Comprehensive study on assessment of Sturgeons population genetic structure in the Caspian Sea

The population genetic structure of five Caspian Sea sturgeon species was investigated. Totally 1121 samples of caudal and unault's fin tissue of the sturgeons (Acipenser persicus, A. gueldenstaedtii, A. stellatus, A. nudiventris and Huso huso) were collected from the Volga River (Russia), Ural River (Kazakhstan), Kura River (Azerbaijan), Sepidrud River and the coastline of the south Caspian in the Iranian waters as well as from the sampling stations selected for the marine survey for sturgeon stock assessment in the Caspian Sea. All samples were stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using phenol-chloroform method. The quality and quantity of DNA was assessed by Agarose gel (1%) electrophoresis and spectrophotometry. The population genetic structure of Ship and Persian sturgeon was studied using both PCR-RFLP (D-loop and ND5/6 gene) and microsatellite technique and that of H. huso, A. stellatus and A. persicus were studied using microsatellite technique. After amplification of genes using PCR, the RFLP technique was used to digest mtDNA using restriction enzyme. The PCR products were electrophoresed on 6% sequencing polyacrylamide gels followed by silver nitrate staining. Data for PCR-RFLP were analyzed using REAP program and those from microsatellite technique were analyzed using Gene Alex. Population genetic parameters including allele frequency, expected and observed heterozygosity, effective allele, Shannon's index were determined. Genetic identity and distance were calculated following Nei criteria and Hardy Weinberg equilibrium was tested based on X2 and analysis of molecular variance (AMOVA) using Reap and Gen Alex at 99% confidence limit. Phylogenetic relationship was determined and drawn using TFPGA program. The population genetic structure and genetic diversity of the 1121 sturgeon specimens were determined. Three independent populations were identified for Acipenser persicus (two populations in the south Caspian in the Iranian waters and one in the north Caspian). Three independent populations were identified for A. gueldenstaedtii (Volga, Ural and South Caspian populations) using the microsatellite technique. Population genetic structure using PCR-RFLP revealed no genetic differentiation among the A. gueldenstaedtii specimens studied from the different regions using ND5/6 gene, while two populations (Ural and south Caspian populations) were detected for this species with the same technique using D-loop genes. Four independent populations (Volga, Ural, Kura and Sepidrud populations) were reported for A. stellatus using the microsatellite technique and four more populations which most probably belong to the autumn and spring races of the above mentioned independent populations were identified for this species. The present study also identified two populations for H. huso; The North Caspian population (in Volga and Ural Rivers) and The South Caspian population (in Golestan and Guilan regions) which were significantly different from each other (P<0.01). The genetic population structure of A. nudiventris was studied using the microsatellite and PCR-RFLP techniques which revealed two populations for this species one in the Ural River and the other in the Sepidrud River (South Caspian). Comparison of the ND5/6 and D-loop genes studies in Russian sturgeon revealed that the D-loop gene is better than the ND5/6 genes in population's differentiation and is therefore strongly recommended for population genetic studies on sturgeons in the Caspian Sea. Genetic diversity studied using microsatellite technique was higher and more accurate as compared to that using RFLP. Nevertheless the RFLP technique was able to introduce molecular markers for the population’s species pacific identification. On developing suitable primers these studies can be speeded up and the cost of such studies can be cut down. However the drawback in using microsatellite technique for population genetic studies is that it cannot introduce a molecular marker for the identification of populations. The present study was able to introduce molecular markers to differentiate the ship sturgeon population in the south Caspian from that in the Ural River using the PCR-RFLP technique Based on the results obtained it is strongly recommended that all activities related to restocking and rehabilitation of sturgeon stocks in Iran be conducted on the basis of genetic principles. Also serious and immediate measures should be taken for the restoration and conservation of rare population of native species of Iran particularly in the Sepidrud region using genetic markers before they are become extinct.

Iranian Fisheries Science Research Institute

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