Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium.
Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
PLoS One. 2024 Sep 19;19(9):e0306905. doi: 10.1371/journal.pone.0306905. eCollection 2024.
Transgenic lines engineered through wild type Rhizobium rhizogenes 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 (Osteospermum fruticosum 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 (rol) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (aux) were present in a single Ri line. Visualization of pRiA4 and pRi1724 rol and aux insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of rol integrated loci varied from 1 to 3 loci. In contrast, the different TR-gene copies were confined to a single locus which consistently co-localized with a TL locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 rol 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.
通过野生型根瘤农杆菌工程改造的转基因品系表现出一种称为 Ri 表型的改变表型。这种表型包括更紧凑的植物习性，这已被证明对获得需要更少化学生长调节剂的更紧凑品种有用。在这里，我们开发了一种用于海角雏菊（Osteospermum fruticosum Norl.）Ri 系的分子和细胞遗传学特征分析的方法，以便预测 pRi T-DNA 基因的分离。通过数字 PCR 进行的拷贝数变异 (CNV) 分析表明，插入的根癌基因座 (rol) 基因的拷贝数存在很大差异，从 1 个到 15 个以上。此外，在单个 Ri 系中存在多达 9 个生长素生物合成基因 (aux)。通过荧光原位杂交 (FISH) 在 4 个 Ri 系中观察到 pRiA4 和 pRi1724 rol 和 aux 插入。整合的 rol 基因座的数量从 1 个到 3 个不等。相比之下，不同的 TR 基因拷贝局限于一个单一的基因座，该基因座始终与 TL 基因座共定位，这是首次证明。基于 CNV 和 FISH，选择了一个携带 7 个 pRi1724 rol 基因拷贝分散在 3 个整合基因座中的 Ri 系进行繁殖。在 2、3、4 和 5 个 pRi1724 拷贝的 R1 后代中证实了拷贝数分离，表明可以通过 CNV 和 FISH 评估第一代 Ri 系的育种值。
