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Duration of . Cast of American Beauty (1999) is made . IMDB rating of American Beauty (1999) is 8.5. IMDB is made by .1997](#ece33553-bib-0046){ref-type=”ref”}) and were used as maternal plants. Seeds from the progeny were planted in the field and morphological identification was performed at all generation.
For the experiment, three generation selfed and outcrossed lines were included to test the effects of selfing and outcrossing on the genetic diversity and frequency of LS alleles. In each experiment, a control (two generations of selfing) and an outcross (two generations of outcrossing) were included. Seeds of the control lines were collected and planted in the field. The progeny of the selfed lines were also collected and planted in the field as the second generation, resulting in the control and the outcrossed lines in each experiment. Seeds of the outcrossed lines were collected and planted in the field as the third generation, resulting in three generations of selfed and outcrossed lines for each experiment.
4.2. Molecular identification of LS alleles {#ece33553-sec-0010}
——————————————-
We carried out PCR using a series of primer pairs corresponding to regions of the *waxy* locus. These primer pairs were designed on the basis of the sequences of three LS loci (*r1, r2*, and *r3*; Morrissette, Scarth, & Durka, [1999](#ece33553-bib-0040){ref-type=”ref”}; Durka et al., [2002](#ece33553-bib-0015){ref-type=”ref”}; Gignoux et al., [2013](#ece33553-bib-0019){ref-type=”ref”}). PCR reactions were performed using a MyCycler™ System (Biorad, Veenendaal, The Netherlands) as described by Gignoux et al. ([2013](#ece33553-bib-0019){ref-type=”ref”}) with the exception that the cycling parameters were as follows: 94°C for 3 min followed by 35 cycles of 45 s at 94°C, 45 s at 60°C, and 90 s at 72°C, and a final step of 72°C for 10 min. The size of the PCR products was determined by agarose gel electrophoresis and visualized under UV light using the SmartLadder™ system (Biorad). The size of the PCR products was estimated on the basis of the migration of the marker designed for *E. influenza* (Muñoz‐Bertomeu, Luco, & Flors
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