Techniques for the identification of yeast artificial chromosomes (YACs) in a YAC-library with RFLP-markers closely linked to genes of interest
Techniques for the identification of yeast artificial chromosomes (YACs) in a YAC-library
with RFLP-markers closely linked to genes of interest

Michalek, W., G. Wenzel and A. Jahoor
Lehrstuhl für Pflanzenbau und -züchtung, TU München, 85350 Freising-Weihenstephan

The analysis of complex genomes involves a multitude of techniques with varyious degrees of resolution. Until recently a gap existed between the genetic analysis of genomes and the possibility to convert the genetic distances (cM) into physical distances (bp). Whereas the development of the Pulsed Field Gel Electrophoresis offered the chance to solve this problem, the invention of cloning large pieces of foreign DNA as artificial chromosomes in yeast (Burke et al. 1987) established the basis for efforts to clone genes only by the knowledge of its position on the genetic map.

Previously, a YAC library for barley was constructed in our laboratory consisting of 18000 clones (Kleine et al. 1993). This library was further developed to a size of 34000 clones with an average insert size of 120kb. With very labour intensive techniques, it was possible to obtain clones with sizes up to more than one megabase, although the biggest part of the library (80%) consists of relatively small clones developed by techniques without the employment of any size fractionation steps. The PFGE-treatment of agarose plugs with megabase-size DNA according to Edwards et al. (1992) resulted in slightly larger YACs with an average insert size of approximately 160kb. Only the PFGE-treatment of the ligated DNA (Anand et al. 1989) prior to transformation produced YACs with sizes up to more than one megabase. Figure1 shows the size distribution of the analysed YAC clones (231) in dependence of the production method. The proportion of clones smaller than 99kb is clearly reduced only in case of a repeated size fractionation step of the ligated DNA.The most limiting factor coming along with this procedure is the about 50-fold reduced rate of transformation efficiency compared to the employment of not size-fractionated DNA.

One of the problems screening the big numbers of YAC-clones is the technique employed to detect specific clones of interest, for example the corresponding clone for RFLP-markers linked to genes of interest for a map-based cloning strategy. Cloning a large genome like the barley genome always leads to a high number of clones and therefore we concentrated on pool-based strategies, as outlined in Figure2. Following the growth of 384 clones the colonies were collected and the DNA isolated to produce a so called "DNA-pool". With this pools two


Figure 1: Size distribution of YAC-clones


Figure 2: Scheme for screening procedures applied to the YAC library

strategies were established to identify positive clones. A PCR based strategy as outlined in the left half of the figure, which is already described for barley in Kleine et al. (1993) and a hybridization based method as depicted on the right. The PCR assay has the advantage of higher sensitivity and the fast production of results but it is necessary to develop a reliable assay for each primer pair. This problem does not arise with direct hybridizations of RFLP-markers. The final step in both cases is the hybridization of the relevant colony-filters with the appropriate probe. Future improvements of the screnning techniques will focus on the optimization of the PCR assay with the ultimative aim to eleminate the final hybridization step. This could be achieved by multidimensional pooling strategies (Amemiya et al. 1992) or by replacing the final hybridization step with a PCR assay in a microtiter-plate format.

With both strategies it was possible to identify a clone with RFLP-marker MWG068, which is closely linked to the Mla-locus (Schüller et al. 1992). The clone is 80kb in size and provides the first YAC-clone identified with a marker closely linked to the Mla locus. Figure 3 shows the final analysis of the detected clone through subsequent hybridizations of a filter made from a PFGE-gel containing the positive clone and three anonymus clones. On the left part the filter was hybridized with the RFLP-marker (lane1) and on the right part a vector specific probe (pBR322) was used in a control experiment. Probably more important than this specific clone is the development of a methology which allows the identification of YAC-clones with single copy RFLP-markers. This should offer the possibility to clone barley genes with a map-based approach once the appropriate closely linked markers are available.

References:
Anand, R., A. Villasante and C. Tyler-Smith (1989) Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed field gel elektrophoresis. Nucleic Acids Res 17: 3425-3433

Burke, D.T., G.F. Carle and M.V. Olson (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806-812

Kleine, M., W. Michalek, A. Graner, R.G. Herrmann and C. Jung (1993) Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone. Mol. Gen. Genet. 240: 265-272

Schüller, C., G. Backes, G. Fischbeck and A. Jahoor (1992) RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley. Theor. Appl. Genet. 84: 330-338


Figure 3: Analysis of yWHS134A8; Lane 1: yWHS134A8; Lanes 2 - 4: anonymous clones;