Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Human Molecular Genetics
Genetic Linkage and Gene Mapping

In 1865, Gregor Mendel, based on his experiments with garden peas, formulated the fundamental principles of inheritance. First, he concluded that hereditary units are discrete, occur in pairs, and can exist in alternative forms. Later (in 1905), these units were named genes, and the variants of a single Gene were called alleles. Second, Mendel discovered that only one gene from each pair enters a germ Cell (gamete). Third, he deduced that gene pairs assort independently of one another, meaning that a single genetically significant cross will yield all possible genetic combinations, provided the number of offspring is sufficiently large (Fig. 20.5). This final Conclusion, although unknown to Mendel at the time, holds true only for gene pairs located on different Chromosomes or, at least, at opposite ends of the same chromosome. None of Mendel's crosses involved gene pairs located close together on the same chromosome. Had they been, he would have noticed that these genes do not assort independently; as we now say, they are linked.

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Fig. 20.5. Independent assortment of genes. When crossing a dihybrid individual (AaBb) with an individual homozygous for two recessive traits (aabb), 50% of the offspring will have parental genotypes (AaBb, aabb), and 50% will display novel genotype combinations (Aabb, aaBb), provided the Sample size of offspring is large enough to be representative. This result indicates that genes A and B assort independently, with offspring exhibiting all possible combinations of each parent's Gametes. It should be noted that the percentage ratio of "parental" to recombinant types depends on the parents' genotypes; for example, crossing individuals with genotypes AABB and aabbs will yield offspring that in 100% of cases possess a genotype different from the parents (AaBb).

In principle, with complete genetic linkage, all genes on any given chromosome should be transmitted to Germ Cells as indivisible blocks, without generating new genetic combinations on the chromosomes during Meiosis (Fig. 20.6). In most cases, however, linkage is incomplete. During meiosis, exchange (recombination, Crossing Over) occurs between gene sites (loci), creating novel gene combinations (Fig. 20.7). Because recombination typically occurs more frequently the greater the distance between two specific gene loci, recombination frequency can be used as a measure of distance (genetic distance) between two genes. Thus, by analyzing recombination frequencies in the offspring of parents heterozygous for a series of linked genes, one can construct a genetic map in which genes are arranged in a linear order. The distance between loci reflects only the recombination frequency and is not equivalent to precise physical distance. However, by comparing physical and genetic chromosome maps, researchers have successfully established a correlation between recombination frequency and the number of DNA nucleotide pairs. The unit used in mapping is 1 centimorgan (cM), a value equal to a 1% recombination frequency, which in humans corresponds to approximately 10⁶ nucleotide pairs (bp).

Fig. 20.6. Complete linkage. The alleles under consideration in the dihybrid parent, AB//ab, are in the coupling phase (cis), while the second parent is homozygous for two recessive traits with the genotype ab//ab. In the absence of recombination between loci A and B, all offspring will have parental genotypes: half with the genotype AB//ab and half with ab//ab. Complete linkage does not always imply the absence of new genetic combinations; for example, all offspring from the cross Ab//aB × AB//ab will possess novel genetic combinations, specifically: Ab//AB, Ab//ab, aB//AB, and aB//ab. However, in the absence of recombination, genes on the same chromosome will always remain linked. For convenience, genetic nomenclature uses a single horizontal or slash mark instead of two to denote the linkage of loci in a pair of identical (homologous) chromosomes.

Fig. 20.7. Incomplete linkage. In this example, 20% (i.e., 0.1 + 0.1 = 0.2) of the offspring have genotypes formed As a result of recombination(s) between loci A and B during meiosis. Recombination frequency is independent of parental genotypes. A parent homozygous for two recessive traits produces only one type of gamete, even if recombination occurs. In a testcross, recombinant products of meiosis manifest phenotypically in the offspring.

Several important points concerning genetic linkage and gene mapping should be noted. First, to estimate the frequency of new genetic combinations (recombinants), one of the parents must be heterozygous at least at two loci (AB/ab or Ab/aB). Second, dihybrid genotypes can exist in two configurations (phases). If two linked genes on each chromosome are represented by the same type of alleles (i.e., both dominant, AB, or both recessive, ab), this configuration is termed the coupling phase (cis-phase). Conversely, if two linked genes on each chromosome are represented by Different types of alleles (i.e., one dominant and the other recessive, aB or Ab), the configuration is termed the repulsion phase (trans-phase). Third, recombination between two genes occurs independently of their phase. Genetically, recombination between genes in a homozygous state (e.g., Ab/Ab or AB/AB) does not result in a new genetic combination and, therefore, even if such recombination occurs, it remains undetectable. Fourth, a recombination frequency of 0% indicates complete linkage, whereas 50% indicates that the genes are either on different chromosomes or located too far apart on the same chromosome to detect linkage. To solve the mapping problem for two widely separated genes on the same chromosome, it is necessary to map intervening genes, which helps determine whether they all belong to a single linkage group.

To construct detailed Genetic Maps of certain eukaryotic organisms, such as mice, maize, fruit flies, nematodes, and Yeasts, it is necessary to identify a series of genes, each represented by at least two alleles. Next, crosses must be performed and recombination frequencies counted in A large number of offspring. The results reflect the degree of linkage between the genes. Ultimately, by utilizing multifactorial crosses (involving more than two pairs of linked genes), detailed genetic maps can be obtained.