Zaleśna A, Choleva L, Ogielska M, Rábová M, Marec F, Ráb P
Department of Biology and Conservation of Vertebrates, Zoological Institute, University of Wroclaw, Wroclaw, Poland.
Cytogenet Genome Res. 2011;134(3):206-12. doi: 10.1159/000327716. Epub 2011 May 7.
The Western Palearctic water frogs Pelophylax ridibundus and P. lessonae were identified as parental (sexual) species and P. esculentus as their interspecific, hybridogenetically reproducing hybrid with hemiclonal heredity. We used genomic in situ hybridization (GISH) to identify parental chromosomes of P.lessonae and P.ridibundus in diploid P. esculentus karyotypes (2n = 26). GISH probes were made by fluorochrome labeling of total genomic DNA extracted from the sexual progenitors. The labeled probe from one species was hybridized to chromosomes of P. esculentus in the presence of excess of unlabeled genomic DNA from the other species. Thus, the P. lessonae probe was blocked by P. ridibundus unlabeled DNA, and vice versa. We successfully discriminated each of the 13 respective parental chromosomes in metaphase complements of the hybrids according to species-specific hybridization signals. GISH enabled us to confirm additional differences between parental chromosomes in size (smaller chromosomes belong to P. lessonae) and in the presence of DAPI-positive centromeric heterochromatin (detected in chromosomes of P. ridibundus, but not in P. lessonae). The fact that no visible intergenomic exchanges were found in metaphase chromosomes of diploid P. esculentus provides important information on the genomic integrity of hemiclonal transmission and supports hybridogenesis as a reproductive mode at the chromosome level for the specimens examined.
西古北区的泽蛙（Pelophylax ridibundus）和莱氏蛙（P. lessonae）被确定为亲本（有性）物种，食用蛙（P. esculentus）则是它们的种间杂交种，通过杂交起源进行繁殖，具有半克隆遗传。我们使用基因组原位杂交（GISH）技术，在二倍体食用蛙（2n = 26）的核型中鉴定莱氏蛙和泽蛙的亲本染色体。GISH探针是通过对从有性祖先中提取的总基因组DNA进行荧光染料标记制备的。来自一个物种的标记探针在另一个物种未标记的基因组DNA过量存在的情况下与食用蛙的染色体杂交。因此，莱氏蛙的探针会被泽蛙未标记的DNA阻断，反之亦然。根据物种特异性杂交信号，我们成功地在杂种中期染色体组中区分出了各自的13条亲本染色体。GISH使我们能够确认亲本染色体在大小（较小的染色体属于莱氏蛙）以及是否存在DAPI阳性着丝粒异染色质方面的其他差异（在泽蛙染色体中检测到，但在莱氏蛙染色体中未检测到）。在二倍体食用蛙的中期染色体中未发现明显的基因组间交换这一事实，为半克隆传递的基因组完整性提供了重要信息，并支持杂交起源作为所研究标本在染色体水平上的一种繁殖方式。
