Polymorphic microsatellite loci for marbled rockfish (Sebastiscus marmoratus)
Lu Liu1, Takashi. Yanagimoto2, Chun-hou. Li3, Na Song1, Tian-xiang. Gao4*
1
Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, China
2
National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Japan
3
Key Laboratory for Exploitation & Utilization of Marine Fisheries Resource in South China Sea,
Ministry of Agriculture; South China Sea Fisheries Research Institute, Chinese Academy of Fishery
Sciences, Guangzhou, China.
4
Fishery College, Zhejiang Ocean University, Zhoushan, Zhejiang, China.
*
Corresponding author: Tian-xiang. Gao
Telephone: +86-580-2550671
Fax: +86-580-2550008
Email: gaotianxiang0611@163.com
Abstract
We isolated and characterized 24 polymorphic microsatellite loci in Sebastiscus marmoratus using
next-generation sequencing platform. We found 2 to 11 alleles per locus, with a mean of 5.542. The
observed and expected heterozygosities ranged from 0.242 to 0.917 and from 0.497 to 0.896, with
an average of 0.603 and 0.737 respectively. Deviation from Hardy-Weinberg proportions was
detected at three loci. The evidences for null alleles were found at three loci. These markers will be
useful in further studies to investigate the genetic variation and population structure of this species,
also may give new insights into maintenance and efficient management of marbled rockfish
resources.
Key words: Sebastiscus marmoratus; microsatellite loci; population structure; genetic diversity
Marbled rockfish (Sebastiscus marmoratus) is one of the most common fish species widely
distributed in the western Pacific from the East China Sea to Japan. Marbled rockfish, as a typical
sedentary fish, tend to living in rocky bottoms of coastal zone. This kind of fish usually can be found
staying within narrow home ranges in the daytime and hiding in holes or crevices during the night
(Fuqita, 1998). With the decline of many traditional economical fish resources, S. marmoratus is
becoming a new kind of important economic fish of worldwide fisheries industry gradually. In order
to prevent the reduction of marbled rockfish, just like other traditional economical fish, it’s necessary
to characterize the population structure which may give new insights into maintenance and efficient
management of marbled rockfish resources. Because of highly polymorphic, reproducible, multiallelic, co-dominant and relatively abundant, microsatellite loci are considered as a powerful genetic
marker which has been widely used in population structure analysis. Although twenty microsatellite
markers had been isolated in S. marmoratus previously, it was still not enough for further study due
to their low polymorphism. More microsatellite markers are needed for population genetic analysis
of this species. Here we reported the development of an additional 24 loci that will increase the
power available for analyzing population structure of marbled rockfish.
Twenty four individuals were collected from the coastal waters near Huizhou, China. The samples
were preserved at -20℃ until DNA extraction. DNA was extracted from muscle tissue of S.
marmoratus using QIAamp DNA Mini Kit (Qiagen). Next-generation sequencing platform was used to
obtain microsatellite loci sequences of S. marmoratus. We sent about 5 µg (500 ng/µL) of the total
genomic DNA to Shanghai Personal Biotechnology Co., Ltd. for high-throughput DNA sequencing
using 454 Genome Sequencer FLX plat form (Roche). Subsequently, thousands of DNA sequences
which contained the short tandem repeat fragment from the random sample through the
MSatFinder interface (www.Genomics.ceh.ac.uk/msatfinder) were found. Polymerase chain reaction
(PCR) primer pairs were designed to amplify 40 microsatellite loci with suitable flanking regions using
the PRIMER PREMIER 5 software (Premier Biosoft International, USA).
The designed primers were evaluated using 24 S. marmoratus individuals. PCR was performed on a
Veriti Thermal Cycler (Applied Biosystems, USA) in a total volume of 25 μl containing 0.4 μM of each
primer, 0.2 mM of each dNTP, 1×PCR buffer, 2 mM of MgCl2, 1 unit Taq polymerase (TaKaRa, Japan)
and 10-100 ng DNA. The amplification profile included an initial denaturing at 94℃ for 5 min, 35
cycles of 45 s at 94℃, at the locus-specific annealing temperature for 1 min, and 72℃ for 45 s, and
final period at 72℃ for 10 min. The PCR products were separated on 6% denaturing polyacrylamide
gel, and visualized by silver staining.
The observed and expected heteozygosities together with tests for Hardy-Weinberg disequilibrium
were calculated by GENEPOP 4.0 (Rousset, 2007). Null allele frequencies (Brookfield, 1996) were
calculated by MICRO-CHECKER 2.2.3 (Van Oosterhout et al., 2004). All results for multiple tests were
corrected using Bonferroni’s correction (Rice, 1989).
Twenty-four out of 40 loci were cleanly amplified and shown to be polymorphic. The numbers of
alleles per locus ranged from 2 to 11 with an average of 5.542. The observed and expected
heterozygosities ranged from 0.242 to 0.917 and from 0.497 to 0.896, respectively, with an average
of 0.603 and 0.737 (Table 1). No loci showed significant deviation from Hardy-Weinberg proportion
except for locus Sm94, Sm117 and Sm148 after Bonferroni correction (P < 0.0021). Three loci (Sm58,
Sm117 and Sm148) showed evidences of null alleles (null allele frequency > 5%). No genetic
disequilibrium was detected in each locus pair. These twenty-four new microsatellite markers could
analyze the genetic structure of S. marmoratus and provide important genetic data for the
conservation and recovery of the S. marmoratus resources.
Acknowledgments
The study was supported by National Natural Science Foundation of China (41176117), Ocean Public
Welfare Scientific Research Project of China (200905019), and Key Laboratory for Exploitation &
Utilization of Marine Fisheries Resource in South China Sea, Ministry of Agriculture (No. LSF2014-02).
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