The identification of a liguleless2 genomic clone
--Justine Walsh and Michael Freeling

lg2 mutants are recessive and map to 3L-101. As homozygotes they remove ligule and auricle in the lower leaves of the plant. In subsequent leaves, ligule and auricle begin to appear at the leaf margins and, in later leaves, spread toward the midvein. The ligule and auricle are also often displaced. Thus, this mutation appears to affect the blade-sheath boundary as well as the formation of ligule and auricle. Mosaic analysis has shown that the Lg2+ product acts non-cell-autonomously (Harper, MNL 67:17, 1993).

lg2-R (reference) arose spontaneously and was described by Brink (J. Hered. 24:325, 1933). Seven putative Mutator-induced alleles have been recovered in this lab. lg2-2757 appeared in a screen of families of selfed Mutator-active plants. lg2-902 was recovered from a directed Mutator tag. We were able to increase the number of alleles to seven by identifying five more lg2 alleles (lg2-219, -228, -229.1, -229.2 and -278 ) in a directed Mutator tag completed this past summer.

We found a Mu8 homologous element that cosegregated with the lg2-2757 allele. The element was present in 43 lg2-2757/lg2-R individuals and absent in 36 +/lg2-R individuals. Using a Mu8 probe, a 5.5 kb EcoR1 genomic clone was identified from a bacteriophage library (Fig. 1). A 1.2 kb EcoR1/BamHI probe, representing non-Mu8 DNA, was used to probe genomic  DNA cut with an enzyme that does not cut in Mu8. In lg2-2757 individuals, this probe hybridizes to the same restriction fragment as Mu8.
 
Genomic DNA of the other six putative Mutator-induced alleles and their respective progenitor alleles was also hybridized with the EcoR1/BamHI probe. DNA differences were detected between the new lg2 alleles and their progenitor wildtype alleles. Initial results show that lg2-902, -228, -229.1, -229.2 and -278 carry some form of rearrangement or insertion and that lg2-219 is a deletion; thus, lg2 is cloned.

Fig 1: Restriction map of 5.5 kb EcoR1 liguleless 2 genomic clone.

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