Cloning of three putative defective kernel loci by transposon tagging

--M. G. James, M. S. Scanlon, D. S. Robertson and A. M. Myers

Approximately 75 defective kernel mutants were generated from crosses with Robertson's Mutator stock. Using Mu as a transposon tag, genomic DNA that cosegregates with the mutant phenotype was cloned from three of these mutants (dek*-1047, dek*-NS807, and dek*-NS326) into phage lambda vectors. Mutant dek*-1047 (which has a flattened, nearly empty pericarp) was produced by the cross of standard and Mutator plants, and mutants dek*-NS807 (which has a shrunken, slightly opaque kernel) and dek*-NS326 (which has a nearly empty seed) were gifts from Nancy Shepherd, E. I. DuPont de Nemours & Co. Another mutant received from Nancy Shepherd, dek*-NS95, was found to be allelic to dek*-NS326.

All three putative defective kernel loci contain Mu1-homologous insertions of approximately 1.4kb, most likely Mu1. In at least two instances, sequence analysis of the Mu-element junctions shows 9bp genomic DNA repeats. Genomic fragments flanking the Mu elements of all three clones were subcloned into plasmid vectors and used as hybridization probes. In each instance, this DNA is single-copy and shows cosegregation with the defective kernel phenotype. Restriction maps for each of the cloned fragments are presented in Figures 1-3.

Figure 1. Restriction map of pMS1, containing cloned fragment from dek*-1047. E = EcoRI; N = NotI; B = BamHI; S = StuI; P = PstI; Bg = BglII; Nc = NcoI.

Figure 2. Restriction map of pMJ1 and pMJ2, containing cloned fragment from dek*-807. H = HindIII; S = SstI; P = PstI; N = NotI; X = XhoI; Xb = XbaI; E = EcoRI.

Figure 3. Restriction map of pMJ26 and pMJ27, containing cloned fragments of dek*-326.. H = HindIII; P = PstI; S = SstI; N = NotI.

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