On the way to tagging the Rf2 restorer of cms-T

--John R. Laughnan and Susan Gabay-Laughnan

There are two known nuclear restorers of cms-T, Rf1 in chromosome 3L, and Rf2 in chromosome 9S closely linked with the waxy-1 (wx) locus. Rf1 and Rf2 are complementary in their interaction, that is at least one dose of each is required for restoration of fertility to cms-T. Moreover, unlike the gametophytic mode of cms-S restoration, cms-T restoration is sporophytic in nature, meaning that it is the genotype of the pollen-producing plant rather than the genotype of the pollen grains themselves that governs pollen viability. Hence, a cms-T plant that is heterozygous for both restorers, Rf1 rf1 Rf2 rf2, produces all normal pollen even though only one-fourth of the pollen grains carry both restorers (Rf1 Rf2).

Like others, we are attempting to tag the Rf2 gene because molecular studies on cms-T restoration have so far shed no light on its function. To this end we have searched for exceptional male-sterile offspring among the progeny of the cross: (T) R213: Rf1 Rf1 Wx rf2/Wx rf2 Bz2 Bz2; no Ac X (N) rf1 rf1 Wx:Ac Rf2/wx Rf2 bz2-m/bz2-m. The R213 Rf1 Rf1 rf2 rf2 female parent is an inbred line derived from inbred lines WF9 and Ky21 by Jack Beckett, JB and is male-sterile in cms-T plants. The male parent, kindly provided by Drew Schwartz, D, carries an insertion of Ac (Activator) in the starchy allele (Wx:Ac), and is homozygous for bz2-m (bronze-2 mutable), which derives from an insertion of Ds (Dissociation) into wildtype Bz2, in chromosome 1L. We have determined, by suitable testcrosses, that this male parent has the genotype rf1 rf1 Rf2 Rf2, which means that it carries the sought after cis arrangement of the two closely-linked genes Wx:Ac and Rf2. Since the male parent carries Wx:Ac Rf2 in one chromosome 9, and wx Rf2 in the other, two progeny types are expected with equal frequency. One has the genotype (T) Rf1 rf1, Wx:Ac Rf2/Wx-rf2, Bz2/bz2-m, and the other carries wx in place of Wx:Ac, but is otherwise the same.

Table 1 summarizes the results of our search for male-sterile plants among the offspring of this cross. Fourteen male-parent sources were involved in a total of 89 crosses that produced 8,876 offspring. Four of the pollen sources were tassels of main plants and 8 sources were tassels of tillers, here designated as, for example, "1833-9a" and "1833-9b". We were constrained to use tiller tassels because the main tassels of the male-parent strain were often "shed out" by the time the later-maturing (T) R213 plants were silking. Among the 8,876 offspring, 90 (1.0%) were male-sterile plants. Most of these exserted no anthers, but some exhibited islands of florets, whose anthers contained normal pollen, on tassels that were otherwise sterile.

Table 1. Male-sterile progeny from the cross: (T) R213: Rf1 Rf1, Wx rf2/Wx rf2, Bz2 Bz2; no Ac X (N) rf1 rf1, Wx:Ac Rf2/wx Rf2, bz2-m/bz2-m.
      Male-sterile plants
Male parent No. of crosses Total plants No. %
930-1a 5 310 1 0.3
930-4a 4 332 6 1.8
930-5a 10 1452 4 0.3
931-1a 9 735 0 ---
931-2 2 103 0 ---
931-3 6 642 0 ---
931-6a 8 675 13 1.9
1833-7 1 20 0 ---
1833-7a 2 64 0 ---
1833-8 2 46 0 ---
1833-9 17 2219 66 3.0
1833-9a 11 998 0 ---
1833-9b 9 1046 0 ---
1835-4 3 234 0 ---
TOTALS 89 8876 90 1.0

The distribution of male-sterile offspring among the male-parent sources provides a clue to their origin. Only 5 of the 14 sources produced male-sterile offspring, but at least three of these, 930-4a, 931-6a and 1833-9, have far higher frequencies of male-sterile offspring than would be expected if these were distributed randomly among the total population. This is not likely due to genetic variability in the progeny since both male and female parents are essentially inbred lines whose offspring are expected to be genetically uniform. That the male-sterile plants are not randomly distributed is evident from the progeny of crosses involving 1833-9 and its two tillers, 1833-9a and 1833-9b. Pollen from 1833-9 produced 66 male-sterile offspring in a population of 2,219 plants. The tiller tassels of this plant produced no male-sterile offspring among totals of 998 and 1046 plants, respectively. Clearly, all or most of the 66 male-sterile plants in the progeny of 1833-9 are the product of a somatic (premeiotic) event in the tassel elements of this plant, presumably at about the 32-cell stage of tassel primordia. A similar event may well account for the high frequencies of male-sterile plants from male parent sources 930-4a and 931-6a, but in the cases of the two remaining sources, 930-1a and 930-5a, no such conclusion is warranted.

At this time we have no conclusive evidence that Ac insertion into Rf2 is involved in the occurrence of the male-sterile plants. Alternatively they could be the result of some other kind of forward mutation of either Rf1 or Rf2. For those cases that result from a premeiotic event in the male parent the mutation must involve Rf2, not Rf1, since the male parent genotype is known to be rf1 rf1 Rf2 Rf2.

Genetic studies underway now will determine whether or not Rf2 mutation is involved in the production of the male-sterile exceptions, and whether they have occurred in both types of progeny, only one of which carries Ac. Laboratory investigation is being conducted to determine whether any of these mutations represent Ac-tagged Rf2 alleles.

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