A BAC-Based Physical Map of Zhikong Scallop (Chlamys farreri Jones et Preston)

et al. (2011) A BAC-Based Physical Map of Zhikong Scallop (Chlamys farreri Jones et Preston). PLoS ONE 6(11): e27612. doi:10.1371/journal.pone.0027612 A BAC-Based Physical Map of Zhikong Scallop (Chlamys farreri Jones et Preston) Xiaojun Zhang 0 Cui Zhao 0 Chao Huang 0 Hu Duan 0 Pin Huan 0 Chengzhang Liu 0 Xiuying Zhang 0 Yang Zhang 0 Fuhua Li 0 Hong-Bin Zhang 0 Jianhai Xiang 0 Tongming Yin, Nanjing Forestry University, China 0 1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences , Qingdao , China , 2 Graduate University of the Chinese Academy of Sciences , Beijing , China , 3 Department of Soil and Crop Sciences, Texas A&M University, College Station , Texas , United States of America Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (,5.86 genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs. - Zhikong scallop (Chlamys farreri Jones et Preston) has a wide distribution along the coasts of North China, Korea, Japan, and Eastern Russia. It is a most dominant scallop species for aquaculture, and its production has reached approximately 80% of the total scallop production in China [1]. However, the frequent mass mortality of the species has seriously affected the development of its industry since 1996 [2]. To develop new scallop genotypes to manage the problem, it is necessary to have a better understanding of the molecular mechanisms underlying its economically important quantitative and qualitative traits, such as disease resistance and growth rate. Therefore, many investigations have recently focused on genome research of the species. Chlamys farreri has 2n = 38 chromosomes and its haploid genome size is approximately 1.24 Gb [3,4]. Significant progress has been made recently in its genomics, including development of genetic linkage maps [57], construction of large-insert genomic DNA libraries [1,3], mapping of economically important quantitative trait loci (QTL) [7], and large-scale sequencing of expressed sequence tags (ESTs) [8]. However, there have not been a genome-wide physical map and extensive long-range genome sequence that are crucial to advanced studies of genomics available in scallop. The shortage of such genomics tools and infrastructure in the species has not only limited the molecular cloning and analysis of its genes and QTL, but also prevented the flow of genomic information from the model species and other mollusc such as oyster (Crassostrea gigas) to scallop. Furthermore, it is desirable to have a genome-wide physical map to sequence and assemble the scallop genome using the next-generation sequencing (NGS) technology. Therefore, a comprehensive physical map is needed for advanced research of the scallop genome. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. Although the development of whole-genome shotgun sequence draft maps based on NGS has become cost-effective recently, a physical map remains an important component of genome sequencing projects [9]. For the large regions of repeat sequences and highly heterozygous genomes, sequencing and assembly will not be easily addressed by NGS alone. Additional tools are needed to provide anchor points to link sequence contigs and bridge the large repeat regions [10]. Physical maps, specially the contig physical maps constructed based on restriction fragment fingerprints of bacterial artificial chromosome (BAC) clones, can provide these anchor points, such as BAC end sequences (BESs) and markers, for genome sequence assembling. Moreover, genome-wide integrative physical and genetic mapping is a most efficient and economical approach to fine mapping and positional cloning of genes controlling many phenotypic traits such as QTL. BAC contig-based physical maps have been constructed in several aquaculture species, including Nile tilapia [11], Atlantic salmon [12], catfish [13,14], rainbow trout [15] and Asian seabass [16]. These physical maps have greatly enhanced genome research in the species. Based on the physical maps, integrated genome maps have been developed for the Atlantic salmon [17] and rainbow trout [18]. These genome resources have been proven very useful for the identification of genomic regions containing genes of economically important traits and whole genome sequencing. However, no genome-wide physical map has been reported for a species of mollusca, the largest marine organism phylum containing approximately 85,000 recognized species (http://en.wikipedia.org/wiki/Mollusca). We have previously constructed two BAC libraries from the nuclear DNA of C. farreri, and identified the BACs containing six genes involved in the innate immune system of mollusc [1]. Using the BACs as a tool, we have characterized two of the six genes and mapped them to C. farreri chromosomes using the fluorescent in situ hybridization (FISH) technology [1921]. In this study, we have developed a genome-wide physical map of Zhikong scallop from the BAC libraries, thus providing useful and friendly-used tools for advanced studies of its genomics, particularly whole genome sequencing and map-based cloning of economical genes and QTL in the species. BAC fingerprinting The clones were from the two previously constructed BAC libraries, Scallop-CBE and Scallop-CME [1], all 81,408 clones were used in fingerprinting. BAC clones were inoculated in 96deep well plates, with each well containing 1.0 ml YENB medium (China patent, ZL200610046257.6) with 12.5 mg/ml chloramphenicol, from the 384-well micr (...truncated)