Studies On Formation Of The Different Ploidy-level Hybrids Of Red Crucian Carp(Carassius Auratus Red Var.)(♀)×Topmouth Culter (Erythroculter Ilishaeformis Bleeker)(♂) And Their Biological Characteristics

Polyploidization, the addition of an extra set (or sets) of chromosomes to the genome, is a predominant mechanism for speciation in plants and animals. Polyploidization can occur via somatic doubling, the fusion of unreduced gametes, and by means of a triploid bridge or polyspermy. The current prevailing opinion is that hybridization plays an important role in triggering polyploidization. Polyploid species are particularly frequent in the plant kingdom. However, while it is generally relatively rare in animals, polyploidy has occurred extensively, independently, and is often repeated in many groups of fish. Furthermore, artificially induced polyploidy has been used in aquaculture to produce sterility and to improve production. Polyploidy in fish represents a useful model system with which to test theories about the origin and consequences of polyploidy that have been derived from work on plants. Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae,2n=100)×topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂,Cultrinae,2n=48) were successfully produced. This is the first report on the formation of these viable diploid, triploid and tetraploid hybrids by crossing different parents with a different chromosome number in vertebrates. In addition, studies of these different ploidy-level hybrid offspring on ploidy-level, chromosomal number, karyotype, mean DNA content, appearance, Sox genes, the organization and variation of5S rDNA and location of5S rDNA on the chromosomes were conducted, and the major contents are as follows:1) Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (RCC♀) x topmouth culter (TC♂) were successfully produced. The different ploidy-level and chromosome constitution of these hybrid offspring were determined with chromosome counting and detection of mean DNA content of blood cells. The distribution of chromosome number in RCC, TC,2nRT,3nRT and4nRT hybrids are as follows:For diploid RCC, the chromosomal metaphases had100chromosomes with the karyotype formula of22m+34sm+22st+22t; For diploid TC, the chromosomal metaphases possessed48chromosomes with the karyotype formula of16m+26sm+6st. In2nRT hybrid offspring of RCC♀±TC♂, the chromosomal metaphases had74chromosomes with the karyotype formula of19m+30sm+14st+11t, indicating diploid hybrids possessed74chromosomes with one set from RCC and one set from TC1In3nRT hybrid offspring of RCC♀×TC♂, the chromosomal metaphases had124chromosomes with the karyotype formula of30m+47sm+25st+22t, suggesting triploid hybrids harbored124chromosomes with two sets from RCC and one set from TC; In4nRT hybrid offspring of RCC♀×TC♂, the chromosomal metaphases had148chromosomes with the karyotype formula of38m+60sm+28st+22t, showing tetraploid hybrids had148chromosomes with two sets from RCC and two sets from TC. In addition, the earlier embryonic development of RCC, TC and their hybrid offsrping were detected, and the fertilization rate, hatching rate and adulthood rate were also involved.2) At2years of age, the measurable and countable traits in2nRT,3nRT and4nRT hybrids were examined, which showed the hybrid characteristics. Using RCC and TC as controls, the observation of microstructure and ultrastructure in2nRT and3nRT hybrids was conducted. An artificial propagation of2nRT and3nRT hybrids was also involved during the reproductive season, but neither egg nor sperm were able to be squeezed out from2nRT or3nRT hybrids. Results obtained here indicated that2nRT and3nRT hybrids were sterile. Due to the few number of4nRT hybrids, neither the detection of microstructure and ultrastructure nor an artificial propagation of4nRT hybrids was conducted. The confirmation of fertility in4nRT hybrids needs further study.3) Against the conserved HMG-box of Sox genes, one pair of degenerate primers were designed and synthesized to amplify the genomic DNA of RCC, TC and their different ploidy-level hybrid offspring. There were three DNA fragments of Sox genes (with the length of215bp,617bp and1958bp) in RCC, only one (with the length of215bp) in TC. In the hybrid offspring, there were three DNA fragments (with the length of215bp,628bp and1959bp) in2nRT hybrids, three DNA fragments (with the length of215bp,616bp and1957bp) in3nRT hybrids, four DNA fragments (with the length of215bp,628bp,918bp and1959bp) in4nRT hybrids. A novel918bp DNA fragment was observed in4nRT hybrids.4) The5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric5S rDNA classes (designated class I:203bp; class II:340bp; and class III:477bp) in RCC and two monomeric5S rDNA classes (designated class IV:188bp, and class V:286bp) in TC. In the hybrid offspring,2nRT hybrids inherited three5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC).3nRT hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC).4nRT hybrids gained class II and class III from their female parent (RCC), and generated a new5S rDNA sequence (designated class I-N). The specific paternal5S rDNA sequence of class V was not found in the hybrid offspring.5) Through fluorescence in situ hybridization (FISH), the fluorescence probes amplified from the parental-special5S rDNA fragments of RCC and TC (RCC-340bp, TC-188bp and TC-286bp) were hybridized to the mitotic metaphase chromosomes of RCC, TC,2nRT,3nRT and4nRT hybrids, respectively, and the location of5S rDNA loci on the chromosomes were also involved. The results obtained here indicated that hybridization and polyploidization had a huge impact on the5S rDNA loci, including the appearance of novel5S rDNA loci and lose of5S rDNA loci.