Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes

Abstract

The development of new molecular techniques (array-CGH, M-FISH, SKY-FISH, etc…) led to great 2 advancements in the whole field of molecular cytogenetic, however the application of these methods are still very 3 limited in farm animals. In the present study we report -for the first time-the production of 13 river buffalo (Bubalus 4 bubalis, 2n=50) chromosome-specific painting probes, generated via chromosome microdissection and DOP-PCR 5 procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of 6 river buffalo chromosome/arms namely 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X and Y, using both conventional and 7 late replicating banded chromosome preparations counterstained by DAPI. The provided 'Bank' of chromosome-8 specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for 9 other species of the family Bovidae, such as cattle, sheep and goats, for chromosome abnormality diagnosis, and, more 10 generally, for evolutionary studies.

Sequential Cross-Species Chromosome Painting among River Buffalo, Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

“…For the production of probes via chromosome microdissection, the river buffalo fixed lymphocyte suspension was spread onto a pre cleaned 24×60 mm coverslip, air dried and then treated for GTG-banding. According to Pauciullo et al [17] , the probes corresponding to the biarmed pairs (from 1 to 5) were produced by dissecting out the centromeric area, to avoid unspecific repetitive amplification of the centromeric regions. The probe corresponding to the X chromosome was produced by dissecting the region Xq21–25, analogous to the Xcen region of the bovine chromosome [18] .…”

“…Each probe was labelled separately by using a secondary DOP-PCR using 2 µL of products from the first reaction as template. Labelling scheme was performed according to Pauciullo et al [17] , with Spectrum Orange-dUTP and Spectrum Green-dUTP (Abbott, USA).…”

“…Six sequential rounds of FISH were performed on the same slide. Each round was realized by using two probes simultaneously hybridized on the metaphase plate according to Pauciullo et al [17] , with the exception of the second FISH round in which 3 probes (2p, 2q and 18) were used simultaneously. The labeled probes were mixed and each precipitated in absolute ethanol together with 10 µg salmon sperm DNA and 10 µg calf thymus DNA (both from Sigma).…”

“…In addition, only some laboratories of excellence in the world have probes availability [14] , thus limiting the application of the method to few research groups. For instance, Kubickova et al [15] proposed a tri-colour FISH to resolve rob (14; 20) and rob (16; 20) translocations in cattle, later seven-colour FISH using a specific paint pool was used in camels to identify smaller chromosomes [16] , whereas recently a pool of 13 chromosome-specific painting probes were used to develop a sequential multi-colour FISH in river buffalo to quickly identify submetacentric chromosomes and gonosomes [17] .…”

Characterization of a very rare case of living ewe-buck hybrid using classical and molecular cytogenetics

“…Painting probes corresponding to the goat acrocentric CHI 1, 2, 5 and sheep metacentric OAR 1q, 2q and 3q were produced via chromosome microdissection from river buffalo GTG-metaphases by scraping the following homologous chromosomes BBU 1q, BBU 2q, BBU 4q 38 . Microdissected chromosomes were amplified by DOP-PCR following the protocol of Pauciullo et al .…”