CMS-S restorers-of-fertility from multiple sources cluster on chromosome 2L

--Gabay-Laughnan, S, Chase, CD

In the CMS-S system, expression of a novel chimeric gene in the mitochondria results in the collapse of starch-filling pollen and a male-sterile phenotype. Loss-of-function mutations in nuclear genes required for mitochondrial gene expression behave as restorer-of-fertility alleles, disrupting expression of the CMS-S gene and the male fertility trait. Restoring alleles also disrupt expression of essential mitochondrial genes. These mutations are visible in pollen because it is haploid; they are tolerated in pollen because late-stage pollen development and pollen germination do not require mitochondrial respiration (Wen et al., Genetics 165:771-779, 2003).

The first nuclear gene capable of restoring fertility to CMS-S male-sterile maize plants was designated Rf3 (Duvick, Adv. Genet. 13:1–56, 1965). The rf3 locus has been mapped to the long arm of chromosome 2 (2L) via the use of a chromosome 2 inversion and reciprocal translocations involving 2L (Laughnan and Gabay, Maize Breeding and Genetics, John Wily, New York, pp. 427–446, 1978; Gabay-Laughnan et al., in press). These studies placed rf3 proximal to the 2L.80 breakpoint. Through the use of RFLP markers, rf3 was positioned 4.3cM distal to the whp1 locus and 6.4cM proximal to the bnl17.14 locus (Kamps and Chase, Theor. Appl. Genet. 95:525–531, 1997).

Since Rf3 maps to 2L, this is usually the first chromosome arm that we test for linkage with newly arising CMS-S restorers. New restorers are located on a variety of chromosomes (reviewed by Gabay-Laughnan et al., Advances in Cellular and Molecular Biology of Plants. Volume 2: Molecular Biology of the Mitochondria, Kluwer, Dordrecht, Netherlands, pp. 395–432, 1995; Chase and Gabay-Laughnan, in press); while at least 47 of them map to chromosome arms other than 2L, there is a cluster of spontaneous restorers mapping to this arm. The spontaneously arisen restorers rfl1, rfl2 and rfn1 all map to 2L. The rfl1 locus exhibits linkage to the whp1 locus, thus placing it in the region of the rf3 locus (Wen et al., 2003). Direct tests of allelism have established that rf3, rfl1, rfl2 and rfn1 are distinct genetic loci. One spontaneous Oh51A restorer allele, rfl1-99829, has been mapped to the rfl1 locus while two Oh51A restorers, rfl2-911066 and rfl2-921663, map to the rfl2 locus. An additional Oh51A restorer allele, rfl*00-130, has just recently been mapped to 2L. Its relationship to the other 2L restorers is as yet unknown. In addition, three of the new restorers recovered from transposon-active lines map to 2L; rfl2-99114 from an Ac-Ds screen maps to the rfl2 locus while the unplaced alleles rfv*-991181, also from an Ac-Ds screen, and rfl*-003379 from an I-En (Spm) screen map to 2L. Tests for allelism of the unplaced restorers on chromosome 2L with Rf3, rfl1, rfl2 and rfn1 are underway. Thus, represented among the cluster of restorers on 2L is the entire range of types observed to occur: dominant and recessive; naturally occurring, spontaneously arising and transposon induced; homozygous viable, homozygous lethal and nonfunctional.

By genetic means, we can estimate the distance between Rf3 and the other restorers on 2L. Plants carrying Rf3 are intercrossed with plants carrying one of the new restorers. The resulting kernels are planted and, at maturity, pollen is examined to identify plants carrying both restorers. These are plants exhibiting 100%, or nearly 100%, normal pollen. Such plants are testcrossed with pollen from a normal-cytoplasm nonrestoring tester plant, and at least 200 kernels from each ear are planted. The resulting plants are scored for male sterility vs. male fertility by tassel examination at maturity. Fertile tassels are cut off and sterile or immature tassels are left. The plants are scored at least every other day until all reach maturity. The number of male-fertile plants is determined by counting the detasseled plants. The number of male-sterile plants is determined by counting the plants with tassels remaining.

Through use of this method, we have determined the positions of rfn1, rfv*-991181, rfl1, and rfl2 relative to rf3. rfn1 and rfv*-991181 are very closely linked to rf3. rfn1 and rf3 exhibit about 1.5% recombination, while rfv*-991181 and rf3 exhibit about 1% recombination. We cannot determine whether these two restorers are on the same side of the rf3 locus by this technique. The rfl1 and rfl2 loci each exhibit approximately 10% recombination with rf3. We cannot determine the order of these two loci, but they are on the same side of the rf3 locus.

Interestingly, two restorers for CMS-T maize, Rf8 and Rf*, either one of which can substitute for Rf1 to partially restore fertility, are either alleles or tightly linked genes on 2L near the rf3 locus (Wise and Pring, Proc. Natl. Acad. Sci. USA 99:10240–10242, 2002; D. Pei and R. P. Wise unpublished observations).

We propose to exploit our collection of restorer alleles for CMS-S maize residing on the long arm of chromosome 2 (2L) in order to characterize and clone nuclear genes that function in mitochondrial gene expression. In CMS-S maize, the biological system, the genetics, and the genomics tools come together, providing an unprecedented opportunity to conduct the molecular-genetic dissection of mitochondrial function in a higher eukaryote.