Establishment And Biological Characterization Of The Diploid Gynogenetic Hybrid Clonal Line Of Red Crucian Carp × Common Carp And Future Derivative Generations

A bisexual fertile allotetraploid (AT) population was obtained by crossing red crucian carp (Carassius auratus red var.,♀,2n=100) with common carp(Cyprinus carpio L., (?),2n=100). Gynogenesis was researched on the diploid eggs produced by AT, and a gynogenetic diploid hybrid clone line was established. The diploid eggs produced by gynogenetic diploid hybrid clone line were fertilized with the diploid sperm of AT to produce a new type of tetraploid fish (G×AT,4n=200). Importantly,2% of G1×AT with the obviously high body height to body length were found in G1×AT population. Self-mating of the high-body G1×AT produced three kinds of bisexual fertile diploid fish:high-body red crucian carp, high-body fork-like-tails goldfish and high-body gray common carp. The main contents of this paper included:①Establishment and genetic mechanism of the diploid gynogenetic hybrid clonal line.②In-depth analysis the cell and molecular biological characteristics of AG to provide insight into the evolutionary origin of goldfish.③Application and biological characterization of high-body crucian carp and high-body gray common carp. The major results were presented as follows:1. Based on the formation of the third gynogenetic generation (G3), G4 and G5were obtained by gynogenesis. Thus, a diploid gynogenetic hybrid clonal line (G1-G5) of red crucian carp X common carp was established. For further study of the mechanism of diploid fish produce diploid eggs, we cultivated the early-stage gonadal cells of G5 in vitro. During the gonad proliferation stage, we found the phenomenon of germ cell fusion. While in the control group, grass carp and common carp gonads, there was no phenomenon of cell fusion. These results were of great value to reveal the mechanism why diploid hybrids produce the diploid gametes.2. The morphological characteristics of artificial goldfish (AG) were observed. The results showed that AG and the natural goldfish (NG) with the similar traits in morphological. Compared with original parents RCC and CC, AG showed more similar characteristics with RCC, but it was great different from CC. It can determine the genetic relationship between goldfish and crucian carp from the morphological traits. AG possessed 100 chromosomes with the karyotype 22m+34sm+22st+22t, and the DNA content was same with diploid RCC. Both ovary and testis gonads of AG were able to produce normal mature gametes after one year culture.3. To detect the molecular genetic characteristics of AG, the Sox genes of NG, AG, RCC and CC were detected firstly. AG and RCC amplified the same bands consistent with the-200,-600 and-1900bp, in NG there were only-200 and-600bp bands, and in CC there were only-200 and-900bp bands. PCR amplification of 5S rDNA results showed that AG and NG with the same amplified band, and both of them had a unique band (168bp) different from RCC and CC.5S rDNA fluorescence in situ hybridization results showed that AG has two strong hybridizing signals as RCC and NG, while in CC there was no signal detected.The complete sequence of mitochondrial DNA of AG was 16579bp. Compeared with original parents RCC and CC, the similar rate were 99.5% and 89.4%, respectively, in line with the maternal inheritance of mitochondria. However, AG had the high similarity rate with NG (99.4%), indicating that they may have the same origin.The number of alleles varied from one to eight in AG, and the size of the alleles ranged from 140-300bp. The amplification patterns of 7 pairs of microsatellite primers showed that AG had a higher degree of genetic polymorphism, a greater genetic heterogeneity, indicating that the source of genome was complexity and the gene was highly hybrid.4. The biological characteristics of high-body crucian carp were studied, the results revealed that high-body crucian carp was a diploid and bisexual fertile fish, possessed many improved features such as smaller head, higher body and shorter caudal. An improved triploid was produced by crossing improved tetraploids with improved diploids. The improved triploids showed some features such as high-body, fast growth, infertility, and high nutrient contents.5. The phenotypes, chromosome numbers, gonadal structure and Sox gene of ICC were observed. The results revealed that ICC was a diploid and bisexual fertile fish, and showed the significant features of the high-back compared with CC. It was worth noting that the Sox gene amplification results showed that ICC was a hybrid, which had the same amplified bands with AT (-200,-600,-900 and-1900bp), while CC had-200 and-900bp bands.These results indicated that to improve the distant crossing hybrids by gynogenetic, the offspring can differentiate into different types of new varieties of fish, which appearance and genetic characteristics had major changes. Such of various improved fishes plays an important role in biological evolution and fish genetic breeding. Furthermore the formation of AG provided the direct evidence for the evolutionary origin and diversification of goldfish.