Lipoxygenase catalyzes the oxidation of polyunsaturated fatty acids to fatty acid hydroperoxides. In germinated barley grains, two lipoxygenase isoenzymes have been purified and characterized (Doderer et al., 1992). The two isoenzymes differ in a number of properties, among them the product they form from linoleic acid as a substrate. Lipoxygenase 1 solely forms the 9-hydroperoxide from this fatty acid, while lipoxygenase 2 exclusively produces the 13-hydroperoxide. Furthermore, the two isoenzymes differ in their expression pattern. Lipoxygenase 1 is already present in the quiescent grain, while lipoxygenase 2 appears during germination (Yabuuchi, 1976).

Genetic mapping using 3 different cDNA probes was performed in a progeny comprising 90 F₂ derived F₂ families of an interspecific cross between H. vulgare (cv. Vada) and H. spontaneum (line 1B-87). In order to minimize errors due to the segregation of heterozygous F₂ individuals, equal amounts of leaf material from 10 F₃ individuals were pooled prior to DNA extraction. Wheat-barley addition lines (Islam et al., 1981) carrying disomic additions of individual barley chromosomes of the cultivar Betzes (except for chromosome 5, which is not available in an addition line) in a wheat background (cv. Chinese Spring) were used for the chromosomal assignment of RFLP bands. DNA extraction, Southern analysis and probe labelling followed standard procedures and were performed essentially according to Graner et al. (1990), except that a solution consisting of 0.6 M NaCl and 0.4 M NaOH was used for transfer of DNA onto nylon membranes (Biodyne B, Pall corp.). Linkage analysis was carried out using "Mapmaker" computer software (Lander et al., 1987). The following parameters were preset: two point analysis LOD 3.5, three point analysis LOD 3.0, and a maximum recombination value of 0.4. Recombination values were converted into map distances (centiMorgans, cM) by using the Kosambi function.

RFLP mapping was performed to determine the linkage groups and the chromosomal positions of individual Lipoxygenase (Lox) genes in relation to a set of already mapped RFLP markers, covering nearly the entire barley genome (Graner et al., 1991). Southern hybridization to genomic DNA of the mapping parents, digested with six different restriction enzymes (BamRI, EcoRI, EcoRV, HindIII, SacI, XbaI) revealed one to six discrete bands with each probe. Since most of these restriction fragments were detected by at least two Lox-derived probes, it can be assumed that they were the result of cross hybridization. Further genetic analysis revealed three loci, whose allelic segregation patterns fitted the expected 3:1 (Lox-A, Lox-C) and 1:2:1 (Lox-B) ratio, indicative for single locus segregation. The Lox-C gene maps on chromosome 7 (5H), while both Lox-A and Lox-B map on chromosome 4, where the two loci are only l cM apart (Fig. 1). The common location of Lox-A and Lox-B on chromosome 4 has been further substantiated by hybridization of the respective probes to HindIII digested DNA of wheat-barley addition lines. Here, each probe revealed a different major fragment, which could be assigned to this chromosome. We propose these loci to be designated as Lox-A, Lox-B and Lox-C.

Despite obvious cross hybridization of the respective probes no further loci could be detected, suggesting that the genetic location of all Lox genes, which are present in the mapping parents, has been determined. In contrast to other gene families, e.g., those encoding the storage proteins of the barley grain, the Lox genes are not clustered on a single locus. The occurrence of sequence duplications both within individual chromosomes and between different chromosomes seems to be a rather common event in the barley genome (Heun et al., 1991; Graner et al., 1991). Additional data, especially those of closely linked markers, are required to determine whether this relates to the evolution of this gene family.

Acknowledgement: This paper is ABIN publication no. 101.

Chromosome 4 (4H)
Chromosome 7 (5H)

References:

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