Identification of genetic marker for differentiation of Persian sturgeon (Acipenser persicus) from Russian sturgeon (A. gueldeustadtti)

The Persian sturgeon (Acipenser persicus) is more abundant sturgeon species in the South Caspian Sea and consist the highest proportion of Iranian Caviar, meat as well as bringing maximum foreign currency income, however from systematic point of view and differentiation of this species from Russian sturgeon (Acipenser gueldenstadttii) a serious challenging issues remain, where some Russian scientist are believe that the Persian sturgeon is not as an valid species and consider it as a subspecies of Russian sturgeon. This research conducted with the objective of identification and introducing a molecular marker based on specific DNA for differentiation of two species of Persian sturgeon and Russian sturgeon via a proved molecular marker method. For this purposes 8 different molecular approaches such: Microsatellite, AFLP, RAPD, sequencing of Cytb, 16sDNA, ND5, Growth Hormone gene and finally Single Nucleotide Polymorphism (SNP) were investigated. Based on applied methodology, between 5 to 16 caudal fin tissues were sampled for each species from different region of the Caspian Sea, Sefiedrud River, Ural and Volga rivers. Following DNA extraction, its quality and quantity were determined and the PCR experiment has been conducted using 5-110 primers according to various methods and type of gene. The PCR products were electrophoresed on Polyacrilamid or agarose gels and followed by silver and Ethidium Bromide staining. In RAPD method, polymorphic DNA band was cut on the gel followed by purification and then the segments were cloned in vector in Top10 strain of E.coli, and then sequenced. Meanwhile for Growth Hormone gene in Persian and Russian sturgeon the MEGA 4, Gene runner software were used to design the appropriate primers for PCR amplification. The PCR products were cloned in PTZ57R/T vector and transformed in Top10 E.coli strain and sequenced finally. For all other genes, similar methods were applied for PCR amplification and its products were sequenced and statistical analysis as well as phylogenetical tree was performed. In Single Nucleotide Polymorphism (SNP) method, after genomic library construction, in total 14.4 billion nucleotides were sequenced and similarity/ differentiation analysis of two species were investigated using specific bioinformatic software. Results indicated that Microsatellite and AFLP methods showed high level of genetic variation both within and between species. The Cytb gene, when 4 sample sequences from each species were compared two species were differentiated, however when analysis repeated over 15 samples, the sequence comparison couldn't differentiate two above mentioned species. Full sequence comparison of 16sDNA and mtDNA-ND5 gene showed variation in some nucleotide in both species of Persian and Russian sturgeon but no significant. Results of sequences obtained from cloned segment with RAPD method and also specific primer design based on produced sequences could succeed to discover a variable DNA band that able to differentiate two species from each other. Results of the present study also showed that the growth hormone gene (GH) of Persian and Russian sturgeon consists of 645 nucleotide that translate to 214 Amino Acids. The sequence comparison indicated that the gene coding growth hormone in Persian and Russian sturgeon had the highest similarity with GH of Mammals (71%), Anguilaformes (63%) and less similarity with bony fish (37%). Phylogenetic analysis indicates that Persian and Russian sturgeon in compare to other organism are ancient species and this gene is originated from a common ancestor. At present study the most appropriate results obtained from Single Nucleotide Polymorphism (SNP) method by sequencing 14.4 billion nucleotide from genome of two species of Persian and Russian sturgeon from North and the South Caspian Sea could prove that the Persian sturgeon is a valid and independent specie. This excellent results is the biggest scientific achievement for differentiation of two highly commercial important sturgeon species in the Caspian Sea in last two decades