genotyping to minimise the number of genotyping errors known to affect relationship studies such as parentage analysis (Hoffmanet al. 2005).
Hardy- Weinberg equilibrium
The ability of a locus to conform to Hardy-Weinberg equilibrium (HWE) is tested by determining the level of agreement between observed genotype frequencies and frequencies expected for a population experiencing random mating, no mutation, no drift and no migration (Selkoeet al. 2006). Deviations from HWE may be in the form of heterozygosity excess or heterozygosity deficit. A number of factors may cause deviations from HWE such as selection acting on certain alleles, inbreeding, null alleles (see below), biases towards typing particular genotypes and the presence of a sex-linked locus (Marshall et al.
1998; Selkoeet al. 2006). In addition, large allele drop out is known to cause an observed excess of homozygotes (Selkoe et al. 2006) and ultimately deviations from HWE. Large allele drop-out is observed when smaller PCR fragments are preferentially amplified rather than larger fragments, causing the larger fragments to occur in a lower concentration than the smaller fragments. If the concentration is too low, the result may be the failure of the allele to be detected after PCR. Consequently, the true heterozygous individual may appear to be homozygous at that locus.
Failure of loci to meet HWE is not a characteristic on which to exclude loci from a set of markers for genetic studies, but it does provide valuable information suggesting the need for further checks as to the suitability of the locus for parentage and population studies.
Loci showing significant deviations from HWE should be checked for linkage disequilibrium, presence of null alleles or whether that locus is sex-linked. Loci exhibiting these factors should be excluded from a set of markers to be used in parentage testing as their ability to cause mis-assignment of parentage have been previously identified (Hoffmanet al. 2005;Joneset al. 2003).
Despite deviations from HWE acting as a red flag for loci to be examined further should they used in parentage studies, loci not in HWE can have a positive role to play in populations studies. However, this is only true if the loci showing significant HWE deviations are not as a result of null-alleles or large-allele drop-outs.For example, recent population bottlenecks can be identified by a significant deficiency of heterozygous individuals (Jehle et al. 2002). This is because a heterozygosity deficit can be caused by
inbreeding (Fernandezet al. 2004) as a consequence of a small founder population after the bottleneck. Heterozygosity deficits however may also be caused by inadvertently analysing allele frequencies from two or more genetically distinct groups (Selkoe et al. 2006),
suggesting population structure beyond that which is currentlyrecognised.
Since the level of heterozygosity is also a measure of the population's ability to adapt to different environmental pressures (Amos et al. 2001), the ability to identify populations having a significant deficit of heterozygous individuals can help conservation management strategies in severe cases to increase genetic diversity within the population, such as that seen for the genetic management of captive whooping crane populations (Jones et al.
2002).
Null alleles
A null allele is any allele that is not detected by the assay used to genotype individuals at a particular locus (Marshall et al. 1998). When using microsatellite loci, null alleles arise most commonly when a mutation event has occurred at one or both of the primer binding sites preventing amplification of the allele.·As a consequence, true heterozygotes may appear as a homozygote when genotyped, or true homozygotes may fail to produce any peRproduct. The result is a lower observed heterozygosity estimate than what is expected.
For genetic analyses such as parentage analysis that require low genotyping errors, loci exhibiting a high frequency of null alleles can confound results, and must therefore be identified prior to their use in parentage analyses. The presence of null alleles can be identified either by a significant deficit of heterozygotes, or by the genotypic incompatibility of individuals known to be related. The characterisation process would therefore allow for those loci with a high frequency of null alleles to be identified and assessed for accuracy in parentage analysis using known families.
Mode ofinheritance
Determining the mode of inheritance is necessary during the characterisation process.
Microsatellite markers used for parentage testing and most population genetic analyses can
only be used if the loci which they amplify are inherited in a Mendelian fashion. This pattern of inheritance defines the manner in which the alleles of a locus are passed on from parent to offspring, such that one allele present in an offspring is maternally inherited and the other paternally. Therefore, when many loci are analysed between the offspring and both parental candidates, an adult and an offspring can be identified as being related or unrelated by the presence or absence of shared alleles.
A survey by Selkoe et al. (2006) found an average of one locus out of 15 violated Mendelian inheritance. Therefore, the likelihood of obtaining such loci in this study was high due to the high number of loci tested (n = 28). Loci exhibiting non-Mendelian inheritance may be due to the presence of null alleles, since null alleles have been identified as causing incompatibilities between parent offspring genotypes (Selkoe et al. 2006). In addition, genotyping errors could result in allele mis-scoring which would have the same effect on parentage analyses as null alleles.
Linkage disequilibrium
The calculation of linkage disequilibrium between a pair of loci provides a statistical method of identifying linked loci. If two alleles at different loci were in some way associated, e.g. if situated close together on a chromosome such that they were inherited together, the result would be a pair of loci essentially acting as a single genetic unit.
Consequently, the outcome of many genetic analyses, including parentage analyses would be compromised by the non-independent relationship between some of the loci involved (Falush et al.2003; Jones et al. 2003). Ultimately this would lead to an increase in Type I errors. Therefore, one locus out of a pair displaying linkage disequilibrium is required to be excluded from the set of markers to be used in cases involving for example parentage testing.
Many statistical tests such as those involved in identifying linkage equilibrium involve multiple comparisons. However, analysing the raw data has revealed that the marking of component tests as statistically significant based on their single-test significance values was inappropriate as it yields too many significant results (Rice 1989). Without a correction for
multiple comparisons, a set of P-values would contain an over-estimated number of significant results. In this study,a Bonferroni correction (Rice 1989) was used during the identification of linked pairs of loci. Performing this correction controls for probability of incorrectly rejecting one or more null hypotheses (Ho)while simultaneously maintaining the ability to accurately detect any results rejecting
Ho
corrected for multiple comparisons (Rice 1989).Chromosomal location ofgenetic markers
Loci lying in close physical proximity on a chromosome may be genetically linked.
Genetically linked loci are non-randomly associated and should be avoided during parentage analysis (where markers used are assumed to be randomly associated). For this reason a predicted microsatellite map was constructed to aid the selection of an independent set of markers (Chapter 4), and the results compared to those obtained from linkage disequilibrium analyses to determine the level of agreement between these two methods of determining whether association between pairs of loci exist.
Parentage
Parentage exclusion is the rejection of parental candidates based on parent-offspring incompatibilities found in the genotypic data (Joneset al.2003). Following an example by Jones et al. (2003), if a mother and offspring have the diploid genotypes AlA and AlB, respectively, at a single Mendelian-inherited locus,then males with the genotypes AlC can be excluded whereas those with B/C cannot. The use of highly polymorphic loci, such as microsatellites, provides high powers of exclusion due to the low frequency with which the alleles occur in a population. Therefore, the probability of successful exclusion would therefore increase with an increase in the number of polymorphic loci used during parentage analysis.
In this study, the probability of excluding a single randomly-chosen unrelated individual from parentage was calculated using the software program Cervus 2.0 (Marshall et al.
1998). This program calculates two exclusion powers: the first parent exclusionary power
calculates the combined power of the set of loci to exclude a randomly-selected unrelated candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring; second-parent exclusionary power is the combined power of the set of loci to exclude a randomly-selected unrelated candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and of a known parent of the opposite sex.
Values of exclusionary power are given as a proportion of 1.00, where 0.00 indicates no exclusionary power and 1.00 indicates complete exclusion (Marshallet al. 1998). The aim of this study was to obtain a set of microsatellite markers that achieve both first and second parent exclusionary powers close to 1.00 so that reliable parentage testing can be performed in the blue crane