Mutable alleles, firstly identified in maize, are characterized by frequently reversions to the wildtype in the course of development (Stubbe, 1933). In homozygous lines this fact led to variegations and sectoring in somatic tissue and germinal revertant seeds. Such variegations were also observed in high frequencies by Ono and Suzuki (1957) in barley at the wx-locus after crosses of different varieties, in particular with Hordeum agriocrithon.
In genetic analyses of crosses of several barleys with the mutant line 152 we found variegations and somatic sectoring at different loci (Schreiber and Habekuß, 1994). Various types of variegation patterns were detected at the waxy-character (Fig. 1), in accordance with the results of Ono and Suzuki (1957). But in our investigations the periclinal type was also present in stable waxy-lines. It may be probably due to a small amylose content expressed in the endosperm starch of the most waxy stocks (Ishikawa et al., 1994). Moreover, the size of the blue-stained area is depended on the position of the cut before iodine staining. Therefore, only the sectorial type was used for determining the frequency of mosaic grains (Table 1).
Fig. 1: Types of variegation patterns in the waxy-character
Table 1: Frequencies of F2- and F3-mosaic grains of the crosses Master¹ x Mut. 152 and reciprocally
__________________________________________________________ Generation Number of grains tested _________________________________________ Total Mosaics Frequency __________________________________________________________ F2-1 8012 6 7,5 x 10^(-4) F2-2 697 1 1,4 x 10^(-3) F3-1 9484 3 3,2 x 10^(-4) F3-2 4535 1 2,2 x 10^(-4) F3-3 1852 1 5,4 x 10^(-4) F3-4 2095 1 4,8 x 10^(-4) __________________________________________________________In agreement with the results of Ono and Suzuki (c.f.) the endosperm starch variegation was observed exclusively in heterozygous plants. Investigations on F1-spikes of different crosses of the mutant line 152 and unstable derivatives (TE 2001b) with waxy-stocks have shown a non-randomly distribution of the grains with waxy-endosperm starch in the spike, as it can be expected after regular meiosis (Fig. 2).
Fig. 2: Spike pictures of segregating F1-spikes (F2-grains)
¹ The multiple marker stock 'Master' was kindly provided by Wolfe, Brandon, Canada.
Furthermore, in some cases F1-spikes of single plants showed a different genetic behaviour, e.g. one spike exhibited a 3:1 segregation and another no segregation or an aberrant ratio.
The endosperm variegation, the clustering of waxy-grains and the differences between spikes on heterozygous plants suggest somatic events in different stages of development, causing also the frequently observed aberrant ratios (Schreiber and Habekuß, 1994). It was concluded, that these phenomena of instability at the waxy-locus are due to non-reziprocal mitotic recombination or gene conversion as it was evidenced for the v-lk region on chromosome 2 (Schreiber, 1992). However, in analyses of F3-progenies of reddish-brown stained F2-waxy-grains a high frequency of blue-stained starchy grains could be founded (Table 2).
Table 2: Frequency of revertants on unstable F2-plants of the cross Master x Mut. 152
________________________________________________________ F2-plant No. Number of analysed F3-grains ________________________________________ Brown Blue Frequency ________________________________________________________ 1b-4 327 13 4,0 x 10^(-2) 2a-21 266 14a 5,2 x 10^(-2) 2b-1 201 2a 1,0 x 10^(-2) 3b-2 75 9a 1,2 x 10^(-1) 3b-4 134 8a 6,0 x 10^(-2) 4b-6 23 5 2,2 x 10^(-1) ________________________________________________________These results were emphasized by staining of pollen grains (Table 3) according to the method of Rosichan et al. (1981). In comparison to the waxy-grain test in table 2 the decreased estimates of reversion frequencies of the waxy-pollen test in table 3 can be explained by the somatic nature of the reversion events. Then most of the blue-stained revertant grains of unstable waxy-lines did not inherited the changed character to the next generation. Thus single somatic events were counted in the grain test, whereas in the pollen test not single antheres but the pollen of hole spikes was analysed.
Table 3: Frequencies of reversion events in F3-waxy-lines
____________________________________________________________ F2-plant No. Frequencies of blue-stained pollen grains ____________________________________________________________ 1a-3 11,7 x 10^(-4) 1p-4 1,65 x 10^(-4) 1s-15 1,28 x 10^(-4) 1f-11 16,6 x 10^(-4) ____________________________________________________________Table 4: Frequencies of reversion events in F3-none-waxy-lines
__________________________________________________________________ F2-plant No. Frequencies of reddish-brown-stained pollen grains __________________________________________________________________ 1d-24 0,9 x 10^(-4) 1o-18 1,1 x 10^(-4) 1f-11 0,21 x 10^(-4) 1d-6 0,24 x 10^(-4) 1s-14 0,55 x 10^(-4) __________________________________________________________________Surprisingly not only the waxy-genotype (wxwx) exhibited instability but also the non-waxy genotype (WxWx) showed brown-stained revertants (Table 4). In F3 -progenies of the cross Mut.152 x Master or reciprocal we observed instability of the genotype wxwx in the direction Wx, besides instability of the genotype WxWx in the wx direction and finally aberrant ratios in the heterozygous genotype Wxwx (not shown). This peculiar phenomenon of instability can not be explained by gene conversion. In PCR analyses of the waxy-gene started recently we have not found any polymorphic sequences between waxy-genotypes and revertants up to now (Fig. 3).
Fig. 3: PCR analyses with different primers of wx-gene sequences of genomic DNA isolated from wild genotypes (WxWx) and waxy-lines (wxwx)
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft.