Limnological Institute, SB RAS, Irkutsk
Deutsches Krebsforschungszentrum, Germany, Heidelberg
LIN - M. A.Grachev,
664033 Irkutsk, Ulan-Batorskaya St., 3, Russia
Tel.: +7(3952)460504, Fax: +7(3952)460504
DKFZ - Jorg D.Hoheisel,
Germany, Heidelberg, Im Neuenheimer Feld 506
Tel.: +49(06221)424680 (office), Fax: +49(06221)424682
LIN - T.Ya.Sitnikova, D Yu.Sherbakov, D.Yu.Zubakov, S.V.Kirilchik., L.V.Sukhanova, O.B.Ogarkov
DKFZ - Jorg D.Hoheisel
The goal of the project proposed is the elaboration of a novel approaches for fast evaluation of the genetic diversity. It is proposed to apply a technique of DNA sequencing by hybridization with high-density oligonucleotide arrays (DNA chips) to the comparisons of homologous genes in closely related species. Using this highly efficient method it is planned to carry out a large-scale evolutionary study of the species flocks of Lake Baikal hydrobionths.
Lake Baikal is probably the best natural laboratory for studies of biological speciation. The lake is inhabited by 2491 species and sub-species of aquatic animals, of them more than 2/3 are endemic. Studies of the evolution of species in Lake Baikal become of special interest in view of the fact that its sediments store a continuous high-resolution record of the paleoclimates of Siberia of at least 5 MYR. In a recent years molecular phylogenetic studies of Baikalian species flocks of cottoid fishes, gammarids, molluscs, oligocheats, and flat worms resulted in interesting insights into evolutionary histories of this groups. Time scales of species radiations were estimated and the evolutionary scenarios were inferred in the context of geological and climatic history of the lake. However all this works should be treated as a very initial though necessary and important steps toward understanding of Baikalian biodiversity origin, formation, and current structure. In the immediate future evolutionary investigation of Baikalian animals should require much more extensive DNA sequencing efforts to include broader range of species within species flocks into phylogenetic analysis and to obtain enough information upon population genetic polymorphism. Unfortunately conventional DNA sequencing of PCR products based on dydeoxy method is a tedious and expensive procedure, and possibly would take many decades to estimate the genetic diversity of the more than 1700 Baikalian endemics. Therefore there is an evident need in a novel high-performance DNA sequencing approach.
A flood of papers now reports the development and use of the innovative DNA chip technology for rapid analysis of genomic data. A recent review on this topic has been published in a special issue of Nature Genetics (Supplement volume 21, Jan. 1999). According to this innovative technology, sequence analysis involve combinatorial arrayed libraries of all oligonucleotide species N (typically 8) nucleotides in length to interrogate target DNA of interest (Fig. 1). In theory, such universal arrays could be used to obtain sequence information for virtually any sequence as all possible complementary sequences are present in the array. Technology has impressive achievements like the demonstration of the possibility to resequence human mitochondrial genom with an array of 135.000 oligonucleotides or analyze the whole genom of yeast on a single DNA chip (Fig. 2), etc. To our knowledge, it is not yet systematically applied to molecular phylogenetic problems, however some pilot studies upon this issue were performed in a recent years (e.g. evolutionary sequence comparisons of BRCA exon 11 of higher primates by Hacia et al., 1998).
To avoid the difficultes of heterozygous mutations detection it is planned to use mitochondrial DNA markers, like CO I gene fragment which phylogenetic utility was proven in a previously performed studies of molecular evolution in Baikalian organisms. Principally, the choice of marker is rather arbitrary. It could be single gene or gene fragment or even whole mitochondrial genom depending upon the paricular task. At the initial stage of the project It is planned to use both conventional (dideoxy) and chip techniques of DNA sequencing. Most simple strategy could be proposed for the nucleotide polymorphisms detection within populations: i) all or at least the majority of the haplotypes in the population will be identified by dideoxy sequencing; ii) basing on the known sequences, DNA chips will be constructed and then used for the haplotype typing of vast amount of specimens thus achieving high-resolution population analysis. It should be emphasised that the same array can be used repeatedly with many DNA samples which makes it cost-effective. Screening for all possible sequence changes by array hybridization is more difficult but feasible task. Provided that the reference sequences are obtained in a previous studies it is possible to design a restricted subset of oligonucleotide probes into an ordered array. An array consisting of oligonucleotides complementary to subsequences of a target sequence can be used to determine the identity of a target sequence, measure its amount, and detect differences between a target and a reference sequence. There are several approaches to identification of single base mutations in a target sequence (e. g. Chee et al., 1996). However, the it could be done only when hybridization signal is perferct. To increase hybridization specificity and sensitivity it is planned to use chemically modified probes, e. g. peptide-oligonucleotides.
The introducing of the DNA chip technology into a common practice of molecular evolutionary studies is supposed to be a main result of the project. However the results of phylogenetic and population analyses of some particular species flocks of Baikalian animals also would be of great interest. We expect to obtain a massive amount of sequence information sufficient for making unequivocal conclusions upon the level of genetic polymorphism at different taxonomic levels: from populations to a whole families. The model groups for this study would be white fishes, gammarids, and gastropods. In particular, it is planned to reveal population structure of Baikalian omul (white fish) which could have practical importance when selecting cohorts for artificial reproduction. The problem of bathymetric and geographical segregation would be studied on Baikalian gammarids as a model group. The investigation of sympatric cryptic species and geographicali varietes of Baikalian gastropods is also a very challenging issue. However, selection of particular species and groups of animals would be a matter of special discussion of the participants, and the above list may be changed depending upon the analysis of earlier data.
Grachev M.A., Slobodyanuk S.Ya., Kholodilov N.G., Fyodorov S.P., Belikov S.I., Sherbakov D.Yu., Sideleva V.G., Zubin A.A., Kharchenko V.V. 1992. Comparative study of two protein-coding regions of mitochondrial DNA from three endemic sculpins (Cottoidea) of Lake Baikal. J Mol Evol 34:85-90;
Slobodyanuk S.Ya., Kirilchik S.V., Pavlova M.E., Belikov S.I., Novitsky A. L. (1994) The evolutionary relationships of two families of cottoid fishes of Lake Baikal (East Siberia) as suggested by analysis of mitochondrial DNA. J Mol Evol 40:392-399;
Sherbakov D. Yu. (1999) Molecular phylogenetic studies on the origin of biodiversity in Lake Baikal TREE Vol.14, no. 3: 92-95.
Kirilchik S.V., Slobodyanuk S.Ya., Belikov S.I., Pavlova M.E. (1995) Phylogenetic relatedness of 16 species of Baikal Lake Cottoidei bullhead fishes deduced from partial nucleotide sequences of mtDNA Cytochrome b genes. Molecular Biology 29:471-476;
Sukhanova L.V., Smirnov V.V., Smirnova-Zalumi N.S., Slobodyanuk S.Ya., Skulin V.A., Baduev B.K. (1996) The study of populations of Baikalian omul Coregonus autumnalis migratorius by mtDNA restriction analysis. J Ichtiology (in Russian) 36:667-673;
Zubakov D.Yu., Scherbakov D.Yu., Sitnikova T.Ya. (1997) Phylogenetic relationships of Baikalian endemic molluscs of the family Baicaliidae (Gastropoda, Pectinibranchia) as suggested by analysis of mitochodrial CO I gene fragment nucleotide sequences. Molecular Biology 31:1097-1102;
Sherbakov D. Y., Kamaltynov R. M., Ogarkov O. B., Verheyen E. Pattern of evolutionary change in baikalian Gammarids inferred from DNA sequences (Crustacea, Amphipoda). // Mol. Phy. & Evol. - 1998. - V.10 - P.160-167.
Jan Weiler, Henrich Gausepohl, Nicole Hauser, Ole N. Jensen, and Jorg D. Hoheisel (1997) Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays. Nucleic Acids Res 25:2792-2799.
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Hoheisel J. D. (1994) Trends Genet 10:79-83.