In cereals with large genomes, genetic and physical marker distances may largely differ, i.e., close genetic linkage does not necessarily reflect close physical proximity (e.g. Künzel 1982, Linde-Laursen 1982). This bears serious risks for important RFLP applications. For example, a closely linked RFLP marker might be too far apart from a target gene for marker assisted cloning.
A direct way to integrate genetic and physical chromosome maps is hybridization of genetically mapped DNA probes to chromosomes in situ. However, in situ detection of single copy sequences of only 0.5 - 2.0 kb length, the usual size of DNA fragments which detect RFLPs, is not yet feasible in plants. Therefore, correlating the RFLP maps to cytogenetic landmarks stagnates, and the necessary map integration is still in a very initial state for the barley genome.
To overcome this situation a new strategy has been devised and used for physical localization of genetically mapped RFLP clones to barley chromosomes. Morphologically distinct translocation chromosomes were microisolated and their DNA was used as template for the polymerase chain reaction (PCR) with sequence-specific primers. RFLP clones of the German Barley Mapping Program (Graner et al. 1991) were assigned to cytologically defined segments of chromosome 5. This related, e.g., approximately one third of the map length of linkage group 5 to approximately one fifth of the mitotic metaphase length of chromosome 5 (paper submitted to 'Genome').
Our technique proved to be highly sensitive. The DNA of ten microisolated prophase nuclei or of twenty microisolated chromosomes resulted, after amplification with sequencespecific primers, in detectable fragments indicative for the single copy RFLP probe.
The new technique allows to localize short single copy sequences not yet detectable by in situ hybridization. However, the resolution of this method for physical genome mapping depends on the availability of suitable chromosomal reconstructs. Since in barley nearly 400 break points of reciprocal translocations are cytologically localized to defined segments of Giemsa-banded somatic metaphase chromosomes (Künzel 1993), the resolution of the method is comparable to that obtained by deletion mapping in wheat (Werner et al. 1992, Kota et al. 1993).
In summary, the new and sensitive experimental route, applicable to any sequenced RFLP probe, may substantially contribute to relate recombinative distances obtained in linkage studies with cytomorphological distances measured in somatic metaphase chromosomes of barley. (Supported by the Federal Ministry for Research and Technology, Grant No. 0319960B).
References:
Graner, A., A. Jahoor, I Schondelmaier, H. Siedler, K. Pillen, G. Fischbeck, G. Wenzel, and R. G. Herrmann. 1991. Construction of an RFLP map of barley. Theor. Appl. Genet. 83:250256.
Kota, R. S., K. S. Gill, B. S. Gill, and T. R. Endo. 1993. A cytogenetically based physical map of chromosome 113 in common wheat. Genome 36:548-554.
Künzel, G. 1982. Differences between genetic and physical centromere distances in the case of two genes for male sterility in barley. Theor. Appl. Genet. 64:25-29.
Kanzel, G. 1993. Coordinator's report: Translocations and balanced tertiary trisomics. BGN 22: in press.
Linde-Laursen, 1. 1982. Linkage map of the long arm of barley chromosome 3 using C-bands and marker genes. Heredity 49:27-3 5.
Sorokin, A., F. Marthe, A. Houben, U. Pich, A. Graner and G. Künzel. 1994. PCR-mediated localization of RFLP clones to microisolated translocation chromosomes of barley. Genome, submitted
Werner, I E., T. R. Endo, and B. S. Gill. 1992. Toward a cytogenetically based physical map of the wheat genome. Proc. Natl. Acad. Sci. USA 89:11307-11311.