S1 File -

<div><p>Transgenic lines engineered through wild type <i>Rhizobium rhizogenes</i> display an altered phenotype known as the Ri phenotype. This phenotype includes a more compact plant habit, which has proved useful to obtain more compact varieties that require less chemical growth regulation. Here, we develop a method for the molecular and cytogenetic characterization of Cape daisy (<i>Osteospermum fruticosum</i> Norl.) Ri lines in order to predict segregation of pRi T-DNA genes. Analysis of copy number variation (CNV) by means of digital PCR indicated large variation in the copy number of the inserted root oncogenic loci (<i>rol</i>) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (<i>aux</i>) were present in a single Ri line. Visualization of pRiA4 and pRi1724 <i>rol</i> and <i>aux</i> insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of <i>rol</i> integrated loci varied from 1 to 3 loci. In contrast, the different T<sub>R</sub>-gene copies were confined to a single locus which consistently co-localized with a T<sub>L</sub> locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 <i>rol</i> gene copies dispersed over 3 integration loci, was selected for breeding. Copy number segregation in R1 progeny of 2, 3, 4 and 5 pRi1724 copies was confirmed, indicating that the evaluation of the breeding value of first generation Ri lines is possible through CNV and FISH.</p></div>