Molecular tools for detecting genetic diversity

The diversity that resides within living organisms, often referred to as biodiversity, is essential for sustained existence and provides the raw materials for agriculture, biotechnology and medicine. Both its conservation and utilisation require knowledge of existing variation. Molecular tools provide valuable data on diversity through their ability to detect Variation at the DNA level. Although this has many important applications, the overview presented here focuses on the use of molecular techniques for detecting genetic differences, distinguishing between individuals and for identification. Identification is of fundamental importance in diversity studies in a variety of different ways. For evaluation of species diversity, it is essential that individuals can be classified accurately. The identification of taxonomic units and endangered species, whose genetic constitution is distinct from their more abundant relatives, is important in the development of appropriate conservation strategies. In population studies, being able to identify whether: two individuals are from the mating of specific parents and estimating the degree of relatedness among individuals, is helpful for the determination of social behaviour, reproductive success and mating choice. Identification is of importance in studying clonal organisms and the roles of sexual and asexual reproduction, where the definition of population units and different genetic lines may be difficult. From a more practical standpoint, identification of who breeds with whom is important for the management of small populations. Moreover, being able to detect gene flow, introgression and contamination from unwanted outcrossing (genetic pollution) is crucial for the attainment of purity and has become a central issue in debate over the safety of genetically modified organisms. Identification is also a key issue for forensic purposes, for example, in the confiscation and legal protection of illegally imported, collected or commercially exploited material. A number of different techniques are available for identifying genetic differences between organisms. The choice of technique for any one specific use will depend upon the material being studied and the nature of the questions being addressed. Molecular techniques differ in the way they sample within the genome and in the type of data that they generate. For classification based on known evolutionary history, analysis of DNA sequences is the best approach, whilst multi-locus profiling techniques, such as VNTRs (variable number of tandem repeats) and AFLPs (amplified fragment length polymorphisms), or microsatellite genotyping, have much to offer for fingerprinting individuals and identity assignment. For practical usage, only a few techniques are suitable for applications where different laboratories must be able to reproduce results and analyse them using standardised scoring and data analysis methods. Molecular data may also need to be automated and amenable for entry into databases. The most promising technologies are discussed with reference to an EU-funded biotechnology demonstration project on variety identification and quality assessment. Whilst for most purposes existing methodologies offer powerful solutions to identification problems, they fall short in one specific circumstance which is of relevance here. To date, there is no technique, which guarantees the identification of a single random mutation in the genome, such as may be required for identification of sports and somaclonal variants. Furthermore, some changes induced by tissue culture are not changes in the informational content of the DNA but changes in expression. To detect differences of this kind, modifications of existing techniques are required. Some examples of possible technologies will be discussed, together with their limitations. Specific reference is made to the detection of variation associated with DNA methylation and insertional changes in transposons