Characterization of variability and population structure

K K Vinod

The state of variability within and between populations can be determined by application of both biometrical and molecular procedures. Whilst the former can describe a population in terms of means and variances and the underlying mode of gene action, if an appropriate biometrical design is applied as part of the characterization of a population (Kearsey, 1993), it is only the procedures of molecular genetics which allow a measure of population structure at the level of the gene and genome to be gained. Various molecular tools are available for the characterization of popula­tions. The most widely used to date is electrophoresis of isozymes, but direct DNA methods, including restric­tion fragment length polymorphisms (RFLPs), randomly amplified poly­morphic DNA and amplified fragment length polymorphism (AFLP), are now being exploited. The application of these procedures allows several different parameters of variability within and between populations to be determined. These include the percentage of polymorphic loci, average and effective numbers of alleles per locus, heterogeneity and heterozy­gosity indices and the various measures based upon the F statistics of Wright (1965).

More recently the application of DNA-based technologies, particularly 'fingerprinting', has provided a wealth of infor­mation on the diversity of wild populations and the relatedness of cultivars. For wild species these techniques have been applied to answer­ing specific questions particularly in relation to breeding systems. A com­parison of Plantago spp. using RFLP analysis revealed by the M13 probe showed that the inbreeding P. major possessed little variation within pop­ulations but marked differentiation between populations whilst the out­breeding P. lanceolata possessed high variability within populations but only moderate variability between populations. Similarly, in an analysis of cultivar differentiation in three species of Bermuda grass (Cynodon), using DNA amplification fingerprinting (DAF), were able to distinguish some closely related cultivars. On the basis of this technique's discriminatory power they recommend that it be used as a method for seed certification and registration purposes. These are just two examples of the many that have been carried out on a wide range of species exploiting the 'fingerprinting' capacity of the molecular methods.

In the majority of applications of these DNA-based technologies com­parisons are based upon statistical analyses of the number of 'bands shared'. Such a procedure can be fraught with problems both on a techni­cal and a genetic level. Some of the technical problems and other sources of error which can be encountered in the preparation, running and read­ing of gels have been considered by Weising et al. (1995), who emphasize the need for technical care and caution in scoring closely spaced bands. In addition there is also the question as to whether two DNA fragments which migrate to a common position on a gel are homologous. This may only be ascertained by extraction of the bands and comparative cross-­hybridization. In RAPD analysis of the Lolium/ Festuca complex of species it was found that four out of six amplification products were homologous when tested by Southern hybridization. Although this is a small sample and ranged across genetically diverse species, it does emphasize the need for caution in assuming homology.

The use of individual isozyme loci and similar genetic markers, as measures of variability, is limited in that they give no indication of the genomic associations that are of importance in the maintenance of co-­adapted gene complexes. It is only by looking at combinations of markers that such information can be ascertained. In Avena spp. and Hordeum spp., for example, Allard and his coworkers (Allard, 1990; Allard et al., 1993) have shown by comparisons involving up to 14 discrete loci that popula­tions are made up of individuals containing differing multilocus assem­blages of favourable epistatic combinations of alleles. These have arisen by rare outcrossing followed by inbreeding to near homozygosity. In A. hir­tula, for example, a majority of Spanish populations were found to be poly­morphic for different multilocus genotypes, which suggests that 'interactions at the interplant level may contribute to adaptive significance' (Allard et al., 1993). In addition, in this species, polyploidy is present which, in its own right, can lead to a greater allelic diversity and potential for differing multilocus associations. Here again the breeding system rein­forces the stabilization of such associations by restricting the degree of recombination that takes place.

The current procedures using molecular markers for assessing popula­tion differentiation consider both expressed and non-expressed parts of the genome. Given the ease with which genetic maps may now be con­structed, it seems likely that, in future, measures of population differentia­tion will take into account genome organization and will target those regions of interest. Already this problem is being addressed as a means of determining identity by descent as part of the statutory procedures in determining 'essential derivation' of cultivars (Dillmann et al., 1995).

Recent developments in QTL analysis of crop plants in defined popu­lations, such as F₂s and recombinant inbred lines, are now bringing together the power of genome analysis at the molecular level and biomet­rical procedures which will eventually allow a more detailed understand­ing of the genetic architecture of traits of both agronomic interest and of importance to evolutionary fitness.
These various studies of population structure provide an insight into the manner in which variability is distributed within a species and some of the factors controlling that pattern. From a conservation aspect it is now neces­sary to consider how these mechanisms interact to determine the spatial dis­tribution of variability and their implications for collection and conservation.

References:

Allard, R.W. (1990) Future directions in plant population genetics. In: Brown, AH.D., Clegg, M.T, Kahler, A.L and Weir B.S. (eds) Plant Population Genetics, Breeding, and Genetic Resources. Sinauer Associates, Sunderland, MA, pp. 43-63.

Allard, R.W., Garcia, P., Saenz-de-Miera, L.E. and Perez de la Vega, M. (1993) Evolution of multilocus genetic structure in Avena hirtula and Avena barbata. Genetics 135, 1124-1139.

Dillmann, C, Charcosset, A, Bar-Hen, A, Goffinet, B., Smith, J.S., Datte, Y. and Guiard, J. (1995) The estimation of molecular genetic distance in maize for DUS and ED protocols: optimisation of the information and new approaches of kinship. BMT /3/6. UPOV; Working Group on Biochemical and Molecular Techniques and DNA Profiling in Particular. Upov, Geneva, pp. 2-27.

Kearsey, M.J. (1993) Biometrical genetics in breeding. In: Hayward, M.D., Bosemark, N.O. and Romagosa, I. (eds) Plant Breeding: Principles and Prospects. Chapman & Hall, London, pp. 163-183.

Weising, K., Nybom, H., Wolff, K and Meyer, W. (1995) DNA Fingerprinting in Plants and Fungi. CRC Press, Boca Raton, FL, 322 pp.

Wright, S. (1965) The interpretation of population structure by F statistics with special regard to system of mating. Evolution 19, 395-420. 

Genetic makeup and variability

K K Vinod

The genetic structure of a population determines its capacity for response to selection, both natural and artificial, and as such is of primary consid­eration in the formulation of strategies for the collection and conservation of biodiversity. The structure of the population is controlled by several fac­tors such as its life form, breeding system and effective population size. These factors, which often reflect past selection pressures, all influence the nature and maintenance of genetic variation both within and between populations and in some cases may themselves be subject to genetic deter­mination. In the conservation of biodiversity it is the underlying genetic control of variability that is of major importance in determining appropri­ate strategies.

The phenotype of an organism is controlled by a multitude of genes which act both individually and in concert upon the various stages of develop­ment and are influenced to varying degrees by the environment. Their action leads, in the majority of cases, to a quantitative expression of growth forms which are continuously distributed in nature. The genetic control of these quantitative traits is by sets of genes (polygenes or quantitative trait loci) each of small effect; although these may be difficult to identify individually, they are inherited in a Mendelian manner and show all the properties of major genes, i.e. linkage, dominance, epistasis and the effects of ploidy. The study of such traits has in the past required the application of biometrical procedures utilizing means and variances, but develop­ments in molecular techniques for genome analysis and genetic mapping offer the prospect of more precise identification of single quantitative trait loci.

The behaviour of genes determining quantitative traits in a population is the same as that of major genes. If random mating is the mode of repro­duction, at a single locus level the individual genotypes are to be found in the Hardy-Weinberg proportions, p²:2pq:q², where p and q represent the diallelic frequencies. When extended over many loci it can be seen that the extreme homozygous classes and thus phenotypes are rare in the popula­tion. These genotypes, which represent free variability and are directly fix­able by selection, have the capacity by hybridization and segregation to create all intermediate genotypic classes. In doing so, the majority of individuals produced will be of differing homozygous/het­erozygous genotypic combinations and, as such, will give rise to interme­diate phenotypes. These differing genotypic classes again have the potential, by hybridization and segregation, to release variation. Here, however, this hidden variability is in two states, the homozygotic and het­erozygotic (Mather, 1973). As the number of genes controlling a trait increases, the proportion of variability exposed to the rigours of selection in the homozygous state will decrease.

The mode of gene action will also influence the state and proportion of exposed variability. The effects of dominance will be two-fold depend­ing on the direction of dominance at the individual loci concerned. Firstly, if all dominant alleles are acting in the same direction the effect will be to reduce the number of phenotypic classes observed as the heterozygous classes will be indistinguishable from the dominant homozygotes. As a consequence, selection will be more difficult as potential variability will still be present in the heterozygotes and can only be revealed by progeny testing. The distribution of individuals will be very much skewed in the direction of the dominant expression. Secondly, if dominance is ambidirec­tional, the effect will be to increase the proportion of intermediate pheno­types in the distribution and with it the release of variability and the potential for response to selection. Nonallelic interaction will to some extent reinforce the effects of dominance in leading to a reduction in class frequencies and the mean expression of a trait in a population.

The evolution of the genetic architecture of a trait is governed by the components of the genetic system (Darlington, 1958), namely the creation of new variation by mutation, recombination and the breeding system. These, when coupled with selection and/ or genetic drift, are the major determinants controlling the manner in which variability is organized within a population.

Genetic variation due to mutation

Mutational change at the DNA level of the genome is the basis of new variation and can take several forms such as base pair deletion, duplica­tion or rearrangement. Its effect may be detectable at the molecular level, as for example in the changes that lead to differing forms of an enzyme (allozyme), which in most cases would be neutral in its action, or it may have a gross effect on the phenotype such as in flower form or colour. Most mutational changes are considered to be deleterious in that they generally disrupt the hitherto integrated structure of the gene. However, some may be advantageous, with their subsequent survival and spread in the population being dependent on such factors as selective advantage, population size and genetic drift. If the mutation is recessive, as in most cases, its frequency in the homozygous state will initially be very rare in outbreeding species, hence the likelihood of exposure to the rigours of selection is very low. However, mutations that affect the breeding system can be at an immediate advantage. The occurrence of a mutant incompat­ibility allele in a single-locus gametophytic system, such as occurs in Trifolium repens, would be advantageous in that it is directly exposed in the haploid phase in the pollen grain and can be effective in promoting fertilization. In that it provides a further option for cross-pollination to occur its survival in the population/ species is ensured. This may well account for the very high number of incompatibility alleles that can be found in species with gametophytic systems.

Adaptive change may arise in a population through alterations at the chromosomal level. This may take the form of structural or numerical change such as gross deletions, inversions, interchanges, aneuploidy and polyploidy. The mechanisms and origin of these types of change are well documented (Darlington, 1956). It is their influence on the maintenance and release of variability and the opportunity they provide for new adap­tive forms to arise which are of importance from a conservation aspect. Polyploidy, for example, which may arise by the direct doubling of a chro­mosome set or be coupled with wide hybridization, is well known as a mechanism for maintaining heterozygous combinations of genes (Stebbins, 1950).

Genetic variation due to recombination

The role of recombination in controlling the release and distribution of variation within a population is of fundamental concern in the develop­ment of strategies for the conservation of genetic resources. It has long been established that the mechanisms controlling chromosome pairing, and the frequency and position of crossing over in the genome are under genetic control. The evolution of the Ph (pairing) gene on chromosome 5B of wheat has led to the regu­lar diploid pairing that takes place, and with it the stability and fertility of a diploid as opposed to the instability and sterility of an allopolyploid. Selection, irrespective of whether it be natural or artificial, can lead to marked differences in the rate of recombination between populations. In the outbreeding species Lolium perenne and Festuca pratensis bred cultivars have a higher chiasma frequency than their wild counterparts. This has arisen as a correlated response to selection for varia­bility by the breeder. Similarly the presence of B chromosomes can influ­ence chiasma frequency. The fine-scale collinearity of cereal and grass genomes should enable strategies to be developed for the positional cloning of the gene(s) controlling chromosome pairing in wheat and the forage grasses, opening up the prospect of genetically manipulating the processes of recombination at will and, with it, the range of variation that may be extracted from a population.

Genetic variation due to selection, drift and gene flow

The differentiation of populations depends on the three processes of selec­tion, drift and gene flow. The forces of selection, reflecting the environmen­tal pressures acting upon the population, are instrumental in defining the underlying genetic structure. The differing modes which it may take, such as stabilizing, disruptive or directional, each have their own effect upon the subsequent gene action and organization of the variation (Mather, 1973). Random drift, particularly in small populations, can lead to arbit­rary fixation of genes. The immigration of new genes from distinctive neighbouring subpopulations can increase the extent of both selective response and drift by introducing new alleles; or it can reduce it by repeatedly introducing genes adapted to a different microenvironment and by so doing retard micro-adaptation to local patches. In addition, life form and the persistence of seed banks may all influence the capacity for selection to lead to local adaptation.

Variability in breeding systems

The flow of variability within a species is dependent on its mode of repro­duction. Sexual species, which may be either inbreeders or out-breeders, have the capacity for recombination and as a consequence variability may be exposed to selection. Asexual forms, which reproduce either by apo­mixis or vegetative means, maintain a uniform genotype, which may be well adapted to present selective forces, but lack the ability to respond to changing conditions. The breeding system is often under genetic control and may be associated with specific life forms. Inbreeding, which is pre­dominantly found in annual life forms, often at the limits of a species dis­tribution (Stebbins, 1950), is generally achieved by mechanisms that ensure self-pollination. Pollen may be shed within closed florets (cleistogamy), as in wheat and barley, or flowers may open and be receptive when anther dehiscence occurs (chasmogamy), as in tomato. Although these mecha­nisms are under precise genetic control, breakdown may occur, allowing outcrossing to take place. In barley, for example, Allard and Hansche (1965) showed that up to 1% outcrossing may be found under some envi­ronmental conditions. Novel recombinants will appear offering scope for further selection and evolutionary change.

Outbreeding is generally found in the more perennial species and is often promoted by one or more genetically controlled mechanisms. These may range from timing differences in anther dehiscence and the receptiv­ity of the stigma through to precisely controlled incompatibility systems. The consequence of such processes is the mainten­ance of a high level of heterozygosity within the individual and variability both between individuals within the population and between populations. This aspect of population structure will be considered in more detail in a later section.

The apomictic mode of reproduction, which involves the production of seed by asexual means, is found in many genera, predominantly of the Gramineae and Rosaceae. It is generally associated with polyploidy and can be obligate or facultative. In those cases where sexual relatives are to be found, which allow genetic analysis, it has been shown to be under simple genetic control. For example, in Panicum maximum it appears to be under the control of a single dominant gene whilst in Citrus several genes are involved. Apomixis has the attribute of maintaining well-adapted combinations of genes together but has the disadvantage that there is no flow of variability and as such the species may well be at an evolutionary dead end (Stebbins, 1950).

Truly vegetative reproduction is rare but like apomixis can lead to the widespread distribution of a species. Spartina anglica is now to be found all around the shores of Great Britain having spread from its origins in Southampton Water by the continual breakup of its rhizomes. It is a repro­ductive mode that is often exploited by humans to maintain and distribute a crop species, as in the potato.

Each of these reproductive modes can be under genetic control and thus subjected to the forces of natural selection in the same manner as the genes responsible for other traits of adaptive significance. An insight into their effect on the state of variability and structure of populations can be obtained from the numerous studies of molecular markers in plant popu­lations.

References:

Allard, R.W. and Hansche, P. E. (1965) Population and biometrical genetics in plant breeding. In: Geerts, S.J. (ed.) Genetics Today, vol. 3, Proceedings of XIth International Congress of Genetics, The Hague, Netherlands, 1963. Pergamon Press, Oxford, pp. 665-668.

Darlington, C.D. (1956) Chromosome Botany. Allen & Unwin, London, 186 pp.

Darlington, C.D. (1958) The Evolution of Genetic Systems. Oliver & Boyd, London, 265 pp.

Mather, K. (1973) Genetical Structure of Populations. Chapman & Hall, London, 197pp.

Stebbins, G.L (1950) Variation and Evolution in Plants. Columbia University Press, New York.