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Development of universal techniques for the rapid screening of genetic variation

Cel

The aim of this project is to develop new and efficient DNA techniques for the screening of genetic variation in a wide range of animals including mammals, reptiles, fish and insects. In particular PCR assays allow the analysis of DNA sequences from different parts of the genome, and can use very small amounts of material, such as hair follicles from a bear or an insect leg from a museum collection. Our strategy was to combine a small number (5) of select laboratories already actively engaged in using these technologies.
We had the following major objectives, which each lab developing two or three different types of sequence (mtDNA, rDNA, introns, microsatellites) in their special organisms (whales, sheep, mice, fish, flies, beetles, grasshoppers).
1.Design of PCR primers for chosen intron and mtDNA amplifications.
2.Assessment of primer universality.
3.Development of efficient screening.
4.Isolation of novel microsatellite markers.
5.Genetic structure of chosen populations and species, as revealed by these.
We decided to concentrate on these sequence based techniques (rather than e.g. RAPDs) since their results are more interpretable and can be analysed more profoundly.
All five projects made significant advances and achieved their individual and joint goals.
Arctander's group (Copenhagen) developed simple and robust protocols for the determination of sex, variation in nuclear microsatellite loci and the variable part of the mtDNA control region in whales and dolphins. This involved sex chromosome specific sequences in 8 species, isolating 350 di, tri-tetra and hexamer microsatellite loci and their primers. Some 4200 individuals from 6 species have been analysed for the mtDNA and 6 microsatellite loci, providing powerful measures of variability.
Boursot's group (Montpellier) developed and used Y chromosome intron sequences, microsatellite loci and mtDNA control region in mice to examine variation at the species, population and family levels. Equivalent levels of information are obtained for geographic substructure by mtDNA and RFLP analysis. The Y chromosome reveals strong selection through an absence of intron polymorphism. Microsatellites allowed a detailed study of gene flow in 2 hybrid zones, and of kin selection in mound building.
Hewitt's group (Norwich) have developed and assessed the value of mtDNA and nuclear DNA markers for population studies in insects. Sets of conserved primers have been developed for COI mtDNA gene from an analysis of heterogeneity in nine species; these primers work universally across the class Insecta with both deep and shallow levels of divergence. Both anonymous and 3' noncoding sequence of nuclear DNA are highly variable and reveal interesting population structure in locusts and grasshoppers. Microsatellites, ITS rDNA and COI sequences have been used for colonisation studies in beetles.
Tautz's group (Munich) developed and studied 3 classes of molecular marker, mtDNA sequence, microsatellites and rDNA ITS sequences, in bats, Latimeria, Galapagos Iguanas and Drosophila. Microsatellites and ITS both revealed population subdivision in Drosophila. The extent of ITS variation among chromosomes indicates lower interchromosome exchange while differences between populations indicates low gene flow. Because of its homogenisation this sequence can provide a record of population history.
Bruford and Wayne's group (London) have concentrated on sheep of all sorts. They developed a strategy for isolating clones of single copy DNA which are sequenced, and chosen by their hybridisation with other ungulates. 100 clones were sequenced and 57 are useful. 27 PCR-primer sets are ready and several have been screened by SSCP for variability across sheep breeds and ungulates. MtDNA Cytb sequences have also been produced and this allows comparison between the nuclear and mitochondrial measures of diversity and phylogeny.
MAJOR SCIENTIFIC BREAKTHROUGHS:
-several major advances and conclusions are emerging from our joint work.
1. From the outset we chose a cloning, sequencing and PCR strategy, because it gives a profound understanding of the genetic variability. This has been amply rewarded with a range of variable sequences with the potential power to measure biodiversity.
2. These PCR sequence based techniques can be modified for rapid screening of larger numbers of individuals. We use PCR-RFLP in whales and grasshoppers, PCR-TGGE and SSCP in flies and sheep, PCR-Automatic Sequencing (ABI & ALF) in beetles, mice and sheep, and other labs around the world are developing such rapid methods. Our results clearly demonstrate how rapid efficient large scale molecular analyses of wild populations can be conducted.
3. Microsatellites are becoming the preferred approach for many population studies. Many thousands are spread through the genome so that one can find sufficient variability fairly readily. Work in all our labs, particularly in mice and whales, demonstrates this. These are now readily automated for analysis.
4. The mtDNA control region is another preferred sequence because of its variability, as our work in whales, mice and man demonstrates. However, in insects it has a lower substitution rate, and nuclear noncoding regions may be more variable, in contrast to mammals. MtDNA is very informative for the reconstruction of patterns of colonisation.
5. The COI gene in mtDNA has both variable and conserved sequences and universal PCR primers which work across the range of insects have been developed. This will be of considerable use in studies at both population and species levels.
6. Variable nuclear DNA sequences (anonymous, introns, flanking, ITS) have also been developed and shown to be useful for biodiversity studies in grasshoppers, mice, sheep and flies. However, once again in insects intron primers are not universal due to the ancient divergence among families and orders.
7. The combination of data from different sequences with a range of genetic properties is giving new perspectives on the genetic structure of populations and species in our chosen organisms. In mice the results produced by mtDNA and Y chromosome markers separately do not provide the proper understanding of history and selection in the production of genetic diversity that can be deduced when they are considered together. Furthermore microsatellites show that population systems are far from the mutation/drift equilibrium so often assumed.
8. The project provided new insights into a group of endangered marine mammals - the whales. These include details of their evolution, population structure, social organisation and mating strategies. Such knowledge is of vital importance for the development of sound management strategies.
9. The comparisons of the patterns of variation in different classes of markers across several closely related species has also yielded important information on the modes of evolution of microsatellites.
10. In addition to all the markers we have developed for general use, two other innovations are notable: a) A simple effective sequencing technique ASSURE-B has been developed to overcome the problem of heterozygosity in highly variable nuclear sequences (Hewitt). b) An intergrated PC software GENETIX has been designed for statistical analysis of population structure for genetic data (Boursot) and is about to be released.

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System finansowania

CSC - Cost-sharing contracts

Koordynator

University of East Anglia
Wkład UE
Brak danych
Adres
University Plain
NR4 7TJ Norwich
Zjednoczone Królestwo

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