NOVEL TECHNOLOGY FOR GENETIC MAPPING

摘要

A method for genetic mapping in eukaryotic organisms is described, comprising: multiple artificial DNA oligonucleotides being introduced in neutral positions into the genome of a single strain of the organism to create an artificially marked strain. This strain contains many specific markers, either composed solely of the inserted oligonucleotides or composed of inserted oligonucleotide(s) and part of the adjacent DNA sequence. The artificially marked strain is crossed with another strain displaying one or more distinct traits. The DNA of segregants from the cross displaying a specific trait is pooled and the presence of the artificial markers in the pooled DNA is detected. The genetic map position of all genes involved in establishing the trait is indicated by a drop in signal intensity for the artificial markers located closest to these genes. The method allows the use of isogenic strains for genetic mapping. It also allows to accumulate large numbers of mutations in a single strain until a particular phenotype is generated and subsequently map the mutation(s) relevant for the phenotype of interest. In a further embodiment large numbers of mutations are accumulated in the artificially marked strain until a phenotype of interest is obtained, the multiply mutated artificially marked strain is then crossed with a wild type strain, the DNA of all segregants displaying the wild type phenotype is pooled and the presence of the artificial markers is detected. The genetic map position of all genes required to restore the wild type phenotype is indicated by a drop in signal intensity for the artificial markers located closest to the position of these genes. In a further embodiment of the invention a restriction site for a rare restriction enzyme is added to the artificial marker, which allows to cut the genomic DNA in different fragments each containing a specific tag. These fragments are introduced into a vector to construct a genomic library in a host organism, of which the transformants can be sorted according to the position of the fragment in the genome. After transformation of the library into a recipient strain, all the fragments can be traced with the specific tag using one of several methods available.