Fluorescein 12 dutp

FISH using a telomere probe, GISH, and image analysis were performed according to Akiyama et al. (2010) with slight modifications. Genomic DNA of L. multiflorum and F. arundinacea was directly labeled with fluorescein-12-dUTP (PerkinElmer Co., Billerica, MA, USA) and Texas Red-5-dUTP (PerkinElmer Co.), respectively, by a nick translation kit (Roche, Basel, Switzerland). For the telomere probe, Arabidopsis telomere repeat sequences (Richards and Ausubel 1988 (link)) were PCR labeled with biotin-16-dUTP (Roche) and detected by Streptavidin DyLight 549 (Vector Laboratories, Burlingame, CA, USA) as the first layer of signal amplification, biotinylated anti-streptavidin (Vector Laboratories) as the second layer and again with Streptavidin DyLight 549 as the third layer. Chromosome spreads were observed under an Olympus BX61 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a DP72 CCD camera (Olympus Optical Co., Ltd.). Based on the GISH results, the ratio of the Festuca-specific genomic component to the total festulolium genome (referred to as the f ratio) was analyzed using intensity values in DAPI images by Adobe Photoshop CS6 Extended (Adobe Systems Inc., San Jose, CA, USA) on a Windows 7 platform (Microsoft Corporation, Redmond, WA, USA). At least 6 chromosome spreads were analyzed to determine the f ratio in each sample.

FISH to detect integration of pattBhybrid FVIII was performed on interphase nuclei of genome-modified CLECs with fluorescein-12-dUTP (PerkinElmer, Waltham, MA, USA) random prime labelled probe generated by PCR amplification of neomycin cDNA from pattB hybrid FVIII (817-bp PCR product: forward primer 5′-TTGCACGCAGGTTCTCCGGC-3′ reverse primer 5′-GGCGTCGCTTGGTCGGTCAT-3′). Texas-Red-5-dUTP-labelled human chromosome 8 centromeric probe (Children's Hospital Oakland Research Institute, Oakland, CA, USA) was used to determine colocalization with chromosome 8. Hybridized slides were counterstained with 4,6-diamino-2-phenylindole and visually enumerated for probe signals with an Olympus BX61 epifluorescence microscope (Olympus, Tokyo, Japan).