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Int. J. Mol. Sci. 2014, 15(7), 12952-12958; doi:10.3390/ijms150712952
Article

Molecular Method for Sex Identification of Half-Smooth Tongue Sole (Cynoglossus semilaevis) Using a Novel Sex-Linked Microsatellite Marker

1,2
,
1
 and
1,*
1 Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China 2 Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem of Ministry of Water Resources, Institute of Hydroecology, Ministry of Water Resources and Chinese Academy of Sciences, Wuhan 430079, China
* Author to whom correspondence should be addressed.
Received: 23 January 2014 / Revised: 28 March 2014 / Accepted: 23 April 2014 / Published: 22 July 2014
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Abstract

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most important flatfish species for aquaculture in China. To produce a monosex population, we attempted to develop a marker-assisted sex control technique in this sexually size dimorphic fish. In this study, we identified a co-dominant sex-linked marker (i.e., CyseSLM) by screening genomic microsatellites and further developed a novel molecular method for sex identification in the tongue sole. CyseSLM has a sequence similarity of 73%–75% with stickleback, medaka, Fugu and Tetraodon. At this locus, two alleles (i.e., A244 and A234) were amplified from 119 tongue sole individuals with primer pairs CyseSLM-F1 and CyseSLM-R. Allele A244 was present in all individuals, while allele A234 (female-associated allele, FAA) was mostly present in females with exceptions in four male individuals. Compared with the sequence of A244, A234 has a 10-bp deletion and 28 SNPs. A specific primer (CyseSLM-F2) was then designed based on the A234 sequence, which amplified a 204 bp fragment in all females and four males with primer CyseSLM-R. A time-efficient multiplex PCR program was developed using primers CyseSLM-F2, CyseSLM-R and the newly designed primer CyseSLM-F3. The multiplex PCR products with co-dominant pattern could be detected by agarose gel electrophoresis, which accurately identified the genetic sex of the tongue sole. Therefore, we have developed a rapid and reliable method for sex identification in tongue sole with a newly identified sex-linked microsatellite marker.
Keywords: Cynoglossus semilaevis; sex identification; sex-linked microsatellite marker (SLM); female-associated allele (FAA); multiplex PCR Cynoglossus semilaevis; sex identification; sex-linked microsatellite marker (SLM); female-associated allele (FAA); multiplex PCR
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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Liao, X.; Xu, G.; Chen, S.-L. Molecular Method for Sex Identification of Half-Smooth Tongue Sole (Cynoglossus semilaevis) Using a Novel Sex-Linked Microsatellite Marker. Int. J. Mol. Sci. 2014, 15, 12952-12958.

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