The frequencies of gains or losses for each single clone were compared between the non-GCB and GCB groups.
Using ligation-mediated (LM-) PCR we identified specific integration site (IS) patterns for each single clone in vitro (Figure 2).
After 7 days, single cells were selected from clonal rings and these were seeded on 24 well plates for further propagation of each single clone.
To minimize "stutter" artifacts from PCR amplification of repetitive sequences, independent triplicates of PCR amplification were performed for each single cell clones on each polyG loci, and manual size calling was further performed on each locus to ensure accuracy.
Each replication included 2 clones for each parent and a single clone for each of 10 genotypes in each hybrid family, for a total of 36 plants.
For single clone culture, individual large NS (diameter ≥ 150 μm) was collected and trypsinized, as described above.
For each single-copy clone, the insertion site of the docking site was determined by splinkerette PCR, a PCR protocol devised for the sequencing of sequences flanking P-element insertions (Potter and Luo 2010), and duplicate clones were discarded.
All primer information and PCR conditions for every single clone can be retrieved from our Xq28 database [ 22].
Integrated SYBR signals for each cell of a single clone were usually unique; therefore, a central tendency, henceforth referred to as the mode, of the integrated SYBR signal was determined for each clone using a custom MATLAB script (MathWorks, Natick, MA).
First, to provide additional insurance that each of the three environmental isolates were axenic, we performed multiple rounds of antibiotic washing, dilution, and plating for single clones on tris-phosphate (TP) plates.
