Maria Kucharska
Department of Genetics, Silesian University
Jagiellonska 28, 40-032 Katowice, Poland
The biochemical markers of barley can be used to map agronomically important genes and to transfer those genes to modern cultivars. Genes coding for isozymes have made important contributions to the construction of linkage maps of different crops (Tanksley and Rick, 1980; Foiset et. al., 1996). They also proved to be useful to "tag" desired genes having expression difficult to follow (Rick and Fobes, 1974; McMillin et. al., 1986; Batlle and Alston, 1996). This is not the case for barley, where only six specific isozyme loci have been located in chromosomes (Kleinhofs et. al., 1993; Graner et. al., 1991) on partially integrated genetic map of barley. This is due to low level of polymorphism among barley cultivars.
Two multiple isozyme marker stocks covering three barley chromosomes were developed with help of induced mutants. They differ from one another by Est5 band position. Isozymes patterns obtained from barley seedlings are clear and can be unambiguously scored on 12% starch gel in single buffer system. The electrode buffer is 0.038 M LiOH, 0.188 M Boric pH 8.2 and the gel buffer is 0.045 Tris, 0.007 M Citric Acid, 0.004 M LiOH, 0.019 M Boric Acid, pH 8.1. The buffers and staining procedures are according to Soltis et. al.,1983. The advantage of using marker stocks is the possibility of reducing the number of crosses that need to be examined in linkage studies.
The original mutants were obtained by chemical treatment of several barley cultivars, Karat, Julia, Delisa and Aramir. Mutant allozymes were revealed in the following isozyme loci: Est1, Est5, Got2, Got3, Lap2. Apart from Est5, mutant allozymes in all the other loci are unique, it means such a molecular forms do not occur in barley cultivars. Apart from the Got3 mutant which is "null", most of the mutant allozymes are "shift" type, if compared to common forms. Mutant allozyme of Est1 migrates in electrical field to the opposite pole in the routinely used condition.
The isozyme markers present in the multiple isozyme marker stocks are listed in Table 1. The linkage study involving crosses of mutants with both morphological markers and translocation lines of barley were performed earlier (Kucharska and Maluszynski, 1991; Kucharska et. al., 1998).
The crosses between mutants were followed by isozyme analysis of F2 and then by crosses between recombinants bearing two desired allozymes. All allozymes but "null" in Got3 segregated in codominant manner. Final selection took place in two separate F2 populations. The first segregated in two loci, Got2 and Lap2, having established mutant allozymes for Est1 and Got2 and common allozyme for Est5, the second segregated in all five isozyme loci involved. From the second population, due to large number of segregating isozymes, we were not able to select recombinant plant directly, instead we selfed three plants which were heterozygotes only in one or two loci and homozygotes with unique allozymes in the remaining, than found desired recombinants in F3.
Some of the crosses produced plants carrying disadvantageous characteristics. Along with isozyme, selection for early maturity, semi-dwarf stature, local diseases resistance, lodging resistance and easiness to use as female parent was exercised.
The homozygous lines are being developed by SSD method and they will be maintained as such. At the moment it is F5 generation. Seeds will be made available from:
Department of Genetics
Silesian University
Jagiellonska 28
40-032 Katowice
Poland
Table 1. List of isozyme markers incorporated into multiple isozyme marker stock of spring barley
| Locus | Chromosome | Linked markers* | Mutant allozyme description |
|---|---|---|---|
| Est1 | 3L | - | migrates to opposite pole |
| Est5 | 1S | br, T1-7f | slow |
| Got2 | 6L | o, T6-7i | fast |
| Got3 | 3L | uz, ert c, ari a, T3-7c | null |
| Lap2 | 1L | T1-3b | slow |
References:
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Graner A., Jahoor A., Schondelmaier J., Siedler H., Pillen K., Fischbeck G., Wenzel G., Herrmann R.G. (1991). Construction of an RFLP map of barley. Theor. Appl. Genet. 83: 250-256.
Kleinhofs A., Kilian A., Saghai Maroof M., Byashev R.M., Hayes P.M., Chen F., Lapitan N., Fenwick A., Balkes T.K., Kanazin V., Ananiev E., Dahleen L., Kudrna D., Bollinger J., Knapp S.J., Liu B., Sorrels M., Heun M., Franckowiak J.D., Hoffman D., Skadsen R., Steffenson B.J. (1993). A molecular isozyme and morphological map of barley (H vulgare) genome. Theor. Appl. Genet. 86: 705-712.
Kucharska M., Kaczorowska K., Ali E.S., Maluszynski M. (1998). Linkage of two mutant allozymes for Amp 2 and Aat 2 with marker loci on barley chromosomes 1 and 6. J. Appl. Genet. in press
Kucharska M. and Maluszynski M. (1991). Linkage of two mutant allzymes for Est5 and Aat3 with marker loci on barley chromosomes 1 and 3. Cereal Res. Comm. 19: 165-169.
McMillin D.E., Allan R.E., Roberts D.E. (1986). Association of isozyme locus and strawbreaker foot rot resisrance derived from Aegilops ventricosa in wheat. Theor. Appl. Genet. 72: 743-747.
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