Isolation and characterization of twenty two polymorphic simple sequence repeat markers from AFLP sequences of Crassostr
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TECHNICAL NOTE
Isolation and characterization of twenty two polymorphic simple sequence repeat markers from AFLP sequences of Crassostrea gigas Yun-Guo Liu1 • Ling-Xiao Liu2
Received: 5 April 2015 / Accepted: 4 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract A total of 22 polymorphic simple sequence repeat (SSR) markers have been developed and characterized from AFLP sequences of Crassostrea gigas in order to protect its natural resources. Polymorphisms of these SSR markers were evaluated in a natural population of 30 individuals collected from Shandong peninsula in China. The number of alleles ranged from three to thirteen. Observed and expected heterozygosities varied from 0.50 to 0.97, and from 0.55 to 0.91, respectively. All the SSR loci conformed to Hardy–Weinberg equilibrium after Bonferroni correction. They have showed sufficient level of polymorphisms to estimate the genetic diversity, population structure and species conservation in the C. gigas. Keywords Crassotrea gigas SSR markers Population structure Genetic conservation The Pacific oyster, Crassotrea gigas, naturally occurs in China, Japan and Korea of East Asia, is the most widely cultured marine shellfish because of its fast growth rate, high production and well acclimatization (Li et al. 2011). However, natural fisheries of Pacific oyster have declined Electronic supplementary material The online version of this article (doi:10.1007/s12686-015-0471-1) contains supplementary material, which is available to authorized users. & Yun-Guo Liu [email protected] & Ling-Xiao Liu [email protected] 1
College of Life Sciences and Technology, Xinjiang ¨ ru¨mqi 830046, China University, U
2
Linyi Academy of Agricultural Sciences, Linyi 276012, China
dramatically in these countries, especially in China, due to human activities such as coastal development, over fishing and pollution. Therefore, it is very important to protect the natural resources of C. gigas. In the long time, a good understanding of the genetic diversity, population structure and genetic differentiation of C. gigas is required in order to establish adequate management plans for the conservation of this species. Simple sequence repeats (SSRs), or microsatellites, are of great utility in species conservation and management because of their high polymorphism, abundance, codominance and small length. In the present paper, we isolated 22 polymorphic SSR markers derived from C. gigas for the purpose of protecting its natural populations. SSR-enriched genomic libraries were constructed using the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) protocol (Liu et al. 2014; Zane et al. 2002). A total of 140 randomly selected clones were sequenced using ABI Prism 3730 automated DNA sequencer (ABI). SSRs were screened using SSRHunter software 1.3. A total of 78 microsatellites of C. gigas were found, of which 50 pairs of primers were designed and tested, with 22 of these giving polymorphic loci (Table 1). A homology s
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