MILAN, ITALY
University of Milan

Identification of a RAPD marker associated with Rf3
--Renato Tarchini, Andrea Rossi, Mario Enrico Pè and Mirella Sari Gorla

Cytoplasmic male sterility (cms) is a maternally inherited trait, widely used in many crop plants for the production of hybrid seed because male fertility can be restored by the action of one or more nuclear restorer genes. cms has been extensively studied in maize, where four major cytoplasm types have been described: the normal cytoplasm (N) and three cytoplasm types (designated as C, T and S) causing male sterility, distinguishable on the basis of their interaction with specific nuclear restorer genes.

Rf3, a nuclear gene mapped on the long arm of chromosome 2, is required for the restoration of fertility in cms type S (Laughnan and Gabay-Laughnan, Annu. Rev. Genet. 17:27-48, 1983). Rf3 acts as a dominant gene with gametophytic expression: cms-S plants heterozygous for the restorer locus (Rf3 rf3) produce 50% normal pollen, according to a 1:1 segregation pattern of the two alleles in microspores after meiosis. Little is known about the nature or function of Rf3, or about the mechanism(s) by which male sterility is overcome.

In order to identify molecular markers tightly linked to Rf3, we have analyzed Near Isogenic Lines (NIL) sharing the same Ny821 genetic background by means of RAPD markers: differences in the amplification patterns obtained from NILs should indicate polymorphisms in a region linked to Rf3. Amplifications have been performed on DNA extracted from all four genetic combinations of N and S cytoplasm with the two allelic forms of the restorer in homozygous condition, to avoid misinterpretations due to the amplification of cytoplasmic components. Several decamers from Operon Inc. (Alameda, CA) have been used under the conditions indicated by Williams et al. (in Methods in Enzymology, Academic Press, Orlando, Florida, 1991).

One of the primers tested has revealed an amplification product of approximately 1900 bp, present in the combinations cyt-N Rf3 Rf3 and cyt-S Rf3 Rf3, but not in the combinations cyt-N rf3 rf3 and cyt-S rf3 rf3 (Fig. 1). DNA corresponding to the polymorphic band has been extracted and used as a probe onto genomic DNA in a Southern blot. Our results indicate that this DNA corresponds to sequences present in low or medium number copy in the maize genome. We are now performing cosegregation analysis on a backcross population in order to confirm the linkage of this sequence with Rf3.

This result, although preliminary, is of particular interest because it indicates the possibility of saturating the region surrounding Rf3 with closely linked molecular markers.

Figure 1. RAPD amplification of four Ny821 Near Isogenic Lines. Lanes 1, 6: Ny821 cms-S Rf3 Rf3. Lanes 2, 7: Ny821 cms-S rf3 rf3. Lanes 3, 8: Ny821 normal Rf3 Rf3. Lanes 4, 9: Ny821 normal rf3 rf3. Lanes 5, 10: Ny821 cms-S Rf3 rf3. M: lambda PstI marker.
*polymorphic band observed.


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