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Four mutants made up two complementation groups, Brazil nut and pine nut, and the remaining four mutants were single alleles.
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36% of the isolates were triple mutants, 14% were double mutants and 17% were single mutants.
Among these mutants, most were single nucleotide or short fragment-indels, while long fragment-inserts derived from the backbone of the gRNA vector were also observed.
The rest of the mutant cells were single yeast cells, cells of aberrant shape and also single cells with bent shape of the germ tube were observed (Fig. 4A).
All alleles sequenced were single mutants (data not shown).
Consistent with our conclusion that most adjacent crypts are not recently related, more than 75% of O-acetyltransferase mutant crypts are single crypts, although small clusters (typically two or three mutant crypts) were also observed [ 23].
Double mutants were obtained from single mutants by using the same primers as for the single mutations.
Or7a mutants were verified by single sensillum recordings and PCR.
A third mutant was tested in these cases to resolve which mutants were the true single-gene deletions.
For comparison, 19 of 38 of the other mutants from the screen were single-residue changes (unpublished observations).
Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris.
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