Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.

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THE silkworm, Bombyx mori, is one of the model organisms in genetic research, second among insects only to the fruit fly, Drosophila melanogaster. It is an economically important species with >3000 known strains (YAMAMOTO 2000) and >400 mutations reported for silkworms, corresponding to 230 mapped genes or loci (DOIRA 1983). Linkage groups have been established for gene mutants (FUJII et al. 1998) and densely spaced RAPD (PROMBOON et al. 1995; YASUKOCHI 1998, 1999), RFLP (SHI et al. 1995), and AFLP (TAN et al. 2001) markers. Whole-genome sequencing projects are well under way (MITA et al. 2004; XIA et al. 2004). Nevertheless, knowledge of the karyotype is still in its infancy. The chromosome number (n = 28, KAWAGUCHI 1928; 2n = 56, KAWAMURA 1979) is known and some progress has been made with respect to the identification of the sex chromosomes (TRAUT et al. 1999; SAHARA et al. 2003a) but there has been no general basis for chromosome identification and physical mapping.

Bombyx shares this problem with other moths and butterflies (Lepidoptera). They are cytogenetically characterized by possessing small and numerous holokinetic chromosomes. The chromosomes lack primary constrictions and are rather uniform in size during mitotic metaphase. No banding technique has yet been found to differentiate the chromosomes. Conditions are better for meiotic chromosomes, especially those in the pachytene stage when chromosomes are extended, pairwise synapsed, and display chromomere patterns (TRAUT 1976), but are still insufficient for general mapping purposes.

In this study, we used pachytene chromosome complements and fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH), which has recently been established in B. mori (SAHARA et al. 2003b) to identify all B. mori chromosomes and assign them to respective linkage groups. The basic requirements to achieve this goal were already fulfilled. BAC libraries, together consisting of 36,864 clones, have been constructed from two strains (WU et al. 1999), and dense genetic map data, based on genes and RAPD markers (YASUKOCHI 1998, 1999), were available. We screened the BAC libraries for suitable clones and determined the loci on Yasukochi's RAPD map. Using these BACs we identified all B. mori autosomes and constructed the complete karyotype of B. mori.

Isolation and genetic mapping of BAC clones:

The two-step PCR screening described in YASUKOCHI (2002) was employed to isolate BACs that represent suitable loci of all of the 28 linkage groups of B. mori. A BAC library (WU et al. 1999) constructed from B. mori strain p50 with average insert size of 134.5 kb was used for the PCR screening. Nine STS primer sets were designed to isolate BACs with known genes [M24370 + J04829, AB007831, X04223, AB011497, D85134, D86601, AF287267, AB010825, and B. mori prothoracicotropic hormone (SHIMADA et al. 1994)]. Partial sequencing was performed in another 60 BACs and STS primer sets designed from the resultant sequences. The STS primers amplify polymorphic DNA fragments between p50 and C108. Linkage analysis using these STSs was performed in the manner described previously (YASUKOCHI 1998) to determine the loci of the BACs. We isolated 7 additional BACs and ascertained their map positions by means of two independent but closely linked RAPD markers. All BACs are listed in Table 1.

View this table: In this window In a new window   TABLE 1 B. mori BAC clones and STS primers used in this study

 

Chromosome preparation:

Pachytene chromosome preparations from B. mori strain p50 were carried out according to SAHARA et al. (1999)(2003b). Briefly, the ovaries of last instar larvae were dissected in an insect saline (GLASER 1917) and pretreated in hypotonic solution (83 mM KCl and 17 mM NaCl; MAREC and TRAUT 1993) followed by fixation in Carnoy's fluid (ethanol, chloroform, acetic acid, 6:3:1). Cells were dissociated in 60% acetic acid and spread on a glass slide placed on a heating plate at 50°. The preparations were passed through a graded ethanol series (70, 80, and 98%) and stored in the freezer (–30°) until further use.

Probe labeling and BAC-FISH:

BAC-FISH was carried out according to the method described in SAHARA et al. (2003b) with slight modifications. Briefly, BAC-containing clones were cultured in LB medium containing 20 µg/ml chloramphenicol at 37° for 16 hr. DNA was extracted with a Plasmid Midi kit (QIAGEN, Tokyo). DNA labeling was done by nick translation using the Invitrogen nick translation system (Invitrogen, Tokyo) with Cy3-dCTP (Amersham, Tokyo) or fluorescein-12-dCTP (Perkin Elmer, Boston).

After removal from the freezer, chromosome preparations were passed through an ethanol series and air dried. Denaturation was done at 72° for 3.5 min in 70% formamide, 2x SSC. The probe cocktail for one slide consisted of 100 ng labeled BAC, 25 µg sonicated salmon sperm DNA (Sigma-Aldrich, Tokyo) and 10 µg (for single chromosome identification) or 100 µg (for karyotyping) sonicated B. mori male genomic DNA in 10 µl hybridization solution (50% formamide, 10% dextran sulfate, 2x SSC). After incubation in a moist chamber at 37° for 3 days, slides were washed at 62° in 0.1x SSC containing 1% Triton X-100. The slides were counterstained and mounted in antifade [0.233 g 1,4-diazabicyclo(2.2.2)-octane, 1 ml 0.2 M Tris-HCl, pH 8.0, 9 ml glycerol] containing 0.5 µg/ml DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich).

Image processing and measurement:

Black-and-white images were taken with a Photometrics CoolSNAP CCD camera attached to a Leica DMRE HC fluorescence microscope, through the A, L5, and N2.1 filters of the fluorescence filter set. Pseudocoloring and superimposing of the images were done using Adobe Photoshop, version 7.0. Routinely, red coloring was used for Cy3, green for fluorescein, and light blue for DAPI images.

Chromosome length was measured by using free software, ImageJ (http://rsb.info.nih.gov/ij/index.html). The results presented are average lengths of measurements repeated five times.

Selection of BAC clones:

The B. mori BAC library of WU et al. (1999) was screened to identify suitable clones for BAC-FISH mapping. Sixty-nine BACs were isolated with polymorphic STSs whose loci were confirmed by linkage analysis on the same population of 166 F₂ individuals described in YASUKOCHI (1998), and 7 BACs were isolated with two independent and closely linked RAPD markers (YASUKOCHI 1998). In total, we selected 76 BACs, 2–6 from each of the 28 linkage groups (Table 1).

Identification of individual chromosomes:

To test the system, we hybridized the three BACs 9A5H, 14I7D, and 5H3E from linkage group 1, the Z chromosome, to female pachytene complements. The three BAC probes indeed hybridized to the Z chromosome, which was identified independently as the pairing partner of the W chromosome (Figure 1A). The W chromosome had been painted with genomic in situ hybridization (GISH) (SAHARA et al. 2003b). The result shows that our BAC-FISH procedure reliably identifies the Z chromosome and that the chromosomal sites of the BACs corresponded well with their loci on the RAPD map of linkage group 1 (Figure 1, B and C).

View larger version (26K): In this window In a new window Download PPT slide   FIGURE 1.— Detection of the WZ bivalent (Z is linkage group 1) using Z-BACs 9A5H, 14I7D, 5H3E, and a female-derived whole-genomic probe for the W chromosome (A). Red signals are from Z-BACs and green signals are from the whole-genomic probe. The chromosomes are counterstained with DAPI (light blue). The arrow points to the heterochromatic block of an autosomal bivalent (see text). N, nucleolus. Bar, 10 µm. (B and C) Linkage map with RAPD loci (in black) from YASUKOCHI (1998) and the map positions of BACs (in red) corresponding to the signals on the Z chromosome.

 
In this manner, all 27 autosomes were identified by two to four red or green double-dot signals (one dot per homolog) depending on the number of Cy3- (red) or fluorescein- (green) labeled linkage group-specific BACs used as probes. The chromosomes are shown in Figure 2 with each bivalent arranged together with the corresponding linkage map oriented with position 0 cM at the top. In a few cases, we produced yellow double-dot signals. This was intended when we mixed Cy3- and fluorescein-labeled probe from the same BAC (Figure 2, bivalents of linkage groups 14 and 15). The same effect was caused inadvertently by two differently labeled BACs with overlapping hybridization signals (Figure 2, bivalents of linkage groups 18 and 20). We never detected double-dot signals on other autosomes. The W chromosome, however, displayed extra signals with many of the autosome-specific BAC probes (see below). In most bivalents, there was good correspondence of the labeling pattern on the chromosome with the determined positions on the respective linkage map. Exceptions are BACs 8H2A in LG8, 3A3C in LG13, 5E8C in LG16, and 3H6F in LG18, which mapped in the correct order but not in the expected distance from one another (Figure 2).

View larger version (59K): In this window In a new window Download PPT slide   FIGURE 2.— Identification of individual chromosomes and comparison with the linkage map. Note that the lengths of chromosome bivalents cannot be compared in this figure as they are derived from different cells. Numbering of linkage groups follows FUJII et al. (1998) and YASUKOCHI (1998) (for details see YASUKOCHI et al. 2005). Linkage maps show RAPD loci from YASUKOCHI (1998) (in black), including the markers used to select the BACs for FISH (red, green, or yellow text on the right). The sites of BAC-FISH signals correspond nearly always with those map positions. For the BACs used, see Table 1.

 
The chromosomes were routinely stained with DAPI. We found that two of the 28 bivalents, those corresponding to linkage groups 11 and 24 (Figure 2), could also be reliably discriminated by the DAPI pattern. The bivalent corresponding to linkage group 11 was easily recognizable by the attached nucleolus, which divided the chromosome into two arms (Figure 2). The arm ratio, 2:1 (the end of the long arm corresponding to the proximal end of the linkage map), was similar to that given in previous reports (RASMUSSEN 1976; TRAUT 1976). In the bivalent corresponding to linkage group 24, a segment of 10% of the chromosome length was deeply stained with DAPI. This conspicuous, presumably heterochromatic, segment was located at approximately two-thirds of the chromosome length (Figure 2, LG24). The DAPI-positive segment had already been detected in a previous study when it was recognized as a conspicuous autosomal heterochromatic block strongly painted by GISH (SAHARA et al. 2003b).

Karyotyping B. mori:

For karyotyping, we used a probe cocktail consisting of 62 BACs labeled with Cy3-dCTP and/or fluorescein-dCTP. The respective BACs and their color combination used for discriminating the chromosomes are listed in Table 1. All 28 bivalents of well-spread pachytene nuclei could be discriminated (Figure 3A). We arranged the bivalents of one cell (Figure 3B, cell 4 in Table 2) according to their average length calculated from 12 karyotyped complements. The probe cocktail did not include markers for the chromosomes corresponding to linkage groups 1(Z), 11, and 24. Those corresponding to linkage groups 11 and 24 could be recognized from the DAPI pattern alone. And, Z-specific probes proved unnecessary as the probe cocktail reliably produced extra label on the W chromosome to which the Z chromosome was synapsed in the pachytene stage.

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 3.— BAC-FISH karyotype of B. mori. (A) An oocyte pachytene nucleus. Bar, 10 µm. (B) The bivalents from this nucleus arranged according to their lengths together with their diagnostic representation.

 

View this table: In this window In a new window   TABLE 2 Pachytene bivalents from 12 karyotyped cells ordered according to bivalent length

 
Length measurements of 12 karyotyped complements are listed in Table 2. It is obvious that the various chromosomes occupy similar but not constant positions in the order of length. Systematic individual length changes during pachytene development are not apparent but cannot be excluded. Considering the rather shallow length gradient especially in the middle part of the B. mori complement, however, variation in position may be merely due to measurement error (the median of standard error is 0.101 with the range from 0.019 to 0.340) and/or variation in individual chromosome spreading.

The high number and small size of the chromosomes and the absence of suitable cytogenetic markers like bands and localized centromeres have hitherto inhibited chromosome identification in B. mori. We circumvented the problem by (1) hybridizing selected fluorescence-labeled BACs to the chromosomes (BAC-FISH) and (2) using pachytene instead of mitotic chromosomes. Pachytene chromosomes have the advantage of an extended length (4.3 times longer than mitotic chromosomes on average) and a reduced number (28 bivalents instead of 56 single mitotic chromosomes). In this way, we were able to identify all 28 chromosomes of the B. mori complement individually.

BAC-FISH had previously been applied to fine mapping in extended chromatin fibers (WEIER 2001). The method commonly used to identify chromosomes by FISH is either chromosome painting (CREMER et al. 1988; LICHTER et al. 1988; PINKEL et al. 1988) or by using centromeric chromosome-specific repetitive sequence probes (HIZUME et al. 2002; VISCHI et al. 2003), which are not available in B. mori. Karyotyping of the whole chromosome complement has been achieved in the human and in the mouse with sophisticated color schemes and probe mixes of five and seven different fluorophores. M-FISH (SPEICHER et al. 1996; JENTSCH et al. 2001) and spectral karyotyping (LIYANAGE et al. 1996; SCHRöCK et al. 1996) are two of these methods. We had decided against chromosome painting. Probes for chromosome painting are usually generated from sorted chromosomes (VAN DILLA et al. 1986; COLLINS et al. 1991; VOOIJS et al. 1993), from microdissected chromosomes (GUAN et al. 1994), or from a densely spaced chromosome-specific BAC array (LYSAK et al. 2001). Sorting chromosomes is probably not possible in Bombyx due to the similarity of chromosome sizes (see Table 2). Microdissection of chromosomes is confronted with the same problem if one does not wish to rely on a single microdissected anonymous chromosome. A collection of 28 sufficiently dense chromosome-specific BAC arrays would have been a feasible alternative for probe generation. But this would have been a rather expensive alternative with respect to costs and experimental work and it would have given less information. The obvious advantage of using only a few BACs and two different fluorophores per chromosome is the generation of anchor points that relate to the genetical map. Besides mere identification of the chromosome, they provide a framework for physical mapping of other BACs and allow us to distinguish between the two chromosome ends.

The two B. mori whole-genome shotgun sequencing projects, MITA et al. (2004) and XIA et al. (2004), presently under way have already achieved 3x and 6x coverage, respectively, of the genome. The karyotype and the anchor points provided in this article afford a basis to map contigs directly or with little experimental effort to the respective chromosomes and linkage groups. Our results already disclose that the map of 28 linkage groups mainly based on RAPD markers (YASUKOCHI 1998) truly reflects the chromosome status in B. mori.

Since the BACs have been proven to give chromosome-specific signals, identification of individual mitotic (as opposed to meiotic pachytene) chromosomes by BAC-FISH will probably also be possible in B. mori. BAC-FISH karyotyping of mitotic chromosomes or mapping other probes relative to our anchor points, however, will be difficult or impossible due to the small size of the mitotic chromosomes. On the other hand, the BAC-FISH karyotyping method is probably easily adaptable to other species with similar problems of chromosome recognition.

We thank Walther Traut (Luebeck, Germany) for critical discussions and Yasuhiro Yamada for technical assistance. This study was partially supported by Grant-in-Aid for Scientific Research no. 15380227 from the Japan Society for the Promotion of Science (K.S. and Y.Y.) and by the Insect Technology Project from the Ministry of Agriculture, Forestry and Fisheries in Japan, no. 2102 (Y.Y.) and no. 2013 (K.S.).

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