Studies On Mitochondrial DNA In Different Ploidy Level Fish And Sox Genes In Triploid Crucian Carp

As a unique cytoplasmic genetic system, mitochondrial DNA (mtDNA) was a small, self-replicating and diverse genome. The study on the mitochondrial genome was significant in the many biological issues, such as gene structure and function, gene expression regulation on time and space, nucleo-cytoplasmic interaction, the dynamics of molecular evolution, the origin of mitochondria, the origin and evolution of species, and so on. The formation of tetraploid stock of the red crucian carps (Carassius auratus red var., ♀, 2n=100) and common carps (Cyprinus carpio L., ♂, 2n=100) provided an important platform for gaining the unique fish materials with different ploidy levels including diploids, triploids and tetraploids. It was the first time to study the molecular genetic relationships among the above different ploidy level fish. In the present study, through studies of the molecular structures of the diploids, triploids and tetraploids, the mtDNA molecular genetic relationships between the different ploidy level fish and other related fish were illuminated. We have produced the triploid crucian carps by mating F3-F11 hybrids (♂) with Japanese crucian carps (Carassius auratus cuvieri) (♀). The triploid crucian carps have showed several enhanced performances such as sterility, fast growth rate, and good flesh quality. The Sox family was a kind of controlling development genes, and it taked the SRY/Sry gene as the basal member. How were the Sox genes in the sterile triploid crucian carps? It was worth to studying this question. The major results in this paper were presented as follows:1. The complete mtDNA sequences of the red crucian carp, triploid crucian carp and allotetraploid were determined. The characters of the molecular structure and gene organization of the mtDNA genomes in the above different ploidy level fish were also described. Through the comparative genomic analysis, we noticed that after 9 generations from F3 to F11 hybrids, the F11 allotetraploid possessed the stable maternal inheritance characteristic. Thus the tetraploid stockpossessed the good base to form a new tetraploid species in the future. Subsequently, the analysis of the mtDNA genetic variability in the triploid common carp (T3), its female parent allotetraploid and its male parent common carp was made. The result indicated that triploid common carp (T3) also possessed the strict maternal inheritance characteristic. However, in the triploid ciucian carp, we discovered the mtDNA recombination from the tRNA-Val gene to the NADH 5 gene with the paternal inheritance of the mitochondrial genome. The above results indicated that there were the mtDNA stable maternal inheritance characteristic, and the unique examples of the mtDNA paternal inheritance have also occurred in the different ploidy level fish.2. The mtDNA control region sequences from diploids, such as the red crucian carp, common carp, Japanese crucian carp, and zebrafish to polyploids, such as the allotetraploid, triploid crucian carp, and triploid common carp, were compared and analyzed. Based on the comparative analysis of the mtDNA control regions in cyprinids with different ploidy levels, the consensus sequences of functional conserved units and the borderline in the different regions of the mtDNA control regions in cyprinids were identified. These results provided the useful and important experiment data for the further study of the mtDNA control region structure in fish and the molecular evolution theory in vertebrate.3. The sequences of the mitochondrial tRNA-Thr gene, tRNA-Phe gene and partial control region in the allotetraploids and triploid crucian carps were analysed. The results showed the allotetraploid stock had the genetic diversity after 9 generations from F3 to Fn hybrids, and triploid crucian carp population possessed the abundant genetic diversity. Such results provided the important molecular biology references for the breeding, genetic conservation, and rejuvenation of the tetraploid stock and triploid crucian carp population.4. The Sox9 gene was a transcription factor required for cartilage formation and testis determination in mammals. We cloned two Sox9 genes from the triploid crucian carp, termed TCSox9a (full length) and TCSox9b (HMG-box).