Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 07 September 2007
Accepted: 25 March 2008
Published: 25 March 2008
Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC) library and the mapping of BAC clones to the linkage map.
This BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group.
We have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing.
Barramundi (Lates calcarifer), also called Asian seabass or the giant sea perch, belonging to the family Latidae is widely distributed in the coast and freshwater of the tropical Indo-west Pacific, from the Persian Gulf to India and Northern Australia [1, 2]. Because of good meat quality and relatively high market value of L. calcarifer, it has become an attractive commodity of both large and small-scale aquaculture enterprises. It is commercially cultivated in Thailand, Malaysia, Singapore, Indonesia, Hong Kong, China and Australia in both brackishwater and freshwater ponds, as well as in cages in coastal waters. The global annual production of L. calcarifer was 400,000 MT according to FAO statistics. However, detailed breeding programs for genetic improvement of Asian seabass are still quite rare . Identification of genomic regions and genes responsible for economically important traits could facilitate genetic improvement through marker-assisted selection , which is of importance for future aquaculture of L. calcarifer.
Linkage and physical maps are indispensable tools needed to identify genomic regions responsible for traits of interest. The genome of L. calcarifer is very compact (only 700 Mb) consisting of 24 chromosome pairs . The first linkage map for L. calcarifer containing 240 microsatellite markers and genes on 24 linkage groups  was applied to mapping QTL for growth traits . Libraries with large genomic DNA inserts are essential for physical mapping and positional cloning, particularly for higher eukaryotes . The BAC (bacterial artificial chromosome) cloning system has become an invaluable tool in genomics studies because of its ability to stably maintain large DNA fragments and its ease of manipulation . Genomic inserts in BAC clones have been shown to be very stable in E. coli and thus serve as ideal templates in generating whole-genome physical maps by DNA fingerprinting, developing sequence-tagged connectors and shotgun sequencing [10–12]. These features make the BAC cloning system a popular choice for high-throughput genomics studies . BAC libraries have been developed for many economically important animal species such as cattle , pig , and sheep  and the highly endangered giant panda . Only currently, BAC libraries were produced for some commercially important fish species such as salmon , catfish , rainbow trout, carp and tilapia .
Here, we describe the construction and characterization of a BAC library covering 6.9 times L. calcarifer haploid genome and mapping of 86 BAC clones to the linkage map. The BAC library and the improved linkage map of L. calcarifer will facilitate the integration of physical and linkage maps, fine mapping of QTL and identification of genes located in QTL of interest, maker-assisted selection and genome research.
A BAC library of L. calcarifer was constructed using the HindIII cloning site in commercially prepared pCC1BAC vector (Epicentre, MD, USA). The BAC library consisted of a total of 49,152 clones, which were manually arrayed into 128 384-well plates.
Insert size distribution
BAC library screening
Screening of the BAC library with 24 microsatellites located on each of the 24 linkage groups of L. calcarifer
GenBank accession no
Forward primer (5'-3')
Reverse primer (5'-3')
PCR Ta (°C)
Positive clone number
Screening of the BAC library with 15 genes/ESTs of L. calcarifer
GenBank accession no
Primer (5'-3', forward)
Primer (5'-3', reverse)
PCR Ta (°C)
PCR product length (bp)
Positive clone number
Microsatellite isolation from BAC clones and linkage mapping
Microsatellites isolated from BAC clones and mapped on the linkage map of L. calcarifer
GenBank accession no
Forward primer (5'-3')
Reverse primer (5'-3')
PCR Ta (°C)
Product Length (bp)
Location in the library (384plate-96plate-well)*
A critical tool for genomic studies in fish species is the availability of deep-coverage large-insert genomic libraries, such as BAC libraries that can be used for physical mapping, integration of linkage and physical maps, positional cloning, comparative genomic studies and genome sequencing . We constructed the first BAC library for L. calcarifer containing 49,152 clones with an average insert size of 98 kb ranging from 45 to 200 kb, indicating that this BAC library provided 6.9 × coverage of the L. calcarifer haploid genome. We have noticed that 50% of the inserts in our BAC library were under 100 kb. It is common that insert size of 50% of BAC clones was smaller than the size of DNA fragments recovered from gels. This phenomenon has been seen in several BAC libraries, such as the BAC library of tomato . The reason for this is that smaller fragments could be included in larger fragments during electrophoresis, and during ligation, the relatively smaller fragments were preferentially ligated to the vectors. PCR screening of the library with 24 markers each from one of 24 LG and 15 randomly selected ESTs/genes demonstrated that the BAC library provided good coverage of the L. calcarifer genome. Whether the BAC clones with large inserts were of hybrid origin remains to be examined.
A second generation linkage map of L. calcarifer is under construction by integrating new markers including microsatellites, ESTs and candidate genes onto the first generation map. Low polymorphism of ESTs and candidate genes was a bottleneck to map them to the linkage map . Using highly polymorphic microsatellites located in BAC clones harboring interesting genes and ESTs, these interesting genes and ESTs could be mapped onto the linkage map as shown in this experiment. By employing highly polymorphic microsatellites in BAC clones, we have mapped 86 BAC clones to the linkage map of L. calcarifer. At least one BAC clone has been anchored on each LG, which can be used to integrate linkage and physical maps in the future. The number of markers on the linkage map of L. calcarifer increased to 302 by mapping 62 novel microsatellites located in 62 BAC clones onto the map. The two male linkage groups (i.e. LG14 and LG18) which were split to two LG on the first generation map respectively  due to the limited number of markers on these LG, were merged to one LG respectively by integrating new microsatellites located in single BAC clones, which improved the quality of the linkage map of L. calcarifer.
The BAC library of L. calcarifer could be also used in constructing a physical map by BAC fingerprinting [12, 20], sequencing BAC ends and positional cloning of QTL of commercial interests  so as to facilitate selective breeding of L. calcarifer. Eventually, the BAC library can be used in whole genome shotgun sequencing when it becomes necessary.
A first L. calcarifer BAC library with 6.9 × coverage of the haploid genome has been constructed and characterized. Screening the library with 24 markers and 15 ESTs/genes demonstrated good genome coverage of this library. Eighty-six BAC clones were mapped to the first generation linkage map, improving the marker density of the linkage map of L. calcarifer. This BAC library together with the improved linkage map not only supplies an indispensable tool to physical mapping, integration of physical and linkage maps, and positional cloning for genes of importance, but also contributes to comparative genomic studies and eventually genome sequencing.
Preparation of high-molecular-weight DNA
Five hundred microliters of blood was collected from a male individual of L. calcarifer with a heparinized syringe. The concentration of leucocytes was quantified to be approximately 109 cells/ml. Quantities corresponding to 2.14 × 107 cells were embedded in 40 μl of 2% InCert agarose (in PBS) for DNA extraction. The mixture was then transferred into ice-cold plug moulds (Bio-Rad, SG, Singapore). Individual plugs were released into cell lysis solution [1% lithiumdodecyl sulfate, 10 mM Tris (pH 8), 100 mM EDTA (pH 8)] that was incubated at 37°C for 1 hr with occasional swirling. The cell lysis solution was replaced with 50 ml of new cell lysis solution and incubated overnight at 37°C. The cell lysis solution was supplanted with 50 ml of 20% NDS. Two ml of proteinase K (20 mg/ml) was added to each 50 ml of 20% NDS consisting of 0.2% N-laurylsarcosine, 2 mM Tris-HCL (pH9.0), 0.14 M EDTA. The solution was incubated at 37°C overnight. Plugs were washed three times with TE50 and 0.05 M EDTA for one hour at room temperature. The plugs were put into a fresh Falcon tube, and washed twice with 50 ml TE50 and 50 μl PMSF (100 mM) at 37°C for 20 min to inactivate proteinase K. The plugs were then washed twice with 50 ml of TE50 in the Falcon tube at room temperature for 30 min to get rid of the PMSF.
Partial digestion of high molecular weight DNA and size selection
Digestion with restriction enzyme HindIII, pulse field gel electrophoresis (PFGE), isolation and purification of high molecular weight (HMW) DNA were performed using the method described previously . Briefly, after displacement of the plugs by 1 × TE buffer, the agarose plugs were soaked in 800 μl of HindIII digestion buffer [0.015% bovine serum albumin (BSA), 75 mM NaCl, 12 mM Tris-HCl (pH 7.50)] and 3 U of HindIII for 16 hours at 4°C, after which, 100 μl of MgCl2 (100 mM) was added and the mixture was incubated at 37°C for one hour to partially digest the genomic DNA. The reaction was stopped by adding 150 μl of 0.5 M EDTA (pH 8.0), 15 μl 20 mg/ml proteinase K and 37.5 μl 20% NDS, and incubating at 37°C for one hour. Plugs were rinsed in TE50 in a Petri dish then transferred to a 15 ml Falcon tube. 15 ml of TE50 and 15 μl of 100 mM PMSF were added to the tube. The tube was incubated at room temperature for 20 min on rotating shaker. The tube with plug was washed twice with 15 ml TE50 at room temperature on a shaker for 30 min each.
Size selection was carried out as described , with minor modifications. In brief, partially digested DNA was separated by PFGE in 0.5 × TBE on a CHEF-DRII apparatus (Bio-Rad, SG, Singapore) under the following conditions: 14°C, 6.0 v/cm, angle = 120°, initial switch time = 5 sec, final switch time = 15 sec, run time = 16 hours and ramping = linear. At the end of this electrophoresis step, the gel portion containing DNA of 50 kb or less in size together with the portion of the gel containing the original plugs was removed. 1% fresh agarose was added to the remaining gel followed by a second electrophoresis step using the same conditions for 18 hours. Gel slices containing size fractionated DNA were obtained by cutting horizontally at 0.5 cm intervals in the size range of 100–250 kb. Each excised gel slice was subsequently inverted and buried in 1% low-melting-point agarose gel. A third electrophoresis step using the same conditions for 18 hours was carried out to concentrate the widely spread DNA fragments in each gel slice into a sharp single band. The band of size selected genomic DNA was then excised and dialyzed in 1 × TAE at 4°C overnight.
Ligation and electroporation
Size fractionated DNA was recovered from each gel band by electroelution in Spectra/Por 7 dialysis bags (Spectrum Laboratories, CA, USA) as described . Partially digested HMW DNA was then ligated to 25 ng of dephosphorylated, HindIII digested pCC1BAC (Epicentre, MD, USA) at a 1:10 molar ratio of insert to vector with 400 units of T4 ligase (NEB, MA, USA) in 50 μl reaction at 16°C overnight. Dialyzed ligation was used to transform ElectroMAX DH10B competent cells (Invitrogen, MD, USA). Electroporation was carried out using a BioRad Gene Pulser (BioRad, CA, USA) at 4 kΩ and 350 V. Cells were incubated in 1 ml SOC medium at 37°C for one hour with shaking and later spread on LB plates containing 12.5 μg/ml Chloramphenicol, 40 μg/ml X-gal and 100 μg/ml IPTG and incubated at 37°C for 24 hours to allow the blue color to develop sufficiently.
Isolation of BAC DNA and estimation of insert size
We isolated BAC DNA from 212 BAC clones randomly chosen using a QIAwell 8 Plasmid Kit (Qiagen, HRB, Germany) following the protocol of the manufacturer. Isolated BAC DNA were digested with the restriction enzyme NotI and then subjected to PFGE for 16 hours using the same PFGE conditions as those for high molecular weight DNA isolation.
Library pooling and PCR screening
White recombinant colonies were manually picked and arrayed to plates (Genetix, Hampshire, UK) of 384-well each containing of 60 μl of LB media and 25% glycerol. Plates were incubated overnight at 37°C and stored at -80°C. The frozen stocks of the primary clones in 384 well plates were recovered and transferred to 4 96-well PCR plates containing 100 μl LB medium supplemented with 15% glycerol and 12.5 μg/ml chloramphenicol, then incubated overnight at 37°C to make a copy of the BAC library.
To establish a hierarchical PCR screening system, the library was divided into 11 superpools each consisting of 12 plates of 384-wells. Each superpool was divided into 48 pools each consisting of one 96-well plate of BAC clones. Cultures from 48 pools were combined to make superpool DNA for the first step PCR screening. Cultures from 48 plates of 96-well BAC clones were combined to make pool DNA for the second step PCR screening. In each pool, cultures from each well of 96 clones from a 96-well plate were used for the third step screening.
For examining the genome coverage of the BAC library, twenty-four microsatellites (Lca318, Lca064, Lca137, Lca171, Lca098, Lca062, Lca130, Lca086, Lca301, Lca002, Lca058, Lca074, Lca253, Lca147, Lca069, Lca367, Lca021, Lca193, Lca220, Lca181, Lca255, Lca040, Lca411 and Lca231) located on each of the 24 linkage groups (Table 1) , and 15 ESTs/genes isolated from cDNA libraries or selected from GenBank were used to screen the library. These 15 ESTs/genes are: PVALB-1, 5-HT, PROL-A, 14KDA-AP, AMY-A, MX, AP, LECT2, LYSO-G, IGF-1, TUB1A, TUB2B, GT7, CYP19A2 and AFPII. Primers (Table 2) were designed in unique regions for each EST/gene using software PrimerSelect (Dnastar, WI, USA). The PCR reaction (25 μl) consisted of 2 μl cultured cells, 1 × PCR buffer (Finnzymes, Espoo, Finland) containing 1.5 mM MgCl2, 200 nM of each primer, 50 μM of each dNTP and one unit DNA polymerase (Finnzymes, Espoo, Finland). PCR was conducted on a PTC-100 PCR machine (MJ Research, CA, USA) using the following PCR program: an initial denaturation at 95°C for 2 min followed by 35 cycles 95°C for 30 sec, 55°C for 30 sec and 72°C for 1–2 min, and a final extension at 72°C for 5 min. PCR products are checked for the presence of PCR products on 2% agarose gels. Positive pools were used to determine a set of addresses corresponding to potential clones, which were subsequently validated by a third PCR analysis of individual clones. PCR products of respective microsatellites and genes/ESTs were confirmed by direct sequencing.
Microsatellite isolation from BAC clones and linkage mapping
DNA was isolated from pool of 300 BAC clones using a QIAwell 8 Plasmid Kit (Qiagen, HRB, Germany). CA- and GA-microsatellites located in the 300 BAC clones were enriched according to a previous protocol  with some modifications . Repeat-enriched DNA fragments of 400–1200 bp in size were cloned into pGEM-T vector (Promega, CA, USA), and transformed into XL-10 blue supercompetent cells (Stratagene, CA, USA). White clones were picked and arrayed into 96-well plates for bidirectional sequencing on an ABI3730 × l DNA sequencer (ABI, CA, USA) using the BigDye V3.0 kit, M13 forward and M13 reverse primers. Redundant and overlapping sequences were grouped using Sequencher (GeneCodes, MI, USA). Unique sequences were compared to known microsatellite sequences of L. calcarifer prior to primer design in order to reduce redundancy. Genotyping and linkage mapping of these microsatellites were performed with the mapping panel described previously . The graphic maps were generated using Mapchart software . To identify the origin of each microsatellite from the 300 BAC clones, these clones were PCR-screened with microsatellite primers. PCR products were checked for the presence of objective bands on 2% agarose gels.
List of abbreviations
bacterial artificial chromosome
quantitative trait loci
pavalbumin beta gene 1
5-hydroxytryptamine type 1 receptor
prolactin gene alpha type
14kDa apolipoprotein gene
amylayse gene alpha type
lysozyme goose type
tublin 1 alpha type
tublin 2 beta type
- GT7-EST containing a (GT)7 microsatellite:
CYP19A2-cytochrome P450 aromatase alpha type 2 and AFPII-type II antifreeze protein.
This study is part of the project "Selective Breeding of Marine Foodfish" funded by AVA, Singapore and the internal research fund from the Temasek Life Sciences Laboratory, Singapore. We would like to thank Dr. Xia J.H. for his comments on the manuscript before submission.
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