Construction Silver Carp (Hypophthalmichthys Molitrix) And Bighead Carp (Aristichthys Nobilis) Genetic Maps And Cloning Of Prion Protein Encoding Gene Of Bighead Carp

Silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are two of the four most important pond-cultured fish species which inhabit the major river basins of China,including Heilongjiang River, Yangtze River and Pearl River.With the increase of human population and the intensification of economical activity,natural spawning and hatching grounds of the two fish are either polluted or altered,accelerating the loss of genetic resource and the decrease of natural fry production accordingly.Due to inappropriate broodstock management,continuous artificial propagation over a long period of time has caused tremendous decrease of genetic diversity and degeneration of disease resistance and growth performance. A set of appropriate markers are needed to detect these problems. Microsatellite DNA genetic linkage map of the two fish have not been constructed until now.Proteinaceous infectious agents (prions), the aggregation of prion protein (PrP), are the causing agents of transmissible spongiform encephalopathies ( TSE ). PrP is highly diverse and has a wide range of distribution. It is highly possible that prion and TSE could form in cultured fish through feed.In this study, microsatellite DNA markers were isolated from silver carp and used to detect genetic diversity of silver carp and cross-species amplification in bighead carp. Genetic linkage map of the two fish were constructed. Prion protein encoding gene of bighead carp was cloned and characterized from bighead carp.The following results were received: A (GT)n enriched library was constructed for silver carp using the FIASCO method. Of the 81 sequenced clones, 96 microsatellite DNA were identified,among them 68 were perfect, 22 were imperfect and 2 were compound. Besides 88 (CA/GT)n microsatellite DNA, other types of microsatellite DNA such as A7,(AG/TC)_n,(AT)₁₂,(ATC)₈,(TCCA)₆ were also been observed. The repeat number ranged from 5 to 103 with an average of 14.5.Sixty-seven pairs of silver carp microsatellite DNA markers were designed. Of them, 44 could get good amplification. All of the 44 microsatellite DNA markers used to investigate polymorphisms of 41 wild silver carps and seven wild bighead carps collected from Yangtze River. In silver carp, 40 markers were polymorphic. A total of 297 alleles were detected at 40 polymorphic loci. The number of alleles per locus varied from two to 16 with an average of 7.4 and the expected heterozygosities of these loci ranged from 0.07 to 0.91 with an average of 0.69. All markers amplified both silver carp and bighead carp DNA.Genetic maps of silver carp and bighead carp were constructed using microsatellite DNA (or simple sequence repeat, SSR) and amplified fragment length polymorphisms (AFLP) markers and two-way pseudo-testcross strategy. The map of silver carp consisted of 271 markers (48 SSR and 223 AFLP) which were assembled into 27 linkage groups, of which 22 contained at least 4 markers. The total length of silver carp map was 952.2 cM, covering 82.8% of the estimated genome size. The map of bighead carp consisted of 153 markers (27 SSR and 126 AFLP) which were organized into 30 linkage groups, of which 19 contained at least 4 markers. The total length of bighead map was 852.0 cM, covering 70.5% of the estimated genome size. Eighteen SSR markers were common to both maps. Two transcripts of bighead carp prion protein encoding gene were cloned and characterized through RT-PCR approach. The deduced amino acid sequences held conservative structure features of prions. One form of bighead carp PrP consisted of 526 residues with an estimated molecular weight of about 55kD, and the pI was about 8.9. The other one consisted of 519 residues with an estimated molecular weight of about 54.6kD,and the pI was about 9.0. They belonged to PrP2 when compared with 3 forms of PrP of Zebrafish. Phylogenetic analysis demonstrated that the homology between freshwater fish is higher than that between freshwater fish and marine fish. The number of tandem repeats decreased in freshwater fish, so it was speculated that the function of PrpC is relevant to salt metabolism.