Mapping zein polypeptides using translocations

This is the first report of an extensive plan to locate and map all of the zein proteins to chromosomes using reciprocal translocations as chromosome arm markers, duplicate-deficient types and segmental aneuploids. The greatly improved method for the separation of zein polypeptides by isoelectric focusing (IEF) in agarose gels (Wilson, Cereal Chem. 61:198-200, 1984) makes our goal possible. At least 36 different zeins have been identified in various stocks and inbred lines. Isopropanol-mercaptoethanol soluble endosperm proteins from four homozygous inbred lines (W23, L317, Oh43 and W64A) were separated by charge in IEF agarose. From each of the four gel patterns, individually stained bands were cut out from the dry agarose gel, boiled in SDS buffer, loaded and separated in 12% acrylamide SDS-6M urea gels. Our numbering system is like that of Wilson (Biochem. Genetics, in press, 1985) in IEF gels, expanded by mol. wt. designations from SDS gels. For example, zein band 10 in IEF agarose from Oh43 and Oh45 has a molecular wt. of 19.6kd in SDS-urea. Our notation is 10-19.6, combining an Rf equivalent (distance from the cathode) with the observed mol. wt.

As observed by others, each inbred line and homozygous translocation stock has a specific zein profile or "fingerprint". IEF band 22 (22-22.5, 22-19.6) is a double band in SDS gels and it is present only in the Oh43 group (Oh43, Oh45, Oh545 and W153R), while band 21.5 (21.5-22.5, 21.5-19.6) is in the SSS group (RSSC, B73, A632, A634) and in L317. These two 22.5 and 19.6kd zein genes appear to be linked and to segregate with 4L.

The maize stocks used in the experiments were fourteen homozygous inbred lines and several homozygous translocation stocks with breakpoints on chromosome 4 and on other chromosomes. The F1s were obtained by crossing homozygous inbred lines to chromosome 4 translocations. The F1s then were backcrossed to several inbred lines to obtain the F2. Homozygosity in the parents was important to obtain a known dosage of genes without extraneous segregating factors. To test for homozygosity, zein extractions from ten seeds from each of the parents were run side by side in IEF agarose gels. No segregation was observed in any of the parents.

Distally located translocation break points often survive on the female side and give duplicate-deficient endosperm. The F1 seed from T4-10f (4L.94; 10L.14) provide such duplicate-deficient progeny. We assume 10L is duplicate and 4L is deficient, since 10L deficient and 4L duplicate seeds are lethal as observed on the F2 ear. With those assumptions, we have tentatively assigned 41-14.3 and 49-19.6 to map at 10L and 22-22.5, 22-19.6 are located distal to 4L between .94-1.0.

Dosage effects were observed in the IEF pattern of hybrids as is expected with the triploid endosperm, since hybrids receive two genes from the female parent and one gene from the male parent. The cross W153R x T4-9g (4S.27; 9L .27) backcrossed to L317 gave two zein bands, 54-19.6 and 53-19.6, that are not visible in any of the parents. These zeins seem to be under the control of a regulatory gene.

The results from the other crosses between the inbred lines and the translocation stocks are summarized as follows:

W23 x T4-9(5974) (4L.80; 9L.87) was backcrossed to N28. Bands 22-22.5, 22-19.6 and 53-19.6 are in coupling, thus linked. Both are located on the long arm of chromosome 4.

B73 x T3-4(5156) (3S.47; 4L. 67) was backcrossed to Oh43. 28-22.5 and 33.5 - 22.5 are in repulsion with one putative crossover observed between these two loci. Both are located on chromosome 4.

N28 x T3-4(5156) (3S.47; 4L.67) was backcrossed to W23; a. 21.5 - 22.5, 21.5 - 19.6 and 53 - 19.6 are in coupling with their location on 4L; b. 28 - 22.5 and 54 - 19.6 are in repulsion and also on 4.

W153R x T4-9g (4S.27; 9L.27) was backcrossed to W64A, 10 - 19.6 and 22 - 22.5, 22-19.6 showed a perfect correlation, like the band pattern of the T4-9g parent.

Laura M. M. Ottoboni and Dale M. Steffensen


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