Plant Genome III Conference

PHYSICAL MAPPING OF RECOMBINATION SITES BETWEEN TIGHTLY LINKED MARKERS ADJACENT TO THE Mla RESISTANCE LOCUS ON BARLEY CHROMOSOME 5.

Field inversion gel electrophoresis (FIGE) and Contour-damped homogenous electric field electrophoresis (CHEF) have been used to separate HMW DNA fragments. Initial experiments analyzed the utility of 16 'rare-cutting' restriction enzymes, I.e., than having an eight-base recognition site or a six-base recognition she that rarely occurs In the barley genome. Electrophoretic conditions were optimized to separate DNA fragments ranging from 1500-50 Kb. Using these conditions, on fragment length polymorphism (RFLPS) were observed in HMW DNA restricted with Mlu 1, Eag 1, Sma 1, Xho 1, Sal 1, Fsp 1, and Bstb 1 an Southern blots probed with both BCD249 and MWG068. No RFLPs were observed in HMW DNA restricted with Pme 1, Not 1, Sfi 1, Sac 11, BssH 11, Rsr 1l, Asc l, Nru 1, and Pac 11 on blots probed with either BCD249 or MWGO68. However, a strong hybridization signal was observed In the limited mobility zone (LMZ) Indicating the probe target DNA is located on a DNA fragment larger than 1500 Kb. Analysis of HMW DNA restricted with Mlu 1 and probed with MWGO68 revealed minor RFLPS at approximately 200 Kb between three of the eight recombinants in the XmwgO68lXbcd249.2 interval. Electrophoretic parameters were optimized for separation of HMW DNA fragments ranging from 500-50 Kb and these recombinants were probed with MWG068. The RFLP appears to result from the shift to a higher molecular weight of one DNA fragment witch hybridizes to MWGO68. A change in molecular weight of a DNA fragment in a recombinant line suggests the recombination site is contained within the DNA fragment. Analysis of recombinant lines restricted with Eag 1 and probed with BCD249 detected RFLPs at approximately 30 Kb between 4 of the nine recombinants tested In the Xbcd249.2lXbcd249.1 interval. In these lines, the RFLP results from the shifting of a HMW DNA fragment to a larger size than present in the parental line, suggesting the recombination site is contained in the larger HMW DNA fragment. Duplicate sequences were discernible an Eag 1 blots probed with BCD249. The DNA fragment which exhibits Me size increase is associated with Xbcd249.1 in the four lines were the polymorphism is detected. CHEF gels have been run with parameters set for separating HMW DNA fragments from 3.5-2.0, 3.0-1.5, 2.5-1.0, and 2.0-0.5 Mb. Ethidium bromide steered gels indicate the "rare cutting" I enzymes yield HMW DNA fragments in excess of 2 MB. Initial results suggest the BCD249 probe hybridizes to fragments larger than 3.5 MB.