Fig. 5

CenH3 immunofluorescence on Beta nuclei and FISH of pBV centromeric satellite on sugar beet. Nuclei were counterstained with 4’,6-diamine-2’-phenylindole-dihydrochloride (DAPI, blue signals) showing chromosome morphology. CenH3 binding was detected by the secondary antibody anti-rabbit coupled to fluorescein isothiocyanate (FITC; green signals). The pBV probe was labelled with biotin-11 UTP and detected by streptavidine-cyanine 3 (Cy3; red signals). Bar = 5 μm. a Localization of CenH3 on sugar beet metaphase chromosomes detected by immuno-FISH using the anti-bvCenH3 antibody and a centromeric satellite pBV probe as control. DAPI staining of an exemplarily chosen sugar beet metaphase shows chromosome morphology (left). Anti-bvCenH3 binding resulted in 36 signals, arranged in pairs corresponding to chromatides (middle left). 18 pBV signals (red) are detected and illustrate the centromeres (middle right). Overlay of all signals (right) reveals co-localization of two CenH3 signals within one large block of pBV on each chromosome (exemplarily indicated by arrowheads in all panels). b CenH3 localization during the sugar beet cell cycle. Anti-bvCenH3 binding displays CenH3 signals (green) in all mitotic cell cycle stages. 18 chromosomes are identified by anti-bvCenH3 binding on all chromosomes. Additional FISH using the centromeric satellite pBV probe (red) exhibits co-localization with CenH3 signals throughout the cell cycle. c CenH3 in species of the genera Beta and Patellifolia. Exemplarily chosen interphase nuclei of different Beta species are shown, including sugar beet, B. maritima, and B. patula, as well as P. patellaris belonging to Patellifolia. Areas exhibiting brighter DAPI staining indicate heterochromatic regions (exemplarily indicated by arrows, upper row). Distinct anti-bvCenH3 signals (green) were detected in sugar beet, B. maritima and B. patula in heterochromatic regions (arrows, middle and lower row). The 18 signals observed represent 2n = 18 chromosomes which is typical for diploid species of the section Beta. No signals were found on P. patellaris nuclei (genus Patellifolia)