Linkage analysis can only be performed on loci that are polymorphic 53 with two or more distinguishable alleles. Naturally occurring polymorphic loci with clear single-gene effects are rarely observed in wild animal populations. In the laboratory, however, it is possible to identify and breed animals with mutations at many different loci (see Chapter 6). Over the last 90 years, thousands of independent mouse mutations have been characterized in various laboratories. In fact, as discussed previously, a primary reason for the initial choice of the mouse as an experimental genetic system was the collection of rare genetic variants present in the hands of the fancy mouse breeders (see Chapter 1). However, even this variation is restricted in its scope and usefulness for geneticists. This is because of the severe limitation in the number of phenotypic markers that can be incorporated into any one cross. Although over 50 independent loci have been characterized with effects on coat color (Silvers, 1979), it is impossible to follow more than a handful at any one time since mutant alleles at any one locus will act to obscure the expression of mutant alleles at other loci. With mutant alleles that affect viability in some way, the problem of sorting out overlapping phenotypes becomes even more severe.
Prerecombinant DNA geneticists were able to circumvent these problems by performing large numbers of different crosses, each of which tested overlapping subsets of phenotypic markers. Thus, as illustrated in Figure 8.1, the loci A, B, and C were mapped in one cross; C, D, and E were mapped in a second cross, and A, D, and F were mapped in a third cross. If linkage was observed in each of these individual crosses among the three loci mapped therein as shown, it was then possible to develop a linkage map that encompassed all six loci even though, for example, the B locus was never mapped directly relative to D, E or F; and A was never mapped relative to E. By extension, it is possible to combine data obtained in hundreds of crosses to map hundreds of phenotypically defined loci to form linkage maps that extend across all nineteen mouse autosomes and the X chromosome.
Mapping in the prerecombinant DNA era was tedious and was generally performed by investigators dedicated to this task alone. However, with the results of the first generation of cloning and sequencing studies, the scientific community became aware of the existence of a hidden level of enormous genetic variation that occurs naturally in all mammalian populations (Botstein et al., 1980). The frequency of DNA variation that exists between chromosome homologs from two unrelated individuals of the same species (including mice and humans) appears to be on the order of one nucleotide substitution or small length change in every 200-500 bp. Since the mammalian genome has a size of 3x109 bp, this frequency implies a total number of genetic differences between any two unrelated individuals on the order of six million per haploid genome set. In a comparison of individuals from separate species, such as M. musculus and M. spretus, the level of variation will be even higher.
In the prerecombinant DNA era, alleles could only be distinguished in terms of an altered phenotype; thus only genes could have alleles, and the demonstration of a genetic locus was dependent on the expression of alternative phenotypes. Today, every variant nucleotide in the genome is a potential locus. To say that DNA variation provides a larger reservoir for use in genetic studies than phenotypic variation is a vast understatement. Furthermore, and of most importance, there is essentially no limit to the number of these loci that can be mapped simultaneously within a single cross.
All simple forms of DNA variation fall into three classes: (1) base pair substitutions; (2) short regions of deletion or tandem duplication; and (3) insertions or translocations. Examples from each of these classes can be detected as restriction fragment length polymorphisms (RFLPs) and/or by PCR-based protocols. These major tools for DNA allele detection will be discussed separately in the following two sections of this chapter ( Section 8.2 and Section 8.3).