Linkages among zein genes as determined by IEF

Seventy different zein polypeptides have been characterized in 18 maize inbreds by serial analysis with isoelectric focusing in agarose (IEF) followed by SDS-PAGE (Wilson, Plant Physiol. 82:196, 1986). Each zein polypeptide is identified by a letter and number related to its relative molecular mass and by a number (between 10 and 60) related to its isoelectric point. A-zein is often called 22 kDa zein, though its true mass is near 27 kDa. B-zein is called 19 kDa zein but is really about 24 kDa. D-zein is called 10 kDa, though its true mass has not been determined. A 1 to 10 scale gives finer discrimination to the A and B zeins. Band identifications were made by running inbreds and individual zeins on gels adjacent to well-characterized "standard" inbred lines.

Eleven different crosses were made among inbreds that had been characterized by serial analysis. Some lines contained su1 as a marker for chromosome 4 and y8 or wx T7-9(4363) for chromosome 7. Backcrosses by one or both parents or the F2s (for the translocation crosses only) were made, and seeds were analyzed for occurrence of zeins. As many as 120 seeds of each class were assayed, so that linkages could be determined (N.T.J. Bailey, Introduction to the Mathematical Theory of Genetic Linkage, 1961).

The results obtained from the cross W64 x A619 are presented here, along with a summary of results from all crosses. Fig. 1 shows the zein patterns obtained with agarose IEF N is the inbred N28, a standard used to check the pattern. The zeins which differ between the two parental inbreds are identified on the right of the gel. Six backcross seeds pollinated by P1 (W64A) are on the left, pollinated by P2 (A619) are on the right of the gel. Note that bands 10 and 22 are present or absent (0 or 2 doses) in BCP1 while they occur in 1 or 3 doses in BCP2 on the right. They appear to be linked. Bands 32 and 33.5 occur or are absent together in 9 seeds, while 3 seeds show apparent crossing-over. It was possible to score 11 zeins for this cross. The results from 240 seeds are given in Table 1.

Assignments to chromosomes 4 and 7 were made with the crosses involving the markers. Chromosome 4 contains both sizes of the major zeins, A and B, and they occur in two regions of the chromosome separated by about 23 cross-over units. Five zein genes are so tightly linked that no confirmed crossovers were detected. Genes for three B-zeins and one D-zein were found on chromosome 7, with the B-zeins being closely linked.

Zeins 32 and 49 gave different recombination values for different crosses, suggesting that the bands represent genetically different zeins in different inbreds. Two inbreds were found to contain both A1/33.5 and A3/33.5 zeins, which could not be distinguished by IEF alone. Large-scale scoring by SDS-PAGE is not practical yet, but examination of a few F3 seeds revealed that A1/33.5 and A3/33.5 can be separated by crossing-over. Band D/55 was linked to wx T7-9(4363) in two crosses. Several bands scored as being D/55 were lifted from the IEF gel and run on SDS-PAGE to confirm their identity. Some inbreds were found to contain a 135/55 zein. Thus, it appears that zeins from different inbreds which have been mapped to the same position on IEF gels may have different genes. Some zeins not resolved by present electrophoretic techniques may also be different genetically. The linkages reported here differ in several instances from those reported by Ottoboni and Steffensen (MNL 59:99, 1985, and Biochem. Genet. 25:123, 1987). Although they used the same methods, they analyzed too few seeds and did not take into account the linkage of some zein genes on chromosome 7. Joint studies are being made to resolve these differences. Work is in progress to use HPLC to further characterize individual zeins and detect double zeins in IEF bands.

Figure 1. IEF of zeins from the cross W64 x A619. N is N28, a standard inbred, P1 is W64A, P2 is A619, and F1 is P1 x P2. Six BC-1 seeds are on the left, six BC-2 seeds on the right. The numbers are the IEF identifications for each band.

Table 1. Zein gene linkages. Recombination frequencies calculated for backcrosses of W64A X A619, with suggested order for zein genes determined from this and 10 other crosses. Zeins are identified by SDS-PAGE mobility, IEF band number, and inbred origin in first column, by IEF number only on the upper row.

C.M. Wilson, T.C. Nelson and G.F. Sprague


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