Sequencing and analysis of four BAC clones containing innate immune genes from the Zhikong scallop (Chlamys farreri) Cui Zhao a, b , Tongwu Zhang c , Xiaojun Zhang a, ⁎, Songnian Hu c , Jianhai Xiang a a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China b Graduate University of Chinese Academy of Sciences, Beijing, China c Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China abstract article info Article history: Accepted 5 April 2012 Available online 20 April 2012 Keywords: LGBP Repetitive sequences Gene cluster Membrane transport genes Transposon The sequencing of BAC clones (~ 100 kb) can reveal some characteristics of a genome that are challenging to obtain based on short sequences. Additionally, although the immune genes of the Zhikong scallop (Chlamys farreri) have been studied widely, few analyses have been conducted at the DNA level. In this study, four C. farreri BAC clones containing innate immune genes, including hsp70, lgbp (lipopolysaccharide and beta- 1,3-glucan binding protein), serine protease and a gene with an immunoglobulin-like domain, were se- quenced and analyzed both to explore the genomic characteristics of C. farreri based on long DNA sequences and to promote the study of C. farreri immune genes at the DNA level. The total length of the four BACs was 389.98 kb. A total of 34 genes were predicted in these sequences, and several features of protein-coding re- gions in the C. farreri genome were inferred based on this information. Two LGBP genes were located close together in a 22-kb region in one BAC clone, indicating the physical linkage of some immune genes in C. farreri. A cluster of membrane transport genes was also observed; these genes might play important roles in eliminating toxins in C. farreri, which lives as a filter feeder. Further analysis showed 15.43% of the BAC se- quence was repetitive. Tandem repeats were the most abundant repeat type, followed by transposable ele- ments. A total of 31 SSRs were predicted in the four BACs. An IS10 family transposon was identified, and a suspected regulatory non-coding RNA gene for this transposon (RNA-OUT) was observed to overlap with it complementarily. This work will promote future studies on the genomics, immune system and non-coding regions of C. farreri. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The Zhikong scallop (Chlamys farreri Jones et Preston 1904) is a marine bivalve that is widely distributed along the coasts of northern China, Korea, Japan and eastern Russia (Qiu et al., 2007; Zhan et al., 2009). As a commercially important cultured bivalve species in China, the sustainable industrial culture of C. farreri has faced many challenges, such as the lack of a high-performance breeding line and the frequent occurrence of diseases, especially summer mortality (Xiao et al., 2005). Efforts to resolve these issues have focused mainly on immunologic and genetic studies. Many genes involved in the innate immune response to pathogens have been cloned and characterized, such as the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) gene (Qiu et al., 2010), the serine protease gene (Zhu et al., 2007) and the c-type lectin gene (Yang et al., 2011). Based on the analysis of these immune genes, some important immune pathways in C. farreri, such as the Toll-like receptor signaling pathway (Qiu et al., 2007; Wang et al., 2011) and the complement-like system (Zhang et al., 2007a, 2008a), have been elucidated. Genetic studies were also con- ducted to promote selective breeding in C. farreri. Many microsatellite/ simple sequence repeat (SSR) markers have been developed (Zhan et al., 2006, 2007, 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). The availability of additional genomic data could provide funda- mental support for further studies. Despite the many achievements in C. farreri immunology and genetics, genomic studies of this organ- ism are still very limited, although they have increased in recent years. To date, two genomic libraries have been constructed, includ- ing one 4.3× fosmid library (Zhang et al., 2007b) and one 9.1× BAC (bacterial artificial chromosome) library (Zhang et al., 2008c). More than 20,000 genome survey sequences have been produced, including Gene 502 (2012) 9–15 Abbreviations: AFLP, amplified fragment length polymorphism; BAC, bacterial artifi- cial chromosome; BES, BAC end sequence; CDS, coding sequence; EST, expressed sequence tag; FES, fosmid end sequence; HSP70, heat shock protein 70; LGBP, lipopolysaccharide and beta-1,3-glucan binding protein; LINE, long interspersed element; LTR, long terminal repeat; mcm6, minichromosome maintenance complex component 6; ndpk, nucleoside diphosphate kinase; NGS, next-generation sequencing; PFGE, pulsed field gel electropho- resis; proPO, prophenoloxidase; QTL, quantitative trait locus; SNP, single nucleotide poly- morphism; SSR, simple sequence repeat. ⁎ Corresponding author. Tel.: + 86 532 82898786; fax: + 86 532 82898568. E-mail address: xjzhang@qdio.ac.cn (X. Zhang). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.009 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene