Studies On Formation Of The Polyploidy Hybrids Of Red Crucian Carp (Carassius Auatus Red Var.)(♀)×Blunt Snout Bream (Megalorbama Amblycephala)(♂) And Their Biological Characteristics

The distant crossing is an effective means to increase genetic variation in the hybrid progeny. With this method, it is possible to form the different ploidy offsprings including the tetraploid hybrids, triploid hybrids, and gynogenetic diploid. In this study, we first obtained many different ploidy offsprings of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂), and their biological characteristics and the formation mechanism were studied. The major results were presented as follows:1. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (2n=100)(♀)×blunt snout bream (2n=48)(♂), which belonged to a different subfamily of fish and had different chromosome numbers in the catalog. The triploid hybrids resulted from the retention of the second polar body of the fertilized eggs and the tetraploid hybrids resulted from the inhibition of the first cleavage of the fertilized eggs.The 2-year-old female tetraploid hybrids reached maturity, and produced unreduced (4n) and reduced (2n) eggs. Pentaploid hybrids were found in the progeny of 4nRB(♀)×BSB (♂). The formation of the unreduced tetraploid eggs was also due to premeiotic endoreduplication, endomitosis, or fusion of germ cells of oogonia. The ploidy levels and chromosome composition of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers.2. The females and males tetraploid hybrids of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂) mated each other to generate the two types of F2(F2-A, F2-B). F2-A and F2-B fertilized with each other to form F3-A and F3-B, respectively. F2-A and F3-A were both tetraploid hybrids with 148 chromosomes with two sets from RCC and two sets from BSB; F2-B and F3-B both were tetraploid hybrids with 200 chromosomes with four sets from RCC. F2-B, F3-B, F2-A, and F3-A matured at the age of one year, and steadily produced diploid gametes. F3-B(♂) were mated with RCC(♀) to produce sterile triple crucian carp (3n=150) with fast-growth.3. Following activation by UV-irradiated sperm of blunt snout bream, without the cold-shocking, tetraploid eggs of the allotetraploid hybrids (red crucian carp (♀)×blunt snout bream (♂)) developed into normal live gynogenetic offsprings. All these gynogenetic progenies were female, including diploids with 100 chromosomes and tetraploid with 148 chromosomes. The gynogenetic progenies with 148 chromosomes matured at the age of one year, which produced tetraploid eggs.4. The females and unexpected males of natural gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (♀,2n=100)×blunt snout bream (♂,2n=48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC1) and another variational gray crucian carps (GGCC). The GRCC and their offsprings were proved to be diploids (2n=100) with one to three microchromosomes by examining the chromosomal metaphases and karyotypes. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish.5. Means of fluorescence in situ hybridization (FISH), Sox-HMG DNA markers, and microsatellite DNA markers were performed in bisexual GRCC and their parents, revealing their molecular genetic relationship and genetic variation, and further indicating the microchromosomes derived from the paternal fish. Our results proved that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish6. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp, blunt snout bream, and their polyploid offspring. The three monomeric 5S rDNA classes of RCC were characterized by distinct NTS types with 83, 220 and 357bp, respectively. In BSB, only one monomeric 5S rDNA was observed, which was characterized by one NTS type with 68bp. In the polyploid offspring, the tetraploid hybrids partially inherited 5S rDNA classes from their female parent; however, they also possessed a unique 5S rDNA sequence with a novel NTS sequence. The characteristic paternal 5S rDNA sequences were not observed. The 5S rDNA of triploid hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization.