MICROSATELLITE LOCI BASED ON THE CHICKEN GENOME
4.1 INTRODUCTION
Microsatellite mapping, comprising linkage maps and predicted maps, is the process of assembling genetic markers in the correct order and position on a chromosome. Linkage maps use linkage statistics such as recombination fractions between pairs of loci to assign each locus to a linkage group. Linkage maps have been constructed for a number of species, with efforts focussed mainly on biological models, as well as biomedically and economically important species such as humans, sheep(Ovis aries), pigs (Sus scrofay and the red jungle fowl (Gal/us gal/us, hereafter referred to as chicken). Ultimately this tool allowed for the study of gene interactions involved in human diseases, as well as quantitative traits of agricultural interest such as body mass and egg production (Burt et al.
1995).Two other species for which linkage maps have been constructed are the great reed warbler (Acrocephalus arundinaceus) (Hanssonet at. 2005) and Japanese quail (Coturnix japonica) (Kayang et al.2004), both important model organisms in biological research.
In contrast to linkage maps, predicted maps use available sequences from an unrnapped species and assign the location of their orthologous sequences in a species for which a map already exists. To date only one published predicted microsatellite map for an order within the class Aves exists: passerine microsatellite map constructed by Dawson et at. (2006).
Until recently, the single limiting factor was the absence of an avian species with an entirely sequenced genome. This made it difficult, if not impossible, to predict the location of sequenced loci within a genome by comparing sequences to those having known chromosomal locations. However, in 2004 the International Chicken Genome Sequencing Consortium completed sequencing a large proportion of the chicken genome (International Chicken Genome Sequencing Consortium 2004), and the sequences made publicly available on the Ensembl sequence database (Birneyet at. 2006).
Although in its infancy, the applications of predictive mapping are the study of quantitative trait loci, understanding karyotype evolution, genome mapping, and the identification of . independent set of microsatellite loci (Dawson et al. 2006). The latter application is extremely useful when selecting loci to be used in parentage testing, where loci must be independently assorted (Jones et al.2003). This was the motivation behind the construction of a predicted microsatellite map for a non-passerine genus;Grus to which the blue crane (G. paradisea) belongs. Creating a map based on the sequence similarity between chicken and Grus species, when proven accurate, would allow for the identification of an independent set of markers to be selected for parentage testing not only in the blue crane, but potentially other species within this genus.
4.1.1 Can predictive mapping be successful?
Genome evolution represented by chromosomal rearrangements occurs over time causing the likelihood of conserved synteny to decrease as a function of genetic distance (Shetty et al. 1999). Consequently, more distantly related species have greater karyotypic differences, as well as differences within the arrangement of genes located on a chromosome.
Therefore, genes that are linked in one species may not necessarily be linked in another species which could lead to erroneous conclusions when analysing results obtained from predictive studies. Inorder for an assessment of the likelihood of predictive microsatellite mapping to be an accurate predictor of locus position within a species, the evolutionary distance between the species/taxonomic group being mapped (cranes for example) and the species on which the map is being based (chicken) must therefore be determined. This will allow for the degree of synteny between the two species to be determined.
One method, fluorescent in situ hybridisation (FISH), has been used to study karyotype evolution (genetic distance) in birds (Derjusheva et al. 2004; Shetty et al. 1999). This technique uses DNA probes from one species, specific for a particular chromosome, and applies this probe to another species to determine the conservation of chromosomal syntenies (the preserved order of genes on a chromosome). Results have shown high homology between two distantly related birds: chicken and emu (Shetty et al. 1999).
Furthermore, a high conservation of syntenies was revealed between the chicken, pigeon
and passerine birds (Derjushevaet al. 2004), as well as between chicken and zebra finch (Itoh et al. 2005). Results taken from these analyses support the idea that predictive microsatellite mapping between chicken and other avian species may be a true reflection of microsatellite locations within the genome of the focal species because of the conservation of avian karyotypes over 80 million years of evolution (Shettyet al. 1999).
A second method of determining genetic similarity employs linkage map comparisons between distantly related species.Although few avian linkage maps have been constructed, comparisons between a chicken and human linkage map indicated that levels of conserved synteny between these two species appears to be very high (Groenen et al. 2000). If this result is extrapolated for genomic evolution in birds, chromosomal synteny between chicken and other avian species is expected to be high because of the smaller genetic distance between avian taxa than chicken and humans (Sibley et al. 1990). Ultimately, linkage map comparisons, when available for more avian species, could be examined to identify the level of agreement between linkage and predicted microsatellite maps, thereby determining the accuracy of the predicted map in assigning loci to the correct chromosomal location. Lastly, cytogenetic analysis of a range of passerine families has shown that passerines (2n = 72-84) have a similar karyotype to chicken (2n = 78) (Dawson et al.
2006). Therefore,loci identified as being linked based on predictive mapping will likely be a true reflection of their status in passerines.
The support of three methods indicating strong homologies between avian taxa as well as the similarity between passerine and chicken karyotypes, suggested that the likelihood for predictive mapping to assign passerine microsatellite loci to the correct chromosomal locations could be high. This provided the motivation for Dawsonet al. (2006) to construct the predictive micro satellite map of the passerine genome based on chicken-passerine sequence similarity. Although cranes are non-passerines, the predicted passerine map is discussed here due to its relevance in providing the basis for the construction of the predictedGrusmicro satellite map.
4.1.2 The predicted passerine map
Passerine species represent a family of birds that are separated by short genetic distances (Sibley et al. 1990). Importantly, the likelihood of successful cross-species amplification increases with decreasing genetic distance, supported by high cross-species utility of passerine microsatellite markers in other passerine species (Primmer et al. 1996). With more than 500 passerine microsatellite sequences available (Dawson et al. 2006), a large selection of loci are available for genetic studies on a passerine species. A requirement in utilising microsatellite loci in population genetic and parentage analyses is the identification and exclusion of linked loci. This can be achieved by selecting loci situated on different chromosomes for use in a set of microsatellite markers.
To identify the location of microsatellite markers within the passerine genome, a predicted microsatellite map was created using the chicken genome as a template. Reasons for using the chicken genome sequence database are highlighted below: (a) no passerine species has had its entire genome sequenced; (b) conserved synteny between chicken and passerine species appears high, with a strong possibility of correct placement of microsatellite loci on passerine chromosomes based on chicken genome sequences.
To test the correct prediction of chromosomal locations of microsatellite loci in passerines, a comparison was made by Dawson et al.(2006) between the predicted passerine map and a linkage map developed by Hansson et
at.
(2005) for the great reed warbler, a passerine.Importantly, these two maps were constructed independently of each other using different methodologies. From this comparison, synteny was shown to be conserved between the predicted passerine map and the great reed warbler map (Dawson et al. 2006), confirming the ability of the passerine-chicken map to correctly predict chromosomal locations of passerine sequences, as well as to identify linkage of passerine microsatellite loci.
4.1.3 Predicted Grus map
The order Gruiformes (non-passerine), to which the blue crane belongs, and Passeriformes (passerines) are separated by a relatively small genetic distance (delta T50H DNA-DNA
hybridisation distance of 20.8, Sibley et al. 1990). As predictive mapping of the passerine genome was shown to be accurate based on chicken-passerine sequence similarity, a predictive map constructed for the Grus genus within Gruiformes would be expected to show a similar level of accuracy in its ability to correctly assign chromosomal locations to microsatellite markers and ultimately indicate the presence of potentially linked loci. In addition, sequences conserved between blue crane and chicken would suggest an increased likelihood of successful cross-species amplification between this blue crane and other more distantly related avian species.
However, one possible shortfall in the Grus map was recognised at the start of this study:
crane and chicken karyotypes differ quite substantially: whooping cranes, 2n = 62 (Blederman et al. 1982); chicken, 2n= 78 (Burtet al. 1995).Itmay therefore be possible that synteny between these two species may not be as conserved as between chicken and passerines. However, Dawson et at. (2006) were able to test the accuracy of the predicted passerine map by comparing it to a linkage map created for a passerine species. The predicted map of theGrus genome will therefore only be able to be proven accurate once a linkage map for this genus has been completed. However, analyses of linkage disequilibrium between Grus microsatellite loci examined in this study (Chapter 5) revealed neither support nor rejection of the predicted Grus map, since chromosomal locations of at least one of the two loci in a linked pair could not be mapped.