Analysis of somaclonal variation-induced zein mutation
--Marilia Gaspar, Sergio T. Sibov, Anete P. de Souza and Laura M. M. Ottoboni

The occurrence of somaclonal variation in plants regenerated from calli established from inbred line Cat-100-6 was observed. One out over two thousand R0 regenerated plants, designated somaclone S1587, presented increased growth rate, tillering and high proliferation capacity. R1 to R5 progenies, obtained by self pollination of S1587, presented a high mutational frequency, giving rise to endosperm mutants, defective seedling and variegated leaves. Most of the mutations were determined by single recessive genes and presented unstable Mendelian segregation along the successive self pollinated generations. However, some mutations were stable.

Molecular analysis of somaclone S1587 was carried out for one of its progeny, designated S1587-17, for the zein genes. Zein proteins were extracted from S1587-17 and Cat-100-6 endosperms and submitted to isoelectric focusing (IEF) analysis. The IEF gels showed that several alterations have occurred in the somaclone S1587-17 zein profile. Since each IEF band corresponds to a single structural gene, it can be concluded that at least 6 zein genes (for proteins Z1, Z2, Z3, Z4, Z5 and Z6) were altered in the somaclone. 2D equivalent analysis of the different zein IEF bands of the two genotypes indicated that five corresponded to the 22 kDa zeins and one to the 19 kDa zeins. To investigate the segregation of the 22 kDa and 19 kDa zein genes, zein was extracted from 112 F2 (Cat-100-6xS1587-17) seeds, and subjected to IEF. Preliminary results indicated that five of the altered zein genes were segregating in a typical Mendelian ratio. Linkage analysis of the segregating zein genes indicated that they are grouped in two clusters (Fig. 1). Southern analysis of the S1587-17 and Cat-100-6 genomic DNA digested with EcoRI using a 22 kDa and a 19 kDa zein cDNA as probes revealed, as expected, several alterations in the organization of the zein genes, with the disappearance, appearance and amplification of bands in the somaclone. Since 5 out of the 6 altered zein bands in the somaclone belonged to the 22 kDa zein genes, and considering that these gene are mainly located on maize chromosome four, experiments using RFLP probes from chromosome four are underway to confirm the localization of the affected zein genes.
Figure 1. Zein proteins from one hundred twelve F2 seeds were analyzed by IEF in order to detect linkage between the Mendelian segregating zein polypeptides: Z1, Z2, Z3, Z4 and Z6. Linkage was determined, using Mapmaker (Lander et al., Genomics 1:174-181, 1987) version 2.0, on an Apple Macintosh computer. Markers were initially assigned to groups using the group command, and loci were mapped with the "three-point" and compare command. A minimum LOD of 3.0 and a maximum recombination frequency of 0.4 was used to verify the linkage orders. The genes for Z2 and Z6 are linked and mapped far away from Z3, Z1 and Z4.

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