leafy mutant stocks

Detlef Weigel Detlef_Weigel at QM.SALK.EDU
Fri Oct 25 11:38:33 EST 1996


We discovered recently that the original =93lfy-6=94 stock contained a
mixture of two lfy alleles, lfy-6 and another allele, now designated
lfy-26.  lfy-26 is a strong lfy allele, in which codon 376 (TGG,
tryptophan) is changed to a stop codon (TGA).  By chance, the two
genomic clones that were recovered from the original =93lfy-6=94 stock an=
d
sequenced corresponded both to the lfy-6 allele, as described (Weigel et
al., Cell 69, 843-59, 1992).  In subsequent backcrosses, again by
chance, the lfy-26 allele became fixed.  Most extant =93lfy-6=94 stocks a=
re
therefore lfy-26.  However, we have been able to reisolate lfy-6 (as
well as lfy-26) from the original stock.

Both alleles can be genotyped by CAPS (cleaved amplified polymorphic
sequence; Konieczny and Ausubel, Plant J. 4, 403-10, 1993) or D-CAPS
(derived CAPS; Alan Pepper and Michael Neff, pers. communication; see
also Jacobson and Moskovits, PCR Methods Appl. 1, 146-8, 1991) markers. =

The lfy-6 mutation introduces a Mae III site into the genomic sequence. =

For lfy-26, genomic DNA is amplified by PCR using two primers, 5=92-TTT
AAC GCT CAT CCT CCA CTC TCT ATT TG-3=92 and 5=92-TTA ATT AGG ATA ACG AC-3=
=92
(corresponding to nucleotides 5220-5248 and 5424-5440, respectively, of
LFY genomic sequence [GenBank accession number M91208]).  The
restriction enzyme Xcm I recognizes the target sequence CCA (N)9 TGG and
will cleave only wild-type DNA, yielding a 191 bp and a 29 bp fragment.

We have submitted new lfy-6 and lfy-26 stocks to the Ohio stock center. =

We thank Xuemei Chen for first alerting us to the presence of the lfy-26
allele, and Alan Pepper and Michael Neff for introducing us to the
D-CAPS technique.

-- =

Detlef Weigel, Ilha Lee, Ove Nilsson
Plant Biology Laboratory
Salk Institute
La Jolla, CA 92037
PH (619) 453-4937
FX (619) 558-6379
EM Detlef_Weigel at qm.salk.edu



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