Isolation and characterization of 22 microsatellite loci in Acrossocheilus wenchowensis
(Cyprinidae, Barbinae)
Yong-Yi Jia1, Shi-Li Liu1,2, Xi-Lian Li1, Zhi-Min Gu1*, Jin-Liang Zhao2, Wen-Ping Jiang1, Qing-Ping Lian1,
Jian-Lin Guo1
1 Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture & Key Laboratory of
Freshwater Aquatic Animal Genetic and Breeding of Zhejiang province, Zhejiang Institute of
Freshwater Fisheries, Huzhou 313001, China;
2 Key Laboratory of Freshwater Fishery Germplasm Resources, Shanghai Ocean University, Ministry
of Agriculture, Shanghai, 201306, China
*Corresponding author: E-mail: guzhimin2006@163.com
Abstract
We isolated and characterized a total of 22 microsatellite loci from the fish Acrossocheilus
wenchowensis, which has unknown conservation status. Loci were screened in 32 individuals from
downstream of Changzhao Reservoir in Zhejiang, China. The average allele number was 10.5 per
locus, ranging from 2 to 25. The observed heterozygosity was from 0.000 to 0.906, and the expected
heterozygosity was from 0.062 to 0.954. 16 of the 22 microsatellites conformed to Hardy-Weinberg
equilibrium and, overall, no linkage disequilibrium was detected. These microsatellite markers will
provide tools for examining population genetic structure, estimating effective population size and
inform fisheries management and conservation.
Keywords Acrossocheilus wenchowensis; Polymorphic Microsatellites; Population genetics; fish
conservation
Acrossocheilus wenchowensis (Cyprinidae, Barbinae) is distributed throughout the Ou, Feiyun,
Taishundong and Aojing River systems. A. wenchowensis lives in small, clean, fast-flowing streams,
eating algae on cobblestones, and spawns adherent eggs among gravel in shallow waters (Shan et al.
2000).
Recently, with increasing impact of human activities on ecological environments, attention has been
focused on the population- and conservation genetics of A. wenchowensis. Artificial propagation,
proliferation and release is a potential means to promote resource recovery. However, fish stocking
may impact on wild resources. It is essential to assess parental genetic background, genetic diversity
of offspring proposed to be released, as well as post-release impact on wild populations, so as to
adjust stocking strategies appropriately. Each of these tasks requires informative DNA markers.
However, current genetic-based research on A. wenchowensis is limited.
Microsatellite markers are employed in co-dominant genetic analysis techniques capable of
providing significant genetic information and have been widely applied in analyzing genetic diversity
of species, phylogenetic analysis, variety identification and evolutionary studies. Although some
progress has been made in development of microsatellite markers for the genus of Acrossocheilus
(Chen et al. 2012), markers targeting A. wenchowensis have now been developed for the first time in
China. In this study, 22 informative microsatellite loci were isolated and characterized, providing a
basis for genetic evaluation of A. wenchowensis and formulation of protection policies.
Enriched partial microsatellite genomic libraries for repeats motifs (CA)12 and (GT)12 were obtained
following Glenn and Schable’s (2005) protocol with minor modifications. Two hundred plasmid
clones with different insert lengths were sequenced. Overall, 152 unique sequences were obtained:
96 contained simple sequence repeats and 66 were suitable for primer design using Primer Premier
6.0 (PREMIER Biosoft International). Diversity detection was performed using eight individuals. PCR
amplification methods were according to Liu (2014). Fifty-four primer pairs stably amplified, of
which 22 pairs revealed apparent polymorphism. We FAM -labelled downstream primers for these
22 pairs and evaluated using 32 A. wenchowensis individuals collected downstream of Changzhao
Reservoir in Zhejiang.
PCR products were separated on an ABI 3730xl genetic analyzer (Applied Biosystems) with ROX-500
size standards, and analyzed using SoftGenetics’ GeneMarker version 2.2. The numbers of alleles
(Na), expected (He) and observed heterozygosities (Ho) as well as conformation to Hardy–Weinberg
equilibrium (HWE) were evaluated using software Genepop version 4.0 (Rousset 2008).
Locus designation, primer sequences, repeat motifs, numbers of alleles (Na), range of PCR product
sizes, and PIC are provided in Table 1. Na ranged from 2 to 25. Ho and He ranged from 0.000 to
0.906 and from 0.062 to 0.954, respectively. Among these polymorphic microsatellite loci, six (Awc
014, Awc 041, Awc 043b, Awc 051, Awc 070 and Awc 071) showed significant deviations (P < 0.05)
from HWE after sequential Bonferroni correction. No evidence was found for linkage disequilibrium
among loci at a 5% significance level. These polymorphic microsatellite markers may be used as
powerful tools for investigating genetic diversity and genetic structure in A. wenchowensis.
Acknowledgments
This work was supported by grants from the Public Welfare Agricultural Research Project (No.
2011C2208), and the Cultivating Innovation Support Project (No. 2014F13307) of Zhejiang Science
and Technology Department.
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