| Chinese sturgeon (Acipenser sinensis) is a large anadromous fish which is listed as the national level for the protection of wild animals in China. Performing controlled breeding are indispensable ways to support the recovery of this endangered species. Although the controlled propagation of Chinese sturgeon has been succeed, it did not get enough attention in study of genetic diversity, genetic structure and pedigree in A. sinensis population. The genetic management for broodstock of Chinese sturgeon is still quite weak.In this study, microsatellite primers were developed in Chinese sturgeon, which were used to study the paternity test and population genetics. The genetic diversity of broodstock was assessed by microsatellite analysis in three Chinese sturgeon cultured populations. The research will not only provide a scientific basis for establishing pedigree and genetic management strategy, but also offer a reference for paternity test and analyzing genetic structure of other sturgeon.(1) We isolated and characterized a total of 24 polymorphic microsatellite loci using Illumina sequencing for Chinese sturgeon. Primers were designed for each of 165 microsatellites with enough flanking sequences and more number of repeats (>6) using Primer Premier 5.0. The PCR was conducted in 24 wild individuals and the PCR products were separated on 12% non-denaturing polyacrylamide gel and visualized by silver staining. The number of alleles (Na) per locus ranged from 2 to 6, expected heterozygosities (He) and Shannon-Wiener Diversity Indices (SW) ranged from 0.235 to 0.786 and from 0.396 to 1.608, respectively. Exact tests revealed that 9 loci showed significant (P<0.01) deviation from Hardy-Weinberg equilibrium (HWE). No (P>0.05) linkage disequilibrium was detected among these loci. The loci tested by PAGE showed that Asi-67123, Asi-77057 and Asi-67648 displayed more than 4 bands; Asi-55961, Asi-52396, Asi-74654, Asi-68632 and Asi-62964 displayed 2 bands; other loci (Asi-72632, Asi-66034, Asi-46902, Asi-68191, Asi-48230, Asi-65194, Asi-75067, Asi-75905, Asi-71347, Asi-71954, Asi-73843, Asi-76964, Asi-72040, Asi-74518, Asi-70421 and Asi-56700) displayed 4 bands in these specimen.(2) 10 novel microsatellite markers were used for assessing genetic diversity of 68 Chinese sturgeon individuals from two families. The results showed that there were 124 alleles in 10 loci. The Na, He and SW per locus ranged from 7 to 22, from 0.733 to 0.873 and from 1.545 to 2.179, respectively. With the criterion that cumulative exclusion probabilities were not lower than 0.99, the 7 high polymorphic microsatellite markers (Asi-75067, Asi-67648, Asi-67123, Asi-73843, Asi-72040, Asi-70421 and Asi-56700) make up the system of paternity test for Chinese sturgeon. The cumulative exclusion probabilities over the 7 loci of single parent, paternity and pair parent were 0.991 232,0.999 503 and 0.999 997, respectively. These high polymorphic microsatellite markers will provide a scientific basis for validating paternity testing efficiently in Chinese sturgeon.(3) The genetic diversity of 215 first filial generation specimens randomly collected from three Chinese sturgeon farms (Taihu (TH), Hengsheng company (HS) and Fenghuangshan (FHS)) in Hubei Province, China were investigated using 12 microsatellite loci. A high level of genetic diversity was observed in all three culture breeds with the Na, He and SW per locus varying from 10.250 to 13.417, from 0.790 to 0.802 and 1.830 to 1.953, respectively. A middle-level genetic differentiation and no inbreeding was observed among samples collected among three cultured farms based on Gst values ranged from 0.037 to 0.193 and Gis -0.018 to 0.095. The Analysis of Molecular Variance (AMOVA) showed that most genetic variation distributed within populations, and very little genetic variation distributed among populations. The 215 individuals were divided into two clusters based on Calinski and Harabasz’pseudo-F and were divided into six clusters based on Bayesian Information Criterion. Thus, the findings of this study will be valuable for protecting the extant population of Chinese sturgeon. At the same time, it is essential to provide baseline data on the state of genetic diversity for evaluating the effect of controlled reproduction and national policy of conversation of this endangered species. |
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