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Modern Genetic Analysis Chapter 17. Population and Evolutionary Genetics
Variation and its Modulation

Population genetics is the translation of Darwin's three principles into precise genetic terms. As such, it deals with the description of genetic variation in populations and with the experimental and theoretical determination of how that variation changes in time and space.

MESSAGE
Population genetics is the study of inherited variation and its modulation in time and space.

Observations of Variation

Population genetics necessarily deals with genotypic variation, but, by definition, only phenotypic variation can be observed. The relation between phenotype and genotype varies in simplicity from character to character. At one extreme, the phenotype may be the observed DNA sequence of a stretch of the genome. In this case, the distinction between genotype and phenotype disappears, and we can say that we are, in fact, directly observing the genotype. At the other extreme lie the bulk of characters of interest to plant and animal breeders and to most evolutioniststhe variations in yield, growth rate, body shape, metabolic ratio, and behavior that constitute the obvious differences between varieties and species. These characters have a very complex relation to genotype, and we must use the methods introduced in Chapter 18 to say anything at all about the genotypes. But, as we shall see in Chapter 18, it is not possible to make very precise statements about the genotypic variation underlying quantitative characters. For that reason, most of the study of experimental population genetics has concentrated on characters with simple relations to the genotype, much like the characters studied by Mendel. A favorite object of study for human population geneticists, for example, has been the various human blood groups. The qualitatively distinct phenotypes of a given blood groupsay, the MN groupare encoded by alternative alleles at a single locus, and the phenotypes are insensitive to environmental variations.

The study of variation, then, consists of two stages. The first is a description of the phenotypic variation. The second is a translation of these phenotypes into genetic terms and the redescription of the variation genetically. If there is a perfect one-to-one correspondence between genotype and phenotype, then these two steps merge into one, as in the MN blood group. If the relation is more complexfor example, as the result of dominance, heterozygotes resemble homozygotesit may be necessary to carry out experimental crosses or to observe pedigrees to translate phenotypes into genotypes. This is the case for the human ABO blood group (see page 170).

The simplest description of Mendelian variation is the frequency distribution of genotypes in a population. Table 17-1 shows the frequency distribution of the three genotypes at the MN blood group locus in several human populations. Note that there is variation between individuals in each population, because there are different genotypes present, and there is variation in the frequencies of these genotypes from population to population. More typically, instead of the frequencies of the diploid genotypes, the frequencies of the alternative alleles are used. The frequency of an allele is simply the proportion of that allelic form of the gene among all copies of the gene in the population. There are twice as many gene copies in the population as there are individuals, because every individual is diploid and homozygotes for an allele have two copies of that allele, whereas heterozygotes have only one copy. So we calculate the frequency of an allele by counting homozygotes and adding half the heterozygotes. Thus, if the frequency of A / A individuals were, say, 0.36 and the frequency of A / a individuals were 0.48, the allele frequency of A would be 0.36 + 0.48/2 = 0.60. Genetics in Process 17-1 gives the general form of this calculation.

Table 17-1 shows the values of p and q for each of the MN blood group populations.

A measure of genetic variation (in contrast with its description by gene frequencies) is the amount of heterozygosity at a locus in a population, which is given by the total frequency of heterozygotes at a locus. If one allele is in very high frequency and all others are near zero, then there will be very little heterozygosity because, by necessity, most individuals will be homozygous for the common allele. We expect heterozygosity to be greatest when there are many alleles at a locus, all at equal frequency. In Table 17-1, the heterozygosity is simply equal to the frequency of the M / N genotype in each population. When more than one locus is considered, there are two possible ways of calculating heterozygosity. First, we can average the frequency of heterozygotes at each locus separately. Alternatively, we can consider each haplotype as a unit as in Table 17-2 and calculate the proportion of all individuals who carry two different haplotypic or gametic forms. This form of heterozygosity is also referred to as haplotype diversity or gametic diversity. The results of both calculations are given in Table 17-2. Note that the haplotype diversity is always greater than the average heterozygosity of the separate loci because an individual is a haplotypic heterozygote if either of its loci is heterozygous. (See the discussion of Hardy-Weinberg equilibrium in Genetics in Process 17-2 on page 545 for the calculation of heterozygosity.)

Simple Mendelian variation can be observed within and between populations of any species at various levels of phenotype, from external morphology down to the amino acid sequence of enzymes and other proteins. Indeed, with the new methods of DNA sequencing, variations in DNA sequence (such as third-position variants that are not differentially coded in amino acid sequences and even variations in nontranslated intervening sequences) have been observed. Every species of organism ever examined has revealed considerable genetic variation, or polymorphism, reflected at one or more levels of phenotype, within populations, between populations, or both. A gene or a phenotypic trait is said to be polymorphic if there is more than one form of the gene or trait in a population. Genetic variation that might be the basis for evolutionary change is ubiquitous. The tasks for population geneticists are to describe that ubiquitous variation quantitatively and to build a theory of evolutionary change that can use these observations in prediction.

There is morphological variation in size and shape in every species, but most of it is the result of the interaction of environmental variation and genetic variation that is difficult to ascribe to separable gene loci (see Chapter 18). Some examples, however, such as certain flower colors in plants, butterfly wing patterns, or shell color and banding patterns in snails, have been shown to result from the segregation of alleles at single Mendelian loci.

It is impossible in this text to provide an adequate picture of the immense richness of genetic variation that exists in species. We can consider only a few examples of the different kinds of Mendelian variation to gain a sense of the genetic diversity within species. Each of these examples can be multiplied many times over in other species and with other traits.

Chromosomal polymorphism.

Although the karyotype is often regarded as a distinctive characteristic of a species, in fact, numerous species are polymorphic for chromosome number and morphology. Extra chromosomes (supernumeraries), reciprocal translocations, and inversions segregate in many populations of plants, insects, and even mammals. Table 17-3 gives the frequency of supernumerary chromosomes and translocation heterozygotes in a population of the plant Clarkia elegans from California. The "typical" species karyotype would be hard to identify.

Immunological polymorphism.

A number of loci in vertebrates code for antigenic specificities such as the ABO blood types. More than 40 different specificities on human red cells are known, and several hundred are known in cattle. Another major polymorphism in humans is the HLA system of cellular antigens, which are implicated in tissue graft compatibility (Chapter 16). Table 17-4 gives the allelic frequencies for the ABO blood group locus in some very different human populations. The polymorphism for the HLA system is vastly greater. There appear to be two main loci, each with five distinguishable alleles. Thus, there are 52 = 25 different possible gametic types, making 25 different homozygous forms and (25)(24)/2 = 300 different heterozygotes. All genotypes are not phenotypically distinguishable, however; so only 121 phenotypic classes can be seen. L. L. Cavalli-Sforza and W. F. Bodmer report that, in a sample of only 100 Europeans, 53 of the 121 possible phenotypes were actually observed.

Protein polymorphism.

In recent years, studies of genetic polymorphism have been carried down to the level of the polypeptides coded by the structural genes themselves. If there is a nonredundant codon change in a structural gene (say, GGU to GAU), this change will result in an amino acid substitution in the polypeptide produced at translation (in this case, glycine to aspartic acid). If a specific protein could be purified and sequenced from separate individuals, then it would be possible to detect genetic variation in a population at this level. In practice, this is tedious for large organisms and impossible for small ones unless a large mass of protein can be produced from a homozygous line.

There is, however, a practical substitute for sequencing that makes use of the change in the physical properties of a protein when an amino acid is substituted. Five amino acids (glutamic acid, aspartic acid, arginine, lysine, and histidine) have ionizable side chains that give a protein a characteristic net charge, depending on the pH of the surrounding medium. Amino acid substitutions may directly replace one of these charged amino acids, or a noncharged substitution near one of them in the polypeptide chain may affect the degree of ionization of the charged amino acid, or a substitution at the joining between two a helices may cause a slight shift in the three-dimensional packing of the folded polypeptide. In all these cases, the net charge on the polypeptide will be altered because the net charge on a protein is not simply the sum of all the individual charges on its amino acids but is dependent on their exposure to the liquid medium surrounding them.

To detect the change in net charge, protein can be subjected to the method of gel electrophoresis. Figure 17-1 shows the outcome of such an electrophoretic separation of variants of an esterase enzyme in Drosophila pseudoobscura, where each track is the protein from a different individual. Table 17-5 shows the frequencies of different alleles for three enzyme-coding loci in D. pseudoobscura in several populations: a nearly monomorphic locus (malic dehydrogenase), a moderately polymorphic locus ( a -amylase), and a highly polymorphic locus (xanthine dehydrogenase).

The technique of gel electrophoresis (or sequencing) differs fundamentally from other methods of genetic analysis in allowing the study of loci that are not segregating, because the presence of a polypeptide is prima facie evidence of a structural genethat is, a DNA sequence coding for a protein. Thus, it has been possible to ask what proportion of all structural genes in the genome of a species is polymorphic and what the average heterozygosity is in a population. Very large numbers of species have been sampled by this method, including bacteria, fungi, higher plants, vertebrates, and invertebrates. The results are remarkably consistent over species. About one-third of structural-gene loci are polymorphic, and the average heterozygosity in a population over all loci sampled is about 10 percent. This means that scanning the genome of an individual would show that about 1 in every 10 loci is in heterozygous condition and that about one-third of all loci have two or more alleles segregating in any population. This represents an immense potential of variation for evolution. The disadvantage of the electrophoretic technique is that it detects variation only in structural genes. If most of the evolution of shape, physiology, and behavior rests on changes in regulatory genetic elements, then the observed variation in structural genes would be beside the point.

DNA Sequence Polymorphism

DNA analysis makes it possible to examine variation among individuals and between species in their DNA sequences. There are two levels at which such studies can be done. Studying variation in the sites recognized by restriction enzymes provides a coarse view of base-pair variation. At a finer level, methods of DNA sequencing allow variation to be observed base pair by base pair.

Restriction-site variation.

A restriction enzyme that recognizes six-base sequences (a "six-cutter") will recognize an appropriate sequence approximately once every 46 = 4096base pairs along a DNA molecule [determined from the probability that a specific base (of which there are four) will be found at each of the six positions]. If there is polymorphism in the population for one of the six bases at the recognition site, then there will be a restriction length polymorphism (RFLP) in the population, because in one variant the enzyme will recognize and cut the DNA, whereas in the other variant it will not (see pages 344-346). A panel of, say, eight enzymes will then sample every 4096/8 500 base pairs for such polymorphisms. However, when one is found, we do not which of the six base pairs at the recognition site is polymorphic.

If we use enzymes that recognize four-base sequences ("four-cutters"), there is a recognition site every 44 = 256 base pairs, so a panel of eight different enzymes can sample about once every 32 base pairs along the enzyme. In addition to single base-pair changes that destroy restriction-enzyme recognition sites, insertions and deletions of stretches of DNA also cause restriction fragment lengths to vary.

Twenty studies of different regions of the X chromosome and the two large autosomes of Drosophila melanogaster by restriction enzymes have found between 0.1 and 1.0 percent heterozygosity per nucleotide site, with an average of 0.4 percent. A study of the very small fourth chromosome, however, found no polymorphism at all.

Complete sequence variation.

Studies of variation at the level of single base pairs by DNA sequencing can provide information of two kinds. First, by translating the sequences of coding regions obtained from different individuals in a population or from different species, the exact amino acid sequence differences can be determined. Electrophoretic studies show only that there is variation in amino acid sequences but cannot identify how many or which amino acids differ between individuals. So, when DNA sequences were obtained for the various electrophoretic variants of esterase-5 in D. pseudoobscura (see Figure 17-1), it was found that electrophoretic classes differ from each other by an average of 8 amino acids, and the 20 different kinds of amino acids were involved in polymorphisms at about the frequency at which they were represented in the protein. Such studies also show that different regions of the same protein have different amounts of polymorphism. For the esterase-5 protein, consisting of 545 amino acids, 7 percent of amino acid positions are polymorphic, but the last amino acids at the carboxyl terminus of the protein are totally invariant between individuals, probably because of functional constraints on these amino acids.

Second, DNA base-pair variation can also be studied for those base pairs that do not determine or change the protein sequence. This includes DNA in introns, in 5-flanking sequences that may be regulatory, in nontranscribed DNA 3 to the gene, and in those nucleotide positions within codons (usually third positions) whose variation does not result in amino acid substitutions. Within coding sequences, these so-called silent, or synonymous, base-pair polymorphisms are much more common than changes that result in amino acid polymorphism, presumably because many amino acid changes interfere with normal function of the protein and are eliminated by natural selection. An examination of the codon translation table (Figure 318) shows that approximately 25 percent of all random base-pair changes will be synonymous, giving an alternative codon for the same amino acid, whereas 75 percent of random changes will change the amino acid coded. For example, a change from AAT to AAC still codes for asparagine, but a change to ATT, ACT, AAA, AAG, AGT, TAT, CAT, or GATall single-base-pair changes from AATchanges the amino acid coded. So, if mutations of base pairs were random and if the substitution of an amino acid made no difference to function, we would expect a 3:1 ratio of amino acid replacement to silent polymorphisms. The actual ratios found in Drosophila vary from 2:1 to 1:10. Clearly, there is a great excess of synonymous polymorphism, showing that most amino acid changes are subject to natural selection. It should not be supposed, however, that silent sites in coding sequences are entirely free from constraints. Different alternative triplet codings for the same amino acid may differ in speed and accuracy of transcription, and the mRNA corresponding to different alternative triplets may have different accuracy and speed of translation because of limitations on the pool of tRNAs available. Evidence for this latter effect is that alternative synonymous triplets for an amino acid are not used equally, and the inequality of use is much more pronounced for genes that are transcribed at a very high rate.

There are also constraints on 5 and 3 noncoding sequences and on intron sequences. Both 5 and 3 noncoding DNA contains signals for transcription, and introns may contain enhancers of transcription (see Chapter 14).

MESSAGE
Within species, there is great genetic variation. This variation is manifest at the morphological level of chromosome form and number and at the level of DNA segments that may have no observable developmental effects.

Variation within and between Populations

The various examples just given show that there are genetic differences between individuals within a population and that the allelic frequencies differ between populations. The relative amounts of variation within and between populations vary from species to species, depending on history and environment. In humans, some gene frequencies (for example, those for skin color or hair form) obviously are well differentiated between populations and major geographical groups (so-called geographical races). If, however, we look at single structural genes identified immunologically or by electrophoresis rather than these outward phenotypic characters, the situation is rather different. Table 17-6 shows the three loci for which Caucasians, Negroids, and Mongoloids are known to be most different from one another (Duffy and Rhesus blood groups and the P antigen) compared with the three polymorphic loci for which the races are most similar (Auberger blood group and Xg and secretor factors). Even for the most divergent loci, no race is homozygous for one allele that is absent in the other two races.

In general, different human populations show rather similar frequencies for polymorphic genes. Figure 17-2 is a triallelic diagram for the three main allelic classes, IA, IB, and i, of the ABO blood group. Each point represents the allelic composition of a population, where the three allelic frequencies can be read by taking the lengths of the perpendiculars from each side to the point. The diagram shows that all human populations are bunched together in the region of high i, intermediate IA, and low IB frequencies. Moreover, neighboring points (enclosed by dashed lines) do not correspond to geographical races, so such races cannot be distinguished from one another by characteristic allelic frequencies for this gene. The study of polymorphic blood groups and enzyme loci in a variety of human populations has shown that about 85 percent of total human genetic diversity is found within local populations, about 8 percent is found among local populations within major geographical races, and the remaining 7 percent is found among the major geographical races. Clearly, the genes influencing skin color, hair form, and facial form that are well differentiated among races are not a random sample of structural-gene loci.

MESSAGE
In general, the genetic variation between individuals within human races is much greater than the average variation between races.

Quantitative Variation

Not all variation in traits can be described in terms of allelic frequencies, because many characteristics, such as height, vary continuously over a range rather than falling into a few qualitatively distinct classes. There is no allele for being 58 or 54 tall. Such characters, if they are varying as a consequence of genetic variation, will be influenced by several or many genes and by environmental variation as well. Special techniques are needed for the study of such quantitative traits, and Chapter 18 deals with those techniques.

© 1999 by W. H. Freeman and Company. All rights reserved.