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(Sarcocheilichthys Sinensis) Using Microsatellite Markers

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(Sarcocheilichthys Sinensis) Using Microsatellite Markers Aquaculture and Fisheries 5 (2020) 80–85 Contents lists available at ScienceDirect Aquaculture and Fisheries journal homepage: http://www.keaipublishing.com/en/journals/ aquaculture-and-fisheries Original research article Genetic diversity and population structure of the Chinese lake gudgeon (Sarcocheilichthys sinensis) using microsatellite markers ∗ Shuting Gua,b, Rongquan Wangb, Chuanwu Lic, Jiale Lia,d,e, Yubang Shena,d,e, a Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China b Key Laboratory of Conventional Freshwater Fish Breeding and Health Culture Technology Germplasm Resources, Suzhou Shenhang Eco-technology Development Limited Company, Suzhou, 215225, China c Fisheries Research Institute of Hunan Province, Changsha, 412000, China d Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China e National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China ARTICLE INFO ABSTRACT Keywords: The Chinese lake gudgeon is a small benthopelagic freshwater fish. It is presently threatened by human activities Sarcocheilichthys sinensis and environmental factors in China. Understanding the genetic diversity and population structure is funda- Genetic diversity mental for implementation of appropriate conservation measures and a sustainable management program. Population structure However, little is reported about the current genetic diversity and population structure. Here, we used ten Microsatellites microsatellite markers to genotype 175 individuals from six populations. Low levels of genetic diversity were found in all six tested populations. The Xiang river population showed the highest level of genetic diversity. Genetic differentiation was very low but significant among the Changyang, Changqidang and Mayang popula- tions, but the Qiandao lake, Gan river, Xiang river populations all showed significant and strong differentiation from the other three populations. Contemporary gene flow was observed in among Changyang, Changqidang and Mayang populations and between Gan and Xiang river populations, respectively. This is the first genetic study to report the genetic diversity and population structure of S. sinensis and the results will be used to develop management and conservation strategies. 1. Introduction Schmidt, & Finn, 2009). Genetic studies are increasingly used by managers to assess the genetic diversity of wild populations before es- Genetic diversity plays an important role in the ability of a species tablishing management plans. to response to environmental changes and reflects its evolutionary po- The Chinese lake gudgeon, Sarcocheilichthys sinensis, a small ben- tential (Frankham, Briscoe, & Ballou, 2002). In general, populations of thopelagic freshwater fish (Froese & Pauly, 2016), belongs to the family a species with high genetic diversity have higher fitness (Hildner, Soule, Cyprinidae. According to previous literature, this species is historically Min, & Foran, 2003). The level of genetic variation is affected by many widely distributed from the Amur basin to the rivers of Korea and the Xi determinants (Ellegren & Galtier, 2016). It is widely demonstrated that River (Froese & Pauly, 2016). It usually lives in lakes, reservoirs, and demographic history has shaped the current genetic diversity of most streams. In the past decades, the population size of this fish has ex- species (Ellegren & Galtier, 2016). For freshwater wild organisms, hibited significant decline (Zhu et al., 2017a). Furthermore, in China human activities (e.g. overfishing, pollution) (Ma, Cowles, & Carter, conservation and management measures are still not fully im- 2000) and climate changes (Cai & Cowan, 2008) predominantly influ- plemented. The significant decline in this species may be due to over- ence their demographic fluctuations and result in reductions in popu- exploitation, low productivity and water pollution. Several published lation size. However, small populations are at greater risk of extinction studies have focused on captive breeding programs and larval devel- than larger stable populations. Furthermore, organisms living in opment of Chinese lake gudgeon (Song and Ma, 1994, 1996). However, freshwater lakes or rivers compared to marine organisms, become iso- the stocking of hatchery-reared S. sinensis has not been carried out to lated much more easily due to the discontinuous nature of freshwater improve the stock production. Levels of genetic diversity, population systems which restrict the movement of freshwater organisms (Hughes, differentiation and trends in abundance of wild populations remain ∗ Corresponding author. 999 Huchenghuan Road, College of Aquaculture and Life science, Shanghai Ocean University, Shanghai, 201306, China. E-mail address: [email protected] (Y. Shen). https://doi.org/10.1016/j.aaf.2019.06.002 Received 11 November 2018; Received in revised form 13 June 2019; Accepted 13 June 2019 Available online 09 July 2019 2468-550X/ © 2019 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). S. Gu, et al. Aquaculture and Fisheries 5 (2020) 80–85 largely unexplored. Neutral molecular markers frequently used in conservation genetic studies have been developed for S. sinensis, such as the mitochondrial genome (Li et al., 2016) and microsatellites (Shen et al., 2017; Zhu et al., 2017b). Although genotyping by sequencing (GBS) has become the most popular tool for population genetics ana- lysis, microsatellites are still useful (Li et al., 2018; Shen & Yue, 2018). The aim of this study was to use ten microsatellite markers to survey the genetic diversity and population structure of six wild populations of Chinese lake gudgeon. The first goal was to analyze the pattern of ge- netic diversity to discuss the conservation status. Genetic diversity has a key role in evolution by allowing a species to adapt to a new environ- ment and to fight off parasites. Second, we aimed to decipher popula- tion differentiation, which is a direct effect of random genetic drift, mutation and natural selection. This is important information for de- signing conservation and management strategies. This is the first in- vestigation of the genetic structure of S.sinensis, populations and this Fig. 1. Sampling localities of the six populations of Sarcocheilichthys sinensis study will provide useful information for the conservation and man- from the Yangtze River System in China. agement of this species. was used to calculate allele number (A), Observed (Ho) and expected 2. Materials and methods (He) heterozygosity, the inbreeding index (f) and the exact test for linkage and Hardy-Weinberg disequilibrium. Allelic richness (Ar) was 2.1. Sampling and DNA isolation measured using FSTAT v2.9.3.2 (Goudet, 1995), to reduce differences in the number of alleles among populations that was caused by differ- Six wild populations of Chinese lake gudgeon consisting of 175 in- ences in sample size. dividuals were collected between 2014 and 2017. All fish were netted, and after fin tissues were clipped these fish were released. Samples 2.4. Population structure representing the six wild populations were collected from Changyang Lake (CY), Changqidang Lake (CQD), Mayang Lake (MY) and Qiandao The pairwise F for each pair of populations were analyzed with Lake (QD), Xiang River (XJ), Gan River (GJ) (Table 1; Fig. 1). Fin tis- ST Arlequin v3.5 (Excoffier & Lischer, 2010). Statistical significance was sues were sampled and preserved in absolute ethanol at −20 °C. examined using an exact test with 10000 permutations. AMOVA was Genomic DNA was extracted from the fin tissues with the modified used to estimate genetic differentiation using Arlequin v3.5 (Excoffier & salting-out method described by Yue and Orban (Yue & Orban, 2005). Lischer, 2010). Tests for genetic differentiation were performed at three The DNA concentration and purity were checked by running on a 0.8% hierarchical levels of variation: within individuals (F ), among in- agarose gel, then adjusted to 10 ng/μL using the NanoDrop 2000C IT dividuals within populations (F ), among populations within groups spectrophotometer (Thermo scientific, US). IS (FSC) and among groups (FCT). Pairwise FST genetic distance was used to produce an NJ tree with MEGA v7 (Kumar, Stecher, & Tamura, 2016). 2.2. PCR amplification and genotyping Bottleneck hypothesis testing was performed under the infinite allele model (IAM), two-phased model of mutation (TPM) and step-wise Ten pairs of primers previously developed against microsatellites mutation model (SMM) using Bottleneck v1.2.02 (Cornuet & Luikart, (Shen et al., 2017) were used for the present work. The forward primers 1996). These methods are based on heterozygosity excess or deficit to of each pair were fluorescently labelled at the 5’ end. The PCR reaction test for the departure from mutation-drift equilibrium. Genetic re- was as previously described by Shen et al. (2017). Cycling conditions lationships among the populations were evaluated using Structure were as follows: 94 °C for 2 min, followed by 38 cycles at 94 °C for 30 s, v2.3.4 (Pritchard, Stephens, & Donnelly, 2000), then the Bayesian 55 °C for 30 s and 72 °C for 45 s, with a final extension at 72 °C for method was used to assign samples into clusters under an admixture 5 min. Extension products were electrophoresed on a capillary DNA model. We used different values
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