New alleles of Rough sheath1
--Richard Schneeberger and Michael Freeling

Rs1-0 is a dominant neomorph which causes severe developmental abnormalities of the ligule, sheath and blade due to ectopic expression of the Rs1 homeodomain protein in the leaf (Genetics 136:295). We report the description of three new probable Rs1 alleles, Rs*- mu64, Rs*-muC3 and Rs*-mu1429 in addition to one new Rough sheath mutation, Rs*-mu173 , that is unlinked to the Rs1 locus. All four mutants were originally identified in our Mutator selfing blocks and in unrelated directed tagging experiments. Each new isolate was crossed into B73, W23 and Mo17 inbred lines for three generations to obtain good expression and penetrance. All four isolates express well in the B73 background and to a lesser extent in W23 and Mo17. All four mutants closely resemble the Rs1-O phenotype, with expression seen on most leaves starting on the third to fifth leaf and terminating near and sometimes including the flag leaf.

Table 1. Linkage data for four Rs* mutations.
F1 Total Observed Normal (No.) Recombination (%)
Rs1- Z4 /Rs-*muC3 190 1 0.5±0.5
Rs1-Z4 /Rs- *mu64 398 1 0.25±0.25
Rs1-Z4 /Rs-*mu1429  198 0 0
Rs1-Z4 /Rs-*mu173 249 114 46±3

In order to determine if these mutations are linked to the Rs1 locus the new isolates were crossed to a Rs1 tester and the resultant double mutants outcrossed to normal plants. The tester was a homozygous Rs1-Z4 B73 inbred stock which expresses Rs1 phenotype at the seedling stage. Double mutants (e.g., Rs1-Z4/ /Rs*-muC3) were selected in the F1 progeny and outcrossed as females by B73. The F2 progeny were scored for Rs1 and normal phenotypes. The data shown in Table 1 indicate strong linkage of Rs*-muC3, Rs*-mu64 , and Rs*-mu1429 to Rs1-Z4. The cross to Rs*-mu173 showed normal segregants in the F2, indicating that this mutation is unlinked to the Rs1 locus. RFLP analysis using the cloned rs1 gene was performed on 10 mutant and 10 normal individuals from families segregating 1:1 for each of the linked mutations to provide further evidence that the linked Rs* mutations are alleles of the Rs1 gene. In each case a rs1 RFLP was identified that absolutely cosegregated with the mutant phenotype in five enzyme surveys (data not shown). A similar RFLP analysis was also performed on the Rs*-mu173 mutation and no rs1 cosegregating RFLPs were detected. The tight linkage, similar phenotypes (including lack of a mutant dosage effect), and the identification of rs1 RFLPs linked with the mutant phenotpye indicate that these new isolates are probably alleles of Rs1. Therefore we will refer to them as Rs1-C3, Rs1-64, and Rs1-1429 . We are currently mapping the Rs*-mu173 mutation to determine if it represents a dominant mutation of another member of the maize homeobox gene family. 

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