Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer

Library construction

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

To examine the quality of the BAC library, the sizes of 212 BAC clones randomly picked from the library were determined. All the 212 clones contained inserts. The insert size distribution of these 212 clones is shown in Figure 1 and 2. The average insert size was 98 kb, ranging from 45 to 200 kb. The insert size of over 80% of the BAC clones in this library was larger than 80 kb, and the insert size of 50% clones was smaller than 100 kb. This BAC library provides 6.9 time haploid genome equivalent based on a genome size of 700 Mb [6].

BAC library screening

To further assess the quality of this BAC library, 24 microsatellite markers randomly selected from each of the 24 linkage groups were used for hierarchical screening. PCR-screenings with the 24 microsatellite markers resulted in the number of positive clones varying from 3 to 15 with an average of 6.6 (Table 1) (see example of the PCR screening in Figure 3). PCR screening with these 15 ESTs/genes (PVALB-1, 5-HT, PROL-A, 14KDA-AP, AMY-A, MX, AP, LECT2, LYSO-G, IGF-1, TUB1A, TUB2B, GT7, CYP19A2 and AFPII) revealed that the number of positive BAC clones varied from 3 to 14 with an average of 7.3 (Table 2). The actual average number of positive BAC clones detected by screening with microsatellites and ESTs/genes was near to theoretically calculated number of genome coverage (i.e. 6.9 time coverage of the haploid genome). At least 3 positive BAC clones for each of the ESTs/genes were identified from the library.

Linkage group	Locus	GenBank accession no	Forward primer (5'-3')	Reverse primer (5'-3')	PCR Ta (°C)	Positive clone number
LG1	Lca318	DQ290175	TCCCACCCCAGTCCAGAAA	TACCAGAGCCTGAAACACAGTAGG	55	6
LG2	Lca064	AY998856	AGGCATATGCACCTCACAAGAGTG	CCCACGGTTTATTTATCTGTCATTATC	55	15
LG3	Lca137	DQ290039	CGCCTTAAATCTCTACGCTCTGG	TCGCATGCTGTAATTAAGGTGGTA	55	5
LG4	Lca171	DQ290065	ATTGCGTTACCAAGAGGTGAA	TGTCTTTGAAGGCTGAAAACTG	55	8
LG5	Lca098	AY998880	CAAAGGGGCCACTGCACATAAT	CTCCAGCTCACCCAGGTTCACT	55	5
LG6	Lca062	AY998854	AGGATGGCACGCTGAAACTATCG	ATAAGCTTGTACAGGGGCTGAGTGC	55	3
LG7	Lca130	DQ290035	GAGGCTCCCAATCCCAACAA	GGAGGCAGACGAGGAAGGAA	55	13
LG8	Lca086	AY998873	AAATGGCCTTCCTGTCCCTTCAG	GTGTTCCCTTGTTCTGCCACAGTG	55	4
LG9	Lca301	DQ290166	GCCAGTGTGAGGGACAGAGA	GGGCCTTGTTTTGCTTTTG	55	9
LG10	Lca002	AF007943	GCCGCTTGTTTACCAGTAAA	TCCATTTGAGGATTAACAGC	55	5
LG11	Lca058	AY998850	AAACAGGCAGCCAGATAGACAGAG	AAGAGGTGGTGGGACTAATTTGAGA	55	13
LG12	Lca074	AY998863	CATCATTTACACTCTGTTTGCCTCAT	GACAGACAGGTGTTTTAGCCTATTTG	55	6
LG13	Lca253	DQ290129	TGGGGACTTGACTTCCTTTTATG	TACCGAGGTTGGATGGTTTTCT	55	3
LG14	Lca147	DQ290047	TGCCCCTAATGTATTCTTTCCACT	GCTCCCACCTCTCATTCATTATTC	55	5
LG15	Lca069	AY998859	GCCTTTCTGTTTTCTGATTTATCTTCAT	AACACCCCGAAATACTGCTACTACAG	55	4
LG16	Lca367	DQ290206	TGTATTACAATGCCCGTGGTCA	TTAAGCCTTTGGTGTCTCAGTGTG	55	10
LG17	Lca021	AF404083	GTGCCACCTGCCTGACC	GCCATGACTGATTGCTGAGA	55	4
LG18	Lca193	DQ290082	CCTCTGCCTTTTCATCTATATTGC	CACATCGCACAAATGGACTGA	55	9
LG19	Lca220	DQ290104	ATGGCTGTGAAAAGACTGGTATCT	CGCCCCTCACTCAACAGAG	55	5
LG20	Lca181	DQ290073	CACTGGGTGGCGTTTGTATTAGC	CAAGAATTGGGATTTTGCTGTGC	55	8
LG21	Lca255	DQ290131	AGAGACACTTTATACGGGGACATC	GTAAACTGAAGCAAGCCAAACCT	55	7
LG22	Lca040	AF404099	TGAGGAAGCATCAGCTGTAATCA	CAGGACGCAAACACTGAAAT	55	3
LG23	Lca411	DQ290221	GTGGTGCAGCGGTTGCTCTC	CCGACTCATGCTGCTTTTCGTAAT	55	5
LG24	Lca231	DQ290112	GGCCAGGTTAATCAAGAC	ACTAGACTGCAATCAAACACA	55	3

Ta: annealing temperature for PCR.

Locus	GenBank accession no	Primer (5'-3', forward)	Primer (5'-3', reverse)	PCR Ta (°C)	PCR product length (bp)	Positive clone number
PVALB-1	AY688372	ATCGTCCGTCCGTTTCCCATAAAA	TGACCTTTCACCTCCCTCCAGACC	55	261	5
5-HT	EU136181	CTGCTCGGCGCGCTCAT	TCCATCCTGCACCTGTGCG	60	200	8
PROL-A	EU136180	GTGCAGAGCCGTCSCCATCA	TTCAGGAAGCTGTCRATCTTGTG	55	500	5
14KDA-AP	EU136179	CCGGGGACAGACAACTCGCTTTCAGAGA	ACAGGTTGGTGAGCTCCAGTTGGTGTTC	55	500	4
AMY-A	AY007592	GGTCGCTTTCCGTAATGTGGTCAA	ACCGGGCATGCCAGTGTTCA	55	250	9
MX	Ay821518	TCATTGATAAAGTGACAGCATTCA	CCAATATCCTTGAGTTTCTTGACA	55	400	7
AP	AJ888375	GACGCCCTCCTCTCCTCTCA	TTTCGACAGCCATCTCTGAACATA	55	700	4
LECT2	EU136177	TTTTTGATCTGAAGATGAGACGTGTCATC	GATCAGATCCCGAGCAGGTCAATC	55	1000	3
LYSO-G	EU136178	AGAGTCCAGGGCTGGAAAT	CCCTCAGAAACTTTAGTTGTGAAC	55	600	9
IGF-1	EU136176	CAGTGGCATTTATGTGATGTC	CCTCGACTTGAGTTTTTCTG	55	503	3
TUB1A	EU136175	GGCACTACACAATCGGCAAAGAGA	TCAGCAGGGAGGTAAAGCCAGAGC	55	144	11
TUB2B	EU136174	GTACAGACGGGGGAAGGGGACCAT	TTCCGCACCCTCAAACTCACCACA	55	160	13
GT7	EU136172	CAGGGTGATCACGCAGTGC	GGCAATCCGACAGCCAGAG	55	156	6
CYP19A2	AY684259	GCTCACCGCCTATAGCCAAAGAA	GGCCGAGTCCTGCCAAGAAA	55	505	8
AFPII	EU136173	TCCCTCCTGTGAAATTGGTTGG	AGGGACGCTGGCACAGACTG	57	1500	14

Ta: annealing temperature for PCR.

Microsatellite isolation from BAC clones and linkage mapping

In order to map BAC clones to the linkage map for future integrating linkage and physical maps, we isolated microsatellites from 300 BAC clones through enrichment of microsatellites. 864 clones collected from the microsatellite-enriched library were sequenced in both directions. 451 clones contained microsatellites (CA > 7 or GA > 7), yielding 229 unique sequences containing microsatellites. Among the 229 sequences, 218 had enough flanking regions for primer design. Primers were designed for 218 microsatellites, among which 63 within 63 BAC clones were informative in the mapping panel used for linkage mapping. 62 were mapped to 24 LG (Figure 4, 5, 6 and 7) while one marker LcaB044 remained unlinked, making the total number of mapped markers on the L. calcarifer linkage map to be 302. At least one BAC based marker was mapped on each LG. Thus, together with 24 microsatellites located in different BAC clones and used for screening the BAC library, a total of 86 BAC based markers have been anchored on the linkage map with at least one on each LG. The male maps of LG14 and LG18, which were split to two LG on the first generation map respectively [6] due to the limited number of markers, were merged to one LG respectively by integrating new microsatellites located in single BAC clones (Figure 6). Details about primer sequences, GenBank accession number, annealing temperature for PCR, PCR product size and locations of the 63 markers located in different BAC clones are summarized in Table 3.

Orginal order	Linkage group	Locus	GenBank accession no	Motif	Forward primer (5'-3')	Reverse primer (5'-3')	PCR Ta (°C)	Product Length (bp)	Location in the library (384plate-96plate-well)*
1	LG1	LcaB003	EU072356	(GT)14	CCTCATACTTGCATCAACATAATA	ATCAAAACCGGCTTCATCT	55	113	128-P2-A3
2	LG1	LcaB030	EU072367	(CA)27	TTCTCCCCGTGCCCCTTTGTA	AGCCCACTCCCCTGAGATGAGC	55	158	128-P2-B8
3	LG2	LcaB128	EU072400	(GT)16	AGTCGGCCTGTGCAATAAGAT	CAGCAGTTTGGGAATAATGACATA	55	262	128-P1-E4
4	LG3	LcaB002	EU072355	(GT)10	TTGGCTGTATTCCTCCTGTCTTGT	TTGGCTCTTTACGCTCAATACTCA	55	182	128-P2-A4
5	LG4	LcaB012	EU072359	(CA)9	GTGGGGTGTCCTGGCTCCTC	TCCCATCTCCTCCTGCTGTTTCT	55	329	128-P2-B11
6	LG4	LcaB014	EU072361	(GT)16	GCAGACCCGCTTTTTATTCAT	GTCCCCTCTGCTCCAGTGTT	55	181	128-P2-C12
7	LG4	LcaB052	EU072376	(CA)10	ATCATGACCCACAAGAGGAGAG	TCAGGGATAGAGACTTGTGAATGA	55	146	128-P2-A5
8	LG4	LcaB053	EU072377	(GT)18	GAGGCCCCGATGAGAAAACCTG	TGATGTCGGCGGAGGAGTGC	55	319	128-P2-H11
9	LG5	LcaB034	EU072369	(CA)8	TTTGCCTGAATAAAACCCTACACT	AAGCCCTTTGCACAGTATTATTTC	55	171	128-P2-C8
10	LG5	LcaB084	EU072391	(GT)14	GAGCGCTCGGCTGTTTCATC	CAGCCAATCTGTTTACCAGCACAC	55	248	128-P2-C4
11	LG5	LcaB086	EU072392	(CA)12	CAGATGATCTTTGACGAACTGAAA	TTCTTGGTGAAAAATGACAACAAA	55	157	128-P3-C5
12	LG5	LcaB130	EU072401	(CA)26	GGGGGAAAGGAAAAACTGATG	TGTAATGGTAAGATTTTGGGTGTG	55	215	128-P2-F6
13	LG5	LcaB177	EU072409	(GA)13	TTTAATTTTAGCCCCGTGATT	GTGTGCCAGTGGGTTTCTC	55	214	128-P3-C1
14	LG5	LcaB180	EU072410	(TC)13	AGTCTACACCGATTACACCAGTCT	ACTCTAACCGCACCAGAAAAG	55	243	128-P2-C7
15	LG5	LcaB229	EU072417	(GT)14	ACATCGCGTTCTCCTCTGAT	CCAGGGTGTGGTAGTCCTCTC	55	140	128-P3-C8
16	LG6	LcaB065	EU072384	(GT)13	GCATTGTTGGCAAAGTTGAGTAT	TCTTACAGTGGGCATCTGACCT	55	148	128-P3-G1
17	LG6	LcaB188	EU072411	(GA)17	TGATTTGGCTTTTAGGTGAAACA	TGACAAAAGAATGCCTTGCTCT	55	211	128-P3-D7
18	LG7	LcaB010	EU072358	(CA)9	TCCTCCTGGGCTGTTGTATCTTAT	ATGGGGTGGACCTCATTTTCA	55	155	128-P1-G10
19	LG7	LcaB072	EU072385	(GT)10	CAACGTGGGTGAATCTGTGT	TTGGCAGCAAATAATTCAGAGTAT	55	217	128-P1-A11
20	LG7	LcaB114	EU072395	(AC)8	TGTGCCCATGTTTACTAGATACCA	GTGTGCACGCTGCATTTGT	55	172	128-P2-F9
21	LG7	LcaB135	EU072402	(TC)18	CATCCCAGGTTTTCATACCATT	ACTGCGGTTATTAATCCACAAAG	55	123	128-P3-C4
22	LG7	LcaB151	EU072404	(TC)11	TTGTGCGCTTCTGTTTGTTTTTCT	GTAGGGCTATGCTGTTGGCTTTCT	55	311	128-P2-D2
23	LG8	LcaB025	EU072366	(GT)13	AGGGGGCAAAGGGGTCACG	GAGCCGGCAGTTGCACATCTG	55	160	128-P2-B3
24	LG8	LcaB083	EU072390	(GT)12	CGCTGGCATGGCTCTAGTAGTGAT	AGCGGGCTAAAAGCTGCTGTG	55	366	128-P1-H5
25	LG8	LcaB214	EU072413	(GA)12	AGCGGGAGGCTGAGAAGTAA	ACCCCTGCCTCTTGTTCATC	55	239	128-P1-H4
26	LG9	LcaB024	EU072365	(GT)10	AGAAGGGAAAAAGAGATGGGATGT	CAGGGCCGTTTTATTGCTGTAG	55	162	128-P3-B2
27	LG9	LcaB045	EU072373	(GT)26	ACAGGGAACGAATGGGGACAA	AAATTGGCACGCTCATTCAAGAAC	55	149	128-P2-D4
28	LG9	LcaB155	EU072405	(GA)24	TGTGGCCTTTGTGTAAGTGAGAA	TCATTCCCGCAAACAACACA	55	197	128-P3-G11
29	LG10	LcaB160	EU072406	(GT)13	CTTCATCCAGCCCAGTGACAG	GAATGGCCAGCTAAAACATCAAC	55	307	128-P3-A1
30	LG10	LcaB201	EU072412	(TC)16	ATTGCACCAGTCCCGAATGAG	GCAGCGTGCTTGTGGAAAAA	55	210	128-P2-D1
31	LG11	LcaB112	EU072394	(GT)7	TACCTGCCTTGTTTTTGTCCTTA	AAGCCTCCATACACAGCTACATTA	55	113	128-P1-D6
32	LG12	LcaB041	EU072371	(AC)8	AGGTATGTTTTTGGGGCTTTTAGT	CCCCCTACCCCTGTTTTACATA	55	250	128-P1-B5
33	LG12	LcaB058	EU072381	(AC)15	AAACCAAATGCTTACACAGTTACC	TTGAGAGCTATTGGGATTACACAT	55	160	128-P1-A2
34	LG13	LcaB059	EU072382	(AC)18	CCTAGCCAAGTGCAACAGTGTG	AGCTGGGAAACAGGCTGAGAC	55	186	128-P3-A12
35	LG14	LcaB055	EU072379	(AC)12	AGTTGCGGTCTTGTCCAAATGG	ACTGGCAGAGTCAAGCAAAGTGTG	55	325	128-P3-A3
36	LG14	LcaB075	EU072386	(GT)12	TGTCGCACACCGCTGCTTTACTAT	CTTGCTCTCACCCTCTCCCTCTTT	55	131	128-P2-G12
37	LG14	LcaB076	EU072387	(AC)17	CGAAAACGTCGATCCAACTAAA	ACAGTCAGTGCGTGAAGTGTATG	55	135	128-P3-A2
38	LG14	LcaB127	EU072399	(AC)11	AGTTGCAGGGCATGCTGTGAAAC	TCGGCATCAAGCGTGGAAGAG	50	159	128-P2-D6
39	LG15	LcaB174	EU072408	(GT)8(GA)15	CAGCATTAAAAAGATGAGAAAAGT	ATTCCCCCATCTTTGTTACAGTT	55	242	128-P2-D7
40	LG16	LcaB013	EU072360	(AC)15	AGGCCAAGGCTGCTCTGTGTC	CAACCTGGGATGAGGCACTAAAAG	55	127	128-P2-B12
41	LG16	LcaB054	EU072378	(AC)8	TGCAGGAGATAAGACGCTGTG	GAGATCGGCAACCTGACAAA	55	298	128-P3-F4
42	LG16	LcaB062	EU072383	(AC)15	ATGAGGGGTGAACAGTTGTCCT	TCTCCTCGTCCTTTTCGTTACC	55	218	128-P3-F8
43	LG16	LcaB078	EU072388	(GT)13	GTTACCATGCCAACAACCAA	TAGCCTGCTATAGATCCCACTG	55	81	128-P3-A4
44	LG16	LcaB228	EU072416	(AC)21	GAATAGGCCTACCTGGTGAGAGG	TCCCTGCTTAGCTGCCATTATC	55	237	128-P2-B12
45	LG17	LcaB023	EU072364	(GT)13	GCAGCGAGATGAACAGTGATTATT	ACATGATCCTCGCCACCATC	55	326	128-P2-G5
46	LG17	LcaB048	EU072374	(GT)20	TGGAGCTTTATTTGAGTGTGAC	CCCCCTATGTATTCAGTATTCTG	55	180	128-P3-C4
47	LG17	LcaB051	EU072375	(GT)19	TACCCAAAGTAAACCAGCAGCACA	CAACTAGCAGGTTTGCACAACACA	55	104	128-P3-H11
48	LG17	LcaB121	EU072397	(AC)16	CTTTTTGTGCCCCAGATGACG	GGAGCAGAGTGGAGCTTTCAGAA	55	238	128-P1-D9
49	LG18	LcaB019	EU072363	(GT)11	TTGAGTCCCCTGTGCTATGTAACA	CACCGCCTCCACAATTAGTGTC	55	199	128-P1-F10
50	LG18	LcaB081	EU072389	(GT)7(GCA) (GT)3	TGAGGACAGCCACCCCACTTTT	GAGCCGCTATCTCATTCCCACATC	55	126	128-P2-F10
51	LG18	LcaB221	EU072415	(TC)9	AGGGGAGTGCTGCCTCAGTG	TTCCCAACAGATAATGATGCTCAA	55	117	128-P3-A8
52	LG19	LcaB005	EU072357	(AC)22	AGGCGGTGCTGGGGCAGAT	TTACCGCAGCCTGGCTAGAGGTCT	55	300	128-P3-H8
53	LG19	LcaB033	EU072368	(AC)15	ATCCACCTTGAGGTTTCTTTATCA	AACCAAGCCACTCCTATCATCTT	55	190	128-P1-D5
54	LG20	LcaB219	EU072414	(GA)25	AGTTGGCTCTTAAAGCATTTGAAT	TTCCCACACCGTTAGGTTTATCTG	55	155	128-P1-H12
55	LG21	LcaB106	EU072393	(GT)7	CTGGCTGCATGGAGAAAGAAGT	TTGGGTTTTGAGCTCACTGACA	55	311	128-P2-F7
56	LG21	LcaB116	EU072396	(GT)20	CATGGCCTTTCTGGGAAGTTATTG	CAGACGGAGCCACAAGCAAAAC	55	226	128-P3-D6
57	LG21	LcaB169	EU072407	(AC)6(GA)20	CACAAACCAGGCGATCACATATCG	GTAAGCCCGCAGAAATCGACTTCA	55	218	128-P3-E9
58	LG23	LcaB015	EU072362	(GT)11	GAGCGCTCTCCCCTGGTTTC	TGCAGCCGAGCACGACTG	55	221	128-P1-G9
59	LG23	LcaB038	EU072370	(GT)19	TGTGCGCACTCACATACATTAG	TGAAAAATAGATGGTAAGCCTCTC	55	216	128-P1-A3
60	LG23	LcaB056	EU072380	(AC)11	ATGCCGTTTCCTGCTGCTGTC	TGATGCTGTTTCTGGCTGGTGTA	55	141	128-P2-E02
61	LG23	LcaB150	EU072403	(GA)11	TCTAGCGCTCGTCCTCTCCTG	AGGCCTCCTCGTTCTCTGCT	55	178	128-P2-A11
62	LG24	LcaB125	EU072398	(GT)12	AAGCACAAGATACGCCTTCCTT	GTGCCCTGGGCCTCTACAT	55	153	128-P2-C11
63	Unlinked	LcaB044	EU072372	(GT)15	CAGGACGTTTGAATACTTGTGT	TTAAAAGGTGGTGGTATTAGTCAT	55	160	128-P2-A8

Ta: annealing temperature for PCR. * 384plate-96plate-well: name of the 384-well plate, name of 96-well plate and well position in the 96-well plate.

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 [21]. 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 [22], 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 [23]. 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) [6], 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 [24] with some modifications [25]. 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 [6]. The graphic maps were generated using Mapchart software [26]. 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.