Zhikong scallop (C. farreri) trochophore larvae were obtained from an aquatic hatchery in Rongcheng, Shandong Province, China. Metaphase was prepared according to the methods described by Huang et al. (2006). Briefly, larvae were treated with colchicines (0.01%) in seawater for 2 h at room temperature, then exposed to 0.075 M KCl solution for 20 min, and, finally, fixed three times (15 min each) in the fresh Carnoy's solution (ethanol:glacial acetic acid, 3:1 v/v). The fixed larvae were dissociated into a cell suspension using 50% acetic acid and then dropped onto hot-wet slides and air-dried.

The chosen clones for testing signal specificity in FISH studies included microsatellite marker-anchored BAC clones, genes-anchored BAC clones, random BAC clones, repetitive sequences-anchored fosmid clones, and genes-anchored fosmid clones. All BAC clones used in the FISH analysis were screened from the C. farreri HindIII-BAC (BH) and BamHI-BAC (BB) libraries (Cheng, 2010), and the microsatellite markers contained in the BAC clones were selected based on the SSR genetic map of C. farreri (Zhan et al., 2009). All fosmid clones used in the FISH analysis were selected according to the information from pair-end sequencing results of the fosmid library of C. farreri (Zhang et al., 2007b). BAC and fosmid DNA were isolated via the standard laboratory methods (Sambrook et al., 1989) and then labeled with digoxigenin (Dig)-11-2'-deoxyuridine 5'-triphosphate (dUTP) or biotin-16-dUTP by Dig- or biotin-nick translation mix (Roche, American) according to the instructions of the manufacturer.

The FISH experiments were performed following the methods published by Huang et al. (2007b). Chromosome slides were pretreated with RNase A (100 μg/ml) in 2× saline sodium citrate buffer (SSC) at 37°C for 1 h, followed by pepsin (0.005%) in 10 mM HCl at 37°C for 10 min. Chromosome slides were denatured in 70% formamide mixed with 2× SSC at 75°C for 2 min, immediately dehydrated in a chilled ethanol series (70, 90, and 100%) for 5 min each, and then air-dried. Hybridization mixture consisted of 10–15 ng/μl Dig-11-dUTP and/or biotin-16-dUTP labeled BAC or fosmid DNAs, 50% deionized formamide, 10% dextran sulfate, and 2× SSC. The hybridization mixture was denatured at 90°C for 5 min and immediately cooled on ice. A denatured probe was then applied onto the slide, and DNA-DNA in situ hybridization was carried out in a moist chamber at 37°C for 16–18 h. Then, slides were washed in 50% formamide in 2× SSC at 37°C for 10 min, in 1× SSC at 37°C three times (5 min each), and in 2× SSC at room temperature for 5 min. Dig-labeled and biotin-labeled probes were detected using anti-digoxigenin-rhodamine (Roche, American) and fluorescein isothiocyanate (FITC)-conjugated avidin D cell sorter (DOS) (Vector, American), respectively. Chromosomal DNAs were then counterstained with 4,6-diamidino-2-phenylindole (DAPI) or 1.5 μg/ml propidium iodide (PI) at 37°C for 10 min. Hybridization signals were visualized, and images were recorded using a (Leica, Germany) DM4000B microscope equipped with an epifluorescence system and a charge coupled device (CCD) camera. At least 10 metaphases were examined for each probe and karyotype analysis, and the chromosomal pairs marked by clones were classified based on the Levan's criteria (Levan et al., 1964). Then, clones within distinct signals and discernable chromosomal location were selected for co-hybridization with other clones.

For co-hybridization, probes labeled with Dig- and biotin- were pooled for hybridization, and other protocol was the same as that for regular hybridization mentioned earlier.

After screening the BAC libraries of C. farreri, 58 microsatellite marker-anchored BAC clones and 4 genes-anchored BAC clones were obtained. Meanwhile, according to the information of pair-end sequences of the fosmid clones, 22 repetitive sequences-anchored clones and 5 genes-anchored clones were selected. In addition, 38 random BAC clones were also tested as a probe for FISH. In total, 127 BAC and fosmid clones were isolated and hybridized on mitotic metaphase chromosomes. Among them, 72 clones could successfully map on the chromosomes, and a total of 69 clones, including 58 BACs and 11 fosmids, could produce specific FISH signals on one pair of chromosomes. These BAC and fosmid clones producing unambiguous and reproducible FISH signals were used as chromosome-specific markers. As shown in Figure 1A, BAC clone BH1060G11 containing microsatellite marker CFLD034 hybridized to the middle of the long arm of a big pair of subtelocentric chromosomes. BAC clone BH565D6 containing microsatellite marker CFMSM014 was mapped to the long arm of a pair of submetacentric chromosomes, about two-fifth of the long arm away from the centromere (Figure 1B). FISH with BAC clone BB240C9 containing Dmrt4 gene revealed one single locus on the telomeric region of the long arm of a pair of submetacentric chromosomes (Figure 1C). The random BAC clone BH60F12 was located on the long arm of a pair of subtelocentric chromosomes, about four-fifth of the long arm away from the centromere (Figure 1D). In addition, fosmid clone F421B9 containing tandemly repetitive DNAs showed distinct signals on the telomeric region of the short arm of a pair of metacentric chromosomes (Figure 1E). Hybridization with fosmid clone F541H2 containing heat shock protein (Hsp22) gene (Figure 1F) revealed intensive signals on the short arm of a pair of subtelocentric chromosomes. The FISH results of all the isolated chromosome-specific clones were summarized in Table 1.

Three pairs of typical metacentric chromosomes of C. farreri could be identified by their distinct size, as shown in Figure 2. The largest one was named as Chr. 1, then the middle one as Chr. 2, and the smallest one as Chr. 3.

Due to the other 16 pairs of submetacentric and subtelocentric chromosomes appeared similar in morphology and continuous in size, at least 15 chromosome-specific markers are required to distinguish them. Among the 69 clones showing unambiguous unique FISH signals in the earlier results, 15 most reliable representations from the 15 pairs of submetacentric and subtelocentric chromosomes, which were shown to be located on the same type or similar size of chromosomes (Figure 3), were selected for multiple two-color FISH. As shown in Supplementary Figure 1-1a, three clones of F409B11, F420E3, and F458F11 were hybridized to three different pairs of submetacentric chromosomes, and we named the three chromosomes as Chr. 4 (Figure 3A), Chr. 5 (Figure 3B), and Chr. 6 (Figure 3C), respectively. The co-hybridization with the mixed probes containing F409B11, F420E3, F458F11, and BH1049A9 (Supplementary Figure 1-1b) revealed that BAC clone BH1049A9 located another submetacentric/subtelocentric chromosomes, which were different from Chr. 4–6, and we named it as Chr. 7 (Figure 3D). Supplementary Figures 1-1c,d show that BH1304E11 was mapped on one small pair of submetacentric chromosomes differing from Chr. 4–7, and this chromosome was named for Chr. 8 (Figure 3E).

Fluorescence in situ hybridization with BH1060G11, F409B11 (Chr. 4), and F420E3 (Chr. 5) (Supplementary Figure 1-1e) revealed that BH1060G11 was mapped on a big pair of a subtelocentric chromosome instead of submetacentric Chr. 4 and Chr. 5, although these three pairs of chromosomes showed similar size. The subtelocentric chromosome mapped with BH1060G11 was named as Chr. 9 (Figure 3F). Similarly, Supplementary Figures 1-1f,g show that clone F408A12 was assigned to another big pair of subtelocentric chromosomes named as Chr. 10 (Figure 3G) differing from Chr. 9 and Chr. 8. Clone BH1003G1 was located on a pair of submetacentric/subtelocentric chromosomes, and the co-hybridizations of this clone and F458F11 (Chr. 6) (Supplementary Figure 1-1j), this clone and BH1049A9 (Chr. 7) (Supplementary Figure 1-1h), as well as this clone and BH1060G11 (Chr. 9) and F408A12 (Chr. 10) (Supplementary Figure 1-1i) confirmed the different chromosome assignments of BH1003G1, and we named this chromosome as Chr. 11 (Figure 3H). BB32G4, BH60F1, BH1003G1 (Chr. 11), and F408A12 (Chr. 10) were hybridized to four different pairs of chromosomes (Supplementary Figures 1-1k,l), and the chromosome hybridized by BB32G4 was named as Chr. 12 (Figure 3I). BH1291D12 was located on another pair of subtelocentric chromosomes differing from Chr. 9–12 by the co-hybridization of BH1291D12, BH1060G11 (Chr. 9), and BB32G4 (Chr. 12) (Supplementary Figure 1-1m), BH1291D12 and F408A12 (Chr. 10) (Supplementary Figure 1-1n), as well as BH1291D12 and BH1003G1 (Chr. 11) (Supplementary Figure 1-1o), and this pair of chromosomes was named as Chr. 13 (Figure 3J). Supplementary Figures 1-2(a–c) revealed that clones BH60F12, BH1291D12 (Chr. 13), BB32G4 (Chr. 12), BH1003G1 (Chr. 11), and BH60F1 were assigned to different subtelocentric chromosomes, and the BH60F12-locating chromosome was slightly smaller than Chr. 13, and it was named as Chr. 14 (Figure 3K). Clone BH60F1 confirmed its location at another subtelocentric chromosome [named as Chr. 15 (Figure 3L)] by the co-hybridization results of this clone with the other clones mapped to the identified subtelocentric chromosomes, as shown in Supplementary Figures 1-1(k,l), 1-2(c,d). The clone BB239F4 had an assignment of the subtelocentric chromosome, named as Chr. 16 (Figure 3M), which were different from Chr. 9–15, which were revealed by the co-hybridization results of BB239F4 and BH60F1 (Chr. 15) (Supplementary Figure 1-2e), BB239F4 and BH60F12 (Chr. 14) (Supplementary Figure 1-2f), BB239F4, BH1291D12 (Chr. 13), and BH1060G11 (Chr. 11) (Supplementary Figure 1-2g), BB239F4 and BB32G4 (Chr. 12) (Supplementary Figure 1-2h), as well as BB239F4, F408A12 (Chr. 10), and BH1060G11 (Chr. 9) (Supplementary Figure 1-2i). BH64E8 and BH60A1 were readily mapped on two pairs of small subtelocentric chromosomes (Supplementary Figure 1-2o), and the co-hybridization analysis revealed that they could separate these two pairs of chromosomes from the other small subtelocentric chromosomes involving Chr. 16, 15, 14, and 13, as shown in Supplementary Figures 1-2(j–n). These two small subtelocentric chromosomes were named as Chr. 17 (Figure 3N) and Chr. 18 (Figure 3O), respectively. Since 15 of the 16 submetacentric and subtelocentric chromosomes with similar morphology and size had been identified, the remaining one would be automatically distinguished, and it was named as Chr. 19.

Based on the abovementioned data, all the 19 chromosomes of C. farreri were successfully distinguished and named for the first time.

The establishment of representative chromosome-specific cytogenetic markers allowed to construct a cytogenetic map of C. farreri. The main strategy was to do the co-localization analysis of the newly developed chromosome-specific markers and the representative chromosome-specific markers on the same chromosome.

By using this strategy, fosmid clone F418E2, BAC clones BB34D10, BH984B5, and BB105B2 were all hybridized to Chr. 1, and BB34D10 was located on the telomeric region of the short arm of Chr. 1, F418E2 was on the middle of one arm of this chromosome, while BH984B5 and BB105B2 were mapped to about middle and telomeric region of the other arm of Chr. 1, respectively (Figures 4A–C). The co-hybridizations of F458F11, BB34D12, and BB233G7 (Figure 4D), as well as BB34D12 and BH64A4 (Figure 4E), revealed that these clones were all assigned to Chr. 6. Among them, BB34D12 was located near the telomeric region of the short arm, the other three clones were all mapped to the long arm, which were close to the centromere (F458F11 and BH64A4), and about telomeric region of Chr. 6 (BB233G7). As shown in Figure 4F, BH1049A9 and BB240C9 were observed as located near the centromere and at the telomeric region of the long arm of Chr. 7, respectively. Clone BH799B12 was also mapped to Chr. 7, and it was located about midway on the short arm of this chromosome. The co-hybridization of BH1304E11 and BH60B1 confirmed their locations on Chr. 8, near the centromere and about four-fifth of the long arm away from the centromere, respectively (Figure 4G). BH1162H2 was mapped to the location that was near the BH1304E11 on the same chromosome. The chromosomal locations of F541H2, F393C3, BB69B10, BH323B5, and BH1285H8 were analyzed by simultaneously hybridizing with the representative clone of Chr. 10 (F408A12). Five clones of F541H2, F393C3, BB69B10, BH323B5, and F408A12 were all located on the short arm of Chr. 10, and their relative order on the chromosome was not unambiguous on the highly condensed metaphase chromosomes (Figures 4H–J). Clone BH1285H8 produced bright signals on the long arm of Chr. 10, about one-fourth from the centromere (BH1285H8, Figure 4K). BH60F12 and BH 966F2 were hybridized on the same chromosome with BH986B2, and they were all located on the long arm of Chr. 14 (Figures 4L,M). Of which, BH986B2 was mapped to near the centromeric region, BH 966F2 was on the middle of the long arm, and BH60F12 was about three-fourth from the centromere. BAC clones BH60F1 and BB75B6 were both located at the end of the short arm of Chr. 15 (Figure 4N). BH1308E3 was also mapped to Chr. 15 by co-hybridization with BB75B6, and it was hybridized near the centromeric region of the long arm. BH64E8 and BH1002C5 were simultaneously located on the short arm of Chr. 17, and it was difficult to distinguish their chromosomal locations (Figure 4O).

Referring to the FISH results of a single specific probe, 69 clones could produce positive signals on a single chromosome. However, for some of them, especially those producing high background, it was difficult to achieve the co-hybridization of multiple clones because the FISH condition of these clones was different. We determined the chromosomal locations of these clones by the karyotype analysis based on their one-color FISH. In summary, all the available data were used for the construction of the karyotype ideogram of C. farreri, which indicated the FISH mapping of 69 cytogenetic markers (Figure 5). All the information on these 69 markers is provided in Table 1. The cytogenetic markers on different chromosomes were different, with 3.6 as an average number of markers. Of which, Chr. 10 has the most number of markers which was nine, and Chr. 3 has the least number of markers which was one.