SEQUENTIAL HYBRIDIZATION OF SIX SPECIES-SPECIFIC PAINTING PROBES TO MAKE THE FIRST MULTI COLOR FISH IN RIVER BUFFALO (Bubalus bubalis 2N=50)

In recent years, the progress of molecular cytogenetic led to an evolution of the fluorescence in situ hybridization (FISH) methods. Multiplex-FISH (M-FISH), spectral karyotyping (SKY), and combined binary ratio labeling FISH (COBRA-FISH) allow the simultaneous visualization of chromosomes in different colors. Their application in human clinical cytogenetic makes plainer the identification of chromosomal abnormalities. Conversely, in animal cytogenetic the use of these methods is still very limited. In this work we propose the first river buffalo multi-color FISH by the production of a small specific chromosome painting probe library. Six specific river buffalo autosomal painting probes were prepared through conventional microdissection and DOP-PCR. Probes were labeled with digoxigenin-11- and biotin-16-dUTP in a second DOP-PCR. Three sequentially rounds of FISH were achieved for the same slides. Each round was realized using two probes simultaneously hybridized on the mitosis. Biotin-labeled probes were revealed by FITC-avidin, whereas digoxigenin-labeled probes were revealed by antidig-rhodamine. Slides were counterstained with DAPI in antifade. Digital images were captured in gray-scale and pseudo-colored by the computer. Chromosome microdissection and DOP-PCR were used to produce six species-specific probes, painting 3 out of 5 sub-metacentric river buffalo chromosomes (BBU 1p, 1q, 3p, 3q, 4p and 4q). The simultaneous hybridization of these probes allowed to develop the first multi-color FISH in this species. Nowadays, the lack of chromosome-specific probes commercially available for individual animal species represents a limiting factor for cytogenetic investigations in farm animals. Therefore, the creation of a such collection of probes opens further opportunity for clinical cytogenetic applications also in river buffalo. Acknowledgements: This study was supported by CISIA-VARIGEAV project, National Research Council (CNR) of Italy