Studies On Genetic Characteristics And Grass Carp Reovirus Resistance Of F₁Hybrids Between Grass Carp（Ctenopharyngodon Idellus） And Barbel Chub（Squaliobarbus Curriculus）

Grass carp (Ctenophaiyngodon idellus), one of four chinese carps, occupies a pivotal position in conventional freshwater fish industries of China. However, to date, the germplasm resource of grass carp has been declining, along with a lot of diseases. Grass carp hemorrhagic disease, an epidemic disease caused by grass carp reovirus, is still no effective methods to control and seriously restricts the develpoment of aquaclture in China. With the development of biological techonology, enhancing the disease resistance of grass carp by inproving germplasm resource must be a useful approach to slove the tough problem. Distant hybridization, an important way for fish breeding, has been used in many cases and has good effect.Barbel chub (Squaliobarbus curriculus), belonging to Leuciscinae as grass carp, is the ideal hybrid object for grass carp. Not only is barbel chub similar to grass carp in appearance but also the barbel chub possesses outstanding capability of disease resistance. Therefore, we carry out the expriment of distant hybridization between grass carp and barbel chub from2010, and we obtain two populations of hybrids:the F1hybrid of grass carp (♀) x barbel chub (♂)(direct cross F1)and the F1hybrid of barbel chub (♀)×grass carp (♂)(reverse cross F1). To explore the feasibility of hybridization and the heterosis of hybrids, the characteristics of appearance, erythrocyte, growth, genome DNA, mitochondrial DNA and the Grass Carp Reovirus (GCRV) resistance of reciprocal cross F1between grass carp and barbel chub were studied. The major results are as follows:1. Comparative analysis of morphology, growth and cytogeneticsThe somatotype of5months old direct cross F1and reverse cross F1were more similar to barbel chub by visual inspection. With age, the characteristics of somatotype, body color, squamation of two years old reciprocal cross F1were more similar to grass carp. Hybrid indexes indicated that four of the eight measurable and numerable characters of direct cross F1were biased toward grass carp, the others were biased toward barble chub; while five of eight of reverse cross F1were biased toward barble chub, three were biased toward grass carp. The size of erythrocyte was no significant difference among grass carp, barbel chub and their direct cross F1, but the ratio erythrocyte long diameter/short diameter of reverse cross F1was minimum.which indicated the erythrocyte of reverse cross F1was most round.By pond culture, the absolute mean body weight and mean body length of reciprocal cross F1were superior to the F1of grass carp and the F1of barbel chub. The hybrid indexes of weight gain rate of reciprocal cross F1,which were superior to the F1of grass carp but worse than the F1of barbel chub, were intermediate type between two parents2. Comparative analysis of molecular geneticsGenetic diversity of grass carp, barbel chub and their reciprocal cross F1was analyzed by Random Amplified Polymorphic DNA (RAPD). These results illustrated the Nei’s gene diversity and Shannon’s Information index of barble chub were highest, then direct cross F and grass carp, while the Nei’s gene diversity and Shannon’s Information index of reverse cross F1were lowest. The genetic identity and UPGMA analysis indicated the reciprocal cross F1were more similar to barbel chub.Genetic structure and genetic diversity analysis of mitogenomes showed the composition and organization of mitogenomes of the reciprocal cross F1and barbel chub were the same as that of other cyprinid fish, including two ribosomal RNA genes,13protein-coding genes,22transfer RNA genes, and two non-coding control region. The results indicated genetic structure of fish mitogenome was highly conserved sequence.Genetic identity and cluster analysis results showed the reciprocal cross F1were more similar to female parent, although there were some mutations which were mainly base substitution. The genetic identity between Xiangjiang River barbel chub and the lower reaches of Yangtze River (Jiangsu province) barbel chub was99%3. Comparative analysis of the characteristic of GCRV resistanceThe Mx gene cDNA sequences of the direct cross F1and reverse cross F1,2306bp and2330bp in length respectively, were cloned by RT-PCR and improved RACE methods. The length of their open reading frames and amino acid sequences were both1884bp and627aa. respectively. The two Mx genes were found containing DYNc domain in the N-terminal and GED domain in the C-terminal. There are only three variation sites in amino acid sequences between direct cross F1and reverse cross F1. Multiple sequence alignment showed the genetic identity of Mx between hybrids F1and grass carp was higher than that between hybrids F1and barbel chub. After challenge with GCRV, the survival rates of reciprocal cross F1were higher than that of barbel chub and significant higher than that of grass carp, while the survival rate of barbel chub was higher than that of grass carp.each blood physiological and biochemical index showed the similar variation trends in the four kinds of fish within96h, respectively. The concentrations of T-SOD and IL-1β were intermediate type between two parents, while the enzyme activities of C3and IFN-a were more similar to female parent. The results indicated the concentrations of T-SOD of grass carp, barbel chub and their reciprocal cross F1were down-regulated within96h after infection by GCRV, which implied the dismutation of superoxide of the four kinds of fish were reduced. At the time point of96h, the enzyme activities of C3of direct cross F1and the concentrations of IL-1β of direct cross F1and reverse cross F1were significantly increased.The enzyme activities of IFN-a of the four kinds of fish were down-regulated in the first36h after infection, then up-regulated to normal level or higher than normal.qPCR assays showed expression of Mx and IFN transcripts was observed in liver, spleen, intestine, gill, head kidney and muscle of grass carp, barbel chub and their reciprocal cross F1although the levels were different. Within96h after challenge with GCRV, overall pattern of expression of Mx and IFN transcripts was fluctuated in all the tested tissues of four kinds of fish, and the expression of Mx and IFN gene transcripts of reverse cross F1and barble chub were higher than that of direct cross F1and grass carp.The expression pattern of IFN gene of reciprocal cross F1were similar with that of the maternal homologous offspring, but the expression of IFN gene transcripts of direct cross F1was higher than of grass carp in all detected tissues at the time point of96h. It was concluded that Mx gene was inducibly up-regulated by GCRV, but the up-regulated Mx could inhibit the expression of IFN to a certain extent.Comprehensive analysis of the results of the survival rate, blood physiological and biochemical indexes, cloning and expression of anti-virus genes, showed the GCRV resistance of barbel chub was better than that of grass carp, so were the reciprocal cross F1.