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In order to analyze the genetic diversity and genetic differentiation of Gymnocypris chilianensis , D-loop region of the mitochondrial DNA was sequenced in 50 individuals of G. chilianensis obtained from 2 geographic locations (Heihe River and Shule River) and 25 individuals of G. przewalskii (Qinghai Lake). Twenty-five homologous sequences of another G. eckloni (Yellow River) downloaded from GenBank were analyzed together. The sequences were analyzed by using the MEGA (version 7.0) and DnaSP (version 6.0) software. The results revealed that 82 haplotypes were detected among 100 individuals. The haplotype diversity ( Hd ) and nucleotide diversity ( Pi ) of G. chilianensis of the Shule River were 0.963 ± 0.029 and 0.00414 ± 0.00069, which were lower than those of 3 other populations. The genetic distance of G. chilianensis in both Heihe River and Shule River was 0.0013. The genetic distances between the 2 G. chilianensis populations and the G. eckloni were 0.0148 and 0.0141, respectively. Population differentiation values ( Fs t) and gene flow ( Nm ) showed that 4 population had occurred obvious genetic differentiation ( Fst : 0.20811 ∼ 0.98863. p < 0.01; Nm < 1). Compared with G. przewalskii and G. eckloni , the differentiation degree was more significant between Heihe River G. chilianensis and Shule River G. chilianensis (Fst = 0.98863, p < 0.01; Nm = 0.00287). Maximum Likelihood (ML) phylogenetic tree showed that G. chilianensis had further genetic distance with G. eckloni and G. przewalskii . In conclution, G. chilianensis (HH&SL) had lower genetic diversity and further genetic distance than G. przewalskii (QH) and G. eckloni (YL). We suggest strengthen the protection of genetic resources of G. chilianensis .

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