Characterization of 95 novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining

Abstract

In order to construct a simple sequence repeat (SSR)-based genetic linkage map and to promote molecular marker-assisted selection (MAS) in scallop breeding, the methods of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO)-colony hybridization and expressed sequence tag (EST) database mining were modified and used to develop 95 novel microsatellite markers for Zhikong scallop. The SSR-enriched library constructed by the FIASCO method consisted of 830 clones, and 295 (35.5%) positive clones were identified after colony hybridization. One hundred and fifty clones were randomly sequenced and the results showed all clones contained at least one microsatellite. Of 91 primer pairs designed, 72 were amplified scorable polymerase chain reaction (PCR) products and 70 were polymorphic with the allele number range of 3-16 alleles/locus (average 7.0 alleles/locus). When EST database mining was performed, 66 microsatellites containing ESTs were identified from 3467 sequences deposited in GenBank. Based on cluster analysis of length and GC content of the flanking regions, 47 primer pairs were designed and 23 scorable EST SSRs were obtained. Compared with genomic SSRs developed in this study, EST SSRs showed lower genetic variability with an average of 4.2 alleles/locus. The results in the present study demonstrate that modified FIASCO-colony hybridization is an efficient and low-cost method for the isolation of large numbers of microsatellite markers for scallop species.

Methods comparison for microsatellite marker development: Different isolation methods, different yield efficiency

“…It is proven that public database mining is a fast, easy and economic strategy because it eliminates several procedures such as library screening and positive clone sequencing (Zhan et al, 2005(Zhan et al, , 2008. The advantages are well demonstrated in EST-SSR development in Zhikong scallop (Table 1) and bay scallop Argopecten irradians (Zhan et al, 2005).…”

“…We combined and modified the methods of FIASCO and colony hybridization as an integrative unity for developing microsatellite markers for Zhikong scallop (see Zhan et al 2008 for more details). Briefly, approximately 500 ng of AFLP pre-amplification product was denatured for microsatellite DNA capture.…”

Sequencing and analysis of four BAC clones containing innate immune genes from the Zhikong scallop (Chlamys farreri)

“…Genetic studies were also conducted to promote selective breeding in C. farreri. Many microsatellite/ simple sequence repeat (SSR) markers have been developed (Zhan et al, 2006(Zhan et al, , 2007(Zhan et al, , 2008, and linkage maps based on AFLP or microsatellite markers have been constructed (Li et al, 2005;Wang et al, 2005;Zhan et al, 2009). Additionally, some SNP markers were reported recently (Huan et al, 2010;Jiang et al, 2011).…”

Development of polymorphic SSR markers in the razor clam (Sinonovacula constricta) and cross-species amplification

“…SSRs have been developed in a wide number of bivalve species, such as Zhikong scallop Chlamys farreri (Zhan et al, 2008), Pacific oyster Crassostrea gigas (Li et al, 2011), hard clam Meretrix meretrix , and blood clam Tegillarca granosa (Dong et al, 2012). Furthermore, expressed sequence tag-derived SSRs (ESTSSRs) can be associated with a function and linked more easily to a phenotypic trait of interest, making them useful for functional diversity studies (Tranbarger et al, 2012).…”