Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization
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The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33 %. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.
KeywordsBAC Small abalone Haliotis diversicolor Genome Ion Torrent
We acknowledge grant support from National Natural Science Foundation of China (No. U1205121 and 31422057), the National High-Technology Research and Development Program of China (863 program; 2011AA100401), Special Scientific Research Funds for Central Non-profit Institutes of Chinese Academy of Fishery Sciences (2013A03YQ01). The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research (No. RG 1435–012).
Conflict of Interest
The authors declare that they have no competing interests.
PX and CK conceived the study. LJ worked on BAC library evaluation, BAC pooling, BAC screening, BAC sequencing, assembly, and manuscript drafting. WY worked on sample preparation and collection and participated in the manuscript preparation. XZ and KW worked on agar plugs preparation for HMW genomic DNA extraction. ZZ, KW, and BC worked on BAC pooling and screening. JX and YJ worked on BAC sequencing and sequencing analysis. PX wrote the manuscript. YZ, SM, and KAG helped on the manuscript revision. All authors read and approved the final manuscript.
- Cunningham C, Hikima J-I, Jenny MJ, Chapman RW, Fang G-C, Saski C, Lundqvist ML, Wing RA, Cupit PM, Gross PS (2006) New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas). Mar Biotechnol 8:521–533PubMedCrossRefGoogle Scholar
- Geiger DL (1999) Distribution and biogeography of the recent Haliotidae (Gastropoda: Vetigastropoda) world-wide. Boll Malacol 35:57–120Google Scholar
- Lindberg DR, Shepherd SA, Tegner MJ, del Próo Gutzmán SA (1992) Evolution, distribution and systematics of Haliotidae. In: Shepherd SA, Tegner MJ, Gutzmánd del Próo SA (eds) Abalone of the world: biology, fisheries and culture. Blackwell Scientific Publishing, London, pp 3–18Google Scholar
- Liu J, Zeng B, Du T, Rao Y (2013) Low genetic diversity and genetic differentiation among domesticated stocks of the small abalone Haliotis diversicolor in China. Afr J Biotechnol 10:19267–19275Google Scholar
- Sambrook J, Russell DW, Russell DW (2006) The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Somridhivej B, Wang S, Sha Z, Liu H, Quilang J, Xu P, Li P, Hu Z, Liu Z (2008) Characterization, polymorphism assessment, and database construction for microsatellites from BAC end sequences of channel catfish (Ictalurus punctatus): a resource for integration of linkage and physical maps. Aquaculture 275:76–80CrossRefGoogle Scholar
- Takeuchi T, Kawashima T, Koyanagi R, Gyoja F, Tanaka M, Ikuta T, Shoguchi E, Fujiwara M, Shinzato C, Hisata K (2012) Draft genome of the pearl oyster Pinctada fucata: a platform for understanding bivalve biology. DNA Res 19:117–130Google Scholar
- Zhang Y, Zhang X, Scheuring CF, Zhang H-B, Huan P, Li F, Xiang J (2008b) Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system. Mar Biotechnol 10:358–365PubMedCrossRefGoogle Scholar