Establishment And Application Of Hybrid Lineage Between Megalobrama Amblycephala(?)× Culter Alburnus(?) And Its Genetic Improvement

The blunt snout bream(BSB,2n=48,Megalobrama amblycephala)is one of the three major herbivorous fishes in the world,and it is also one of a high-quality freshwater fish in China;the topmouth culter(TC,2n=48,Culter alburnus)is a carnivorous fish with delicious meat and is widely distributed in major river systems of China.These two fishes belong to the Cyprinidae and Cultrinae but different Genera:Megalobrama and Culter,respectively.Distant hybridization is a commonly used method in fish genetic breeding,which can merge different genomes from different species.However,the established hybrid lineage by distant hybridization are of great significance in terms of production applications and species formation.In the past ten years,our laboratory established the hybrid lineage of BSB(?)×TC(?)(BTF₁-BTF₅).In this study,we produced the sixth generation(BTF₆)and an improved hybrid culter by this hybrid lineage.Furthermore,we induced the gynogenesis using this hybrid lineage and successfully obtained gynogenetic groups.In addition,biological characteristics such as genetics in those fish were analyzed.The main research contents were as follows:1.Researches regarding reproduction of hybrid lineage and its genetic characteristic.Based on the established hybrid lineage(F₁-F₅)of BSB(?)×TC(?),BTF₆ was successfully obtained by self-crossing of BTF₅.Through analyses of the appearance,DNA content,and chromosome number of the parents and the hybrid lineage(F₁-F₅),it was found that there is no significant difference between BTF₆ and other hybrids.Ten Hox genes with significant SNPs differences between BSB genome and TC genome were amplified from the DNA of BTF₁ to BTF₆ and used to analyze genetic variation during the establishment of hybrid lineage.The results revealed that the most of heterologous genes can exist stably in hybrid fish,and only a small amount of heterologous genes from TC were losted during this process.The chimeric genes were found in BTF₁,and part of them were propagated to BTF₆.What's more,the gonadal chromosomes of BTF₁ were prepared and observed.We found that heterologous chromosomes derived from BSB and TC exhibited normal pairing and segregating during meiosis.We also found that the Dmc1 and REC8 genes related to meiosis had no significant difference in gene structure between BSB and TC,and the gene expression level were normal in BTF₁.These results indicated that the reason why the offspring of the hybrids can produce haploid gametes normally through meiosis may be related to the high similarity of the homologous genes regarding to meiosis between BSB and TC.These results indicated that this lineage was a new hybrid lineage and can be stably inherited.2.Researches on the formation of hybrid culter by the back-crossing of hybrid lineage and it biological characteristics.We obtained a new type of hybrid culter derived from a hybrid lineage of BSB(?)×TC(?).On the basis of analyses of DNA content,chromosome number,45S r DNA and morphological traits,we determined that the hybrid culter was not only an allodiploid with 48 chromosomes but also similar in appearance to TC but with a greater body width.Furthermore,the new hybrid culter contained fewer IBs(reduced by 5.70%)than that in TC.Expression patterns of homologous genes revealed that up-regulated expression was the major pattern in BTBT,and the gene number whose expression level biased toward the BSB genome was approximately equaled to that in TC genome.Those results revealed that BTBT is an improved culter with higher quality and it is expected to serve as a new resource for the genetic breeding of culter.3.Researches on the establishment of gynogenetic groups from hybrid lineage and it biological characteristics.1)By inducing the gynogenesis of BTF₁,two types of offspring with obvious differences in appearance were successfully obtained,namely,GBTF₁-B and GBTF₁-M,respectively.After analyses of chromosome number,and microsatellite,we determined that both GBTF₁-B and GBTF₁-M were allodiploids.The expression level of homologous genes between GBTF₁-B,GBTF₁-M,BSB and TC were analyzed using liver transcriptome,and the results revealed the expression of GBTF₁-B was biased toward BSB,while GBTF1-M was intermediate between BSB and TC.2)We also induced gynogenesis in BTF₄ derived from the specific-lineage of BSB(?)×TC(?),integrating the technologies of hybridization and gynogenesis,and successfully obtained GBTF₄.We determined that GBTF₄ was a diploid gynogenetic hybrid fish,which had no significant appearance difference to BTF₄.On the other hand,we also investigated whole genomic changes and DNA methylation between GBTF₄ and BTF₄.Although the individuals of GBTF₄ were derived from individuals of BTF₄,they exhibited 12.28%genomic alterations,30.01%methylation pattern alterations and lower methylation level overall than BTF₄.In addition,0.03%of fragments were traced to introgression from UV-irradiated sperm.These results enriched our knowledge regarding the combination of hybridization and gynogenesis and provided new hybrid germplasm resources.To sum up,based on the established hybrid lineage,a series of researches had been carried out in this study:in breeding,we obtained a new generation(BTF₆)by self-crossing of BTF₅ and a new type of hybrid culter by two rounds of back-crossing from this hybrid lineage.In terms of genetic characteristics,the genetic variation during the establishment of the hybrid lineage,genetic component of hybrid culter and the genetic,transcriptional and epigenetic variations of the gynogenetic hybrid fish were analyzed.In terms of the reproductive characteristics,the question regarding hybrid fish produced normal haploid gametes during the meiosis were found and explored.These results are of great significance in the establishment and application of hybrid lineages of fish its genetic improvement.