The Bombyx mori Karyotype and the Assignment of Linkage ...2 22.3 1I12H Fluorescein CACAGGGCTTTTTGGTTCTA GCTTTTATGTTATTCACTCG 2 60.3 5J8G Cy3 AAAACGGCTAACTAACGAAG TGAGAAACAGGAGACTACT

Copyright © 2005 by the Genetics Society of AmericaDOI: 10.1534/genetics.104.040352

The Bombyx mori Karyotype and the Assignment of Linkage Groups

Atsuo Yoshido,* Hisanori Bando,* Yuji Yasukochi† and Ken Sahara*,1

*Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan and†National Institute of Agrobiological Sciences, Tsukuba, 305-3934, Japan

Manuscript received December 26, 2004Accepted for publication February 15, 2005

ABSTRACTLepidopteran 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 andby the absence of suitable markers like centromere position and chromosome bands. In this study, wecarried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using geneti-cally mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to foureither green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocallyeach of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assignedone of the already established genetic linkage groups and the correct orientation in the chromosome wasdefined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. morigenome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition inorganisms where conventional banding techniques are not available.

THE silkworm, Bombyx mori, is one of the model or- apsed, and display chromomere patterns (Traut 1976),but are still insufficient for general mapping purposes.ganisms in genetic research, second among insects

In this study, we used pachytene chromosome com-only to the fruit fly, Drosophila melanogaster. It is an eco-plements and fluorescence in situ hybridization withnomically important species with �3000 known strainsbacterial artificial chromosome probes (BAC-FISH),(Yamamoto 2000) and �400 mutations reported forwhich has recently been established in B. mori (Saharasilkworms, corresponding to �230 mapped genes or lociet al. 2003b) to identify all B. mori chromosomes and(Doira 1983). Linkage groups have been established forassign them to respective linkage groups. The basic re-gene mutants (Fujii et al. 1998) and densely spacedquirements to achieve this goal were already fulfilled.RAPD (Promboon et al. 1995; Yasukochi 1998, 1999),BAC libraries, together consisting of 36,864 clones, haveRFLP (Shi et al. 1995), and AFLP (Tan et al. 2001)been constructed from two strains (Wu et al. 1999), andmarkers. Whole-genome sequencing projects are welldense genetic map data, based on genes and RAPDunder way (Mita et al. 2004; Xia et al. 2004). Neverthe-markers (Yasukochi 1998, 1999), were available. Weless, knowledge of the karyotype is still in its infancy.screened the BAC libraries for suitable clones and deter-The chromosome number (n � 28, Kawaguchi 1928;mined the loci on Yasukochi’s RAPD map. Using these2n � 56, Kawamura 1979) is known and some progressBACs we identified all B. mori autosomes and con-has been made with respect to the identification ofstructed the complete karyotype of B. mori.the sex chromosomes (Traut et al. 1999; Sahara et al.

2003a) but there has been no general basis for chromo-some identification and physical mapping.

MATERIALS AND METHODSBombyx shares this problem with other moths andbutterflies (Lepidoptera). They are cytogenetically char- Isolation and genetic mapping of BAC clones: The two-stepacterized by possessing small and numerous holokinetic PCR screening described in Yasukochi (2002) was employed

to isolate BACs that represent suitable loci of all of the 28chromosomes. The chromosomes lack primary constric-linkage groups of B. mori. A BAC library (Wu et al. 1999) con-tions and are rather uniform in size during mitotic meta-structed from B. mori strain p50 with average insert size of 134.5phase. No banding technique has yet been found to dif- kb was used for the PCR screening. Nine STS primer sets were

ferentiate the chromosomes. Conditions are better for designed to isolate BACs with known genes [M24370 � J04829,meiotic chromosomes, especially those in the pachytene AB007831, X04223, AB011497, D85134, D86601, AF287267,

AB010825, and B. mori prothoracicotropic hormone (Shimadastage when chromosomes are extended, pairwise syn-et al. 1994)]. Partial sequencing was performed in another60 BACs and STS primer sets designed from the resultantsequences. The STS primers amplify polymorphic DNA frag-ments between p50 and C108. Linkage analysis using these1Corresponding author: Division of Applied Bioscience, GraduateSTSs was performed in the manner described previouslySchool of Agriculture, Hokkaido University N9, W9, Kita-ku, Sapporo,

060-8589, Japan. E-mail: [email protected] (Yasukochi 1998) to determine the loci of the BACs. We

Genetics 170: 675–685 ( June 2005)

676 A. Yoshido et al.

isolated 7 additional BACs and ascertained their map positions indeed hybridized to the Z chromosome, which wasby means of two independent but closely linked RAPD mark- identified independently as the pairing partner of theers. All BACs are listed in Table 1.

W chromosome (Figure 1A). The W chromosome hadChromosome preparation: Pachytene chromosome prepa-been painted with genomic in situ hybridization (GISH)rations from B. mori strain p50 were carried out according to

Sahara et al. (1999, 2003b). Briefly, the ovaries of last instar (Sahara et al. 2003b). The result shows that our BAC-larvae were dissected in an insect saline (Glaser 1917) and FISH procedure reliably identifies the Z chromosomepretreated in hypotonic solution (83 mm KCl and 17 mm NaCl; and that the chromosomal sites of the BACs corre-Marec and Traut 1993) followed by fixation in Carnoy’s

sponded well with their loci on the RAPD map of linkagefluid (ethanol, chloroform, acetic acid, 6:3:1). Cells were disso-group 1 (Figure 1, B and C).ciated in 60% acetic acid and spread on a glass slide placed on

a heating plate at 50�. The preparations were passed through a In this manner, all 27 autosomes were identified bygraded ethanol series (70, 80, and 98%) and stored in the two to four red or green double-dot signals (one dotfreezer (–30�) until further use. per homolog) depending on the number of Cy3- (red)

Probe labeling and BAC-FISH: BAC-FISH was carried outor fluorescein- (green) labeled linkage group-specificaccording to the method described in Sahara et al. (2003b)BACs used as probes. The chromosomes are shown inwith slight modifications. Briefly, BAC-containing clones were

cultured in LB medium containing 20 �g/ml chlorampheni- Figure 2 with each bivalent arranged together with thecol at 37� for 16 hr. DNA was extracted with a Plasmid Midi kit corresponding linkage map oriented with position 0 cM(QIAGEN, Tokyo). DNA labeling was done by nick translation at the top. In a few cases, we produced yellow double-using the Invitrogen nick translation system (Invitrogen,

dot signals. This was intended when we mixed Cy3- andTokyo) with Cy3-dCTP (Amersham, Tokyo) or fluorescein-fluorescein-labeled probe from the same BAC (Figure12-dCTP (Perkin Elmer, Boston).

After removal from the freezer, chromosome preparations 2, bivalents of linkage groups 14 and 15). The samewere passed through an ethanol series and air dried. Denatur- effect was caused inadvertently by two differently labeledation was done at 72� for 3.5 min in 70% formamide, 2� SSC. BACs with overlapping hybridization signals (Figure 2,The probe cocktail for one slide consisted of 100 ng labeled

bivalents of linkage groups 18 and 20). We never de-BAC, 25 �g sonicated salmon sperm DNA (Sigma-Aldrich,tected double-dot signals on other autosomes. The WTokyo) and 10 �g (for single chromosome identification) or

100 �g (for karyotyping) sonicated B. mori male genomic DNA chromosome, however, displayed extra signals within 10 �l hybridization solution (50% formamide, 10% dextran many of the autosome-specific BAC probes (see below).sulfate, 2� SSC). After incubation in a moist chamber at 37� In most bivalents, there was good correspondence offor 3 days, slides were washed at 62� in 0.1� SSC containing 1%

the labeling pattern on the chromosome with the deter-Triton X-100. The slides were counterstained and mounted inmined positions on the respective linkage map. Excep-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 �g/ml DAPI tions are BACs 8H2A in LG8, 3A3C in LG13, 5E8C in(4�,6-diamidino-2-phenylindole; Sigma-Aldrich). LG16, and 3H6F in LG18, which mapped in the correct

Image processing and measurement: Black-and-white im- order but not in the expected distance from one an-ages were taken with a Photometrics CoolSNAP CCD camera

other (Figure 2).attached to a Leica DMRE HC fluorescence microscope,The chromosomes were routinely stained with DAPI.through the A, L5, and N2.1 filters of the fluorescence filter

set. Pseudocoloring and superimposing of the images were We found that two of the 28 bivalents, those correspond-done using Adobe Photoshop, version 7.0. Routinely, red col- ing to linkage groups 11 and 24 (Figure 2), could also beoring was used for Cy3, green for fluorescein, and light blue reliably discriminated by the DAPI pattern. The bivalentfor DAPI images.

corresponding to linkage group 11 was easily recogniz-Chromosome length was measured by using free software,able by the attached nucleolus, which divided the chro-ImageJ (http://rsb.info.nih.gov/ij/index.html). The results

presented are average lengths of measurements repeated five mosome into two arms (Figure 2). The arm ratio, �2:1times. (the end of the long arm corresponding to the proximal

end of the linkage map), was similar to that given inprevious reports (Rasmussen 1976; Traut 1976). In

RESULTSthe bivalent corresponding to linkage group 24, a seg-ment of �10% of the chromosome length was deeplySelection of BAC clones: The B. mori BAC library of

Wu et al. (1999) was screened to identify suitable clones stained with DAPI. This conspicuous, presumably het-erochromatic, segment was located at approximatelyfor BAC-FISH mapping. Sixty-nine BACs were isolated

with polymorphic STSs whose loci were confirmed by two-thirds of the chromosome length (Figure 2, LG24).The DAPI-positive segment had already been detectedlinkage analysis on the same population of 166 F2 indi-

viduals described in Yasukochi (1998), and 7 BACs were in a previous study when it was recognized as a conspicu-ous autosomal heterochromatic block strongly paintedisolated with two independent and closely linked RAPD

markers (Yasukochi 1998). In total, we selected 76 BACs, by GISH (Sahara et al. 2003b).Karyotyping B. mori : For karyotyping, we used a probe2–6 from each of the 28 linkage groups (Table 1).

Identification of individual chromosomes: To test the cocktail consisting of 62 BACs labeled with Cy3-dCTPand/or fluorescein-dCTP. The respective BACs andsystem, we hybridized the three BACs 9A5H, 14I7D, and

5H3E from linkage group 1, the Z chromosome, to their color combination used for discriminating thechromosomes are listed in Table 1. All 28 bivalents offemale pachytene complements. The three BAC probes

677BAC-FISH Karyotype of the Silkworm

TABLE 1

B. mori BAC clones and STS primers used in this study

Probes for STS and RAPD primersLocus BAC karyotype Accession

LG (cM) code labeled with Forward Reverse no.

1(Z) 9.8 9A5H AATCTCAAAAATAACCCGTAG AAAAATCAGGAAACTCAACAT1(Z) 68.4 14I7D CGTTGTAACTCGTATGCTTT GCCCTTTATTTGACTGACTC1(Z) 89 5H3E GTGGCAAGCGAGATGGTTAC AGGTGGAGTGGTTAGTTTTA

2 18.7 9D6C Cy3 ACAAACAAACAAAGGCAAAG TGAAAAGTCTACCCATCTCT2 22.3 1I12H Fluorescein CACAGGGCTTTTTGGTTCTA GCTTTTATGTTATTCACTCG2 60.3 5J8G Cy3 AAAACGGCTAACTAACGAAG TGAGAAACAGGAGACTACT2 89.5 12F1E Fluorescein AAGTATCACAGAACGAATG ATATGAAAGCCAACACGA

3 9.2 1A4B Cy3 GAGAAGCCCATAAGAACTAA TACAAAGACAAAGCGAATCA3 38.3 1J6D Cy3 TCATTCATCATCTAAACTCG a AACAAATACGGCATCCACTG a M24370,

J048293 97 6E4G GACCATCCATCCACCACTA TAAAAGCCCATAACATCA

4 0 1D9H Cy3 AGACACAGGGAAGCATTT b GTCGTGGGAGCAGTTGGA b

4 58.5 1G9D; Cy3; CTGGGTTCTGTAGTGTCGTC CTTCTTTTGGTTCGTGGTATC4L5E fluorescein

5 31.2 2C2D Fluorescein CTTTATTTTGAGCCACCTTT AACCGACATTTATTAGCACA5 52.8 7H6C Fluorescein TCAGCCAGTCACCTTGTTTT GTTACTCCTCTACCTTATGT

6 3 5J10A TTCAACAGCCTACAACAGCA TATCCCATCGCCTTACCAAT6 7.1 10L5C Cy3 GATAATGTCAAGTCAAAACG TATGTCAAACGAAGTAAGCA6 56.6 8C9E AACTTCCATCCACTTACTTCG GGTTCTTTATTACTCACTTTGC6 80.8 3A5B Cy3 TCGGGAAACATAGTAGAAGG c AAAAGCAAGTGAACAGTGAG c

6 94.2 17H8F Fluorescein AACGGATAAAAAGAAAACAA c GCTGAAATGAGAAAACACGA c

6 107.1 9L9D Cy3 TAGGCAGACGAGCATACGG TTCAGTGTCAATAGCAATCC

7 26.8 7I6E Fluorescein TTTGATTTTTATTTGTTCG GTTTATTTGCCCCGTGGAC7 79.3 4G9F Fluorescein ATCGTCCCGCCATCACAAAT b CAAAGCAAAGGTTAGAGAAA b

8 100 4B3A Fluorescein TGACAACATTTATTCCCTTAT CATTCTCTTCTCCTTTTCTTC8 116.1 8H2A Cy3 TCTTTGTAATGAGCGGTAT GTAGGGGAAGGGTTTTGTAT8 132.9 2K5B Fluorescein ACTGCCTTTGTTTACTCAC GCTCACCGCTTTATTATTCT

9 8.7 3C6H Cy3 CTTGCCTTTTTATTTACCTC GATTTCCATCCCGTGCTTCA9 52.3 4D8E Cy3 CTATTGCCTACGCCTATTGA GGTTTCTATTCTGGTTATCG9 105.3 3F8D Fluorescein GACCATTAGAAGCATTAGTGT TTGAAGTTGAGAAGTTACCAG AB011497

10 14.9 3F10F Fluorescein ACTGGCCTGA d/GGACAACGAG d ACCGCGAAGG d/AGGGCGTAAG d

10 47.6 4B10D Fluorescein AGCCTCGTGTCTCTTTTGAA AGAGTGATTTTTCGGCTTTT10 92.6 5L8D Fluorescein ATTATTGTGTGACTTTTGACTG GACTCTCTGCTTTCCTTATTC

11 96.6 7H8B TGATTAGACCGCAACGAGTA CAGTAGATAGATTTAGACACAT11 115.1 7E3G TCCTTACCTGCTATTTATCG a TACTTATTATCTTGCCTTGT a AB007831

12 26.3 5I3D Cy3 CTTTATTTTCGTGTTTTGTC TCATTATGGTAGGGTTGTTC12 51.7 8L6H Fluorescein GCCAAGATAAACAGTAGCATT TTTCAGCGATAAGATAAGTAAG12 60.8 8A3H Cy3 CGTAATAATCGCAATAATCT TGTCAATAAATCTCGTGGTT

13 6 8H12G Fluorescein TTCTTCGTAAACCCACACTA GAACAAATCGCAGGACACTT13 71.9 3A3C Cy3 CCGTGACCGAAATAGGAAGA AGATGCGATGTAGACCAGAG13 100.6 6K10B Fluorescein ACGGGAATAGGATAGAACC CTGGCGATTGCTGTAAAAC

14 20.5 3H1H Fluorescein AACAGCATTACAGTCATCAA TATCATTTTTCATTTCAGTG14 48.7 4H6C Cy3 ATCTGTTTACTACGGTTTTC CAAGCGGTCGGTTTCACTAT

(continued)


TABLE 1

(Continued)

Probes for STS and RAPD primersLocus BAC karyotype Accession

LG (cM) code labeled with Forward Reverse no.

14 81.5 2H2F Fluorescein AAGGTCAATAAACAACAGC TAGCGTATCACAAACAAATCG

15 101.5 3K9A Cy3 � TAATGAAAAATGGCTACCG TCGCAAATCAGGCAAAATfluorescein

15 126.5 7J1A Fluorescein GCTTTTGTTGGTTGATTTTA CTGGATTGTGGCTTTGACTA15 133.8 2I12E ATTATCCGTTCACCATCGTT GCCCTTGCTTCCTCTTCGTA

16 26 4A3D Cy3 CGAGAATAAATGAATAGATGTGA CTTTGGTGTCCGAGGTTATTA D8513416 27.2 5E8C Cy3 TTATTGGTATTGGCATTATC CTTCAGAGGTTTTGTCGTAT16 67.7 6A9C Cy3 GTTGTGTTTTATTTGTTCG TGATGACGCTTGTATTAGGA

17 53.1 1D2A ACATAACTCAACGCAAAAGCA TGACTACGGACACTACCAAAC17 71.2 3I2C Fluorescein TAGATAACTCGCAATGGTGAA CAAAATAAGCAATAACAGACT

18 34.9 1D1D Cy3 CAAACGTCGGd/AGTCGTCCCCd GGGGTGACGAd/GGTCCCTGACd

18 45.1 3H6F Fluorescein CAAGGGCAGAd/TTTGCCCGGTd GTGACGTAGGd/GGTGAACGCTd

18 77.3 1G2F Cy3 CCGCCTAGTCd/TTTGCCCGGTd CCAACGTCGTd/CCATTCCCCAd

19 18.8 4D12F Cy3 ATGCTATTGTTTCGCTTTTCe CTCTATTAGTGTCTGTTTGGc X0422319 62 1D12C Cy3 CCTGAAGAAGAATGGTAGAT TCAAACGAAACAAAATAGAA D8660119 83.4 8A3F Fluorescein TCCACTCCTGd/CCGCCTAGTCd AATGCCGCAGd/AGGGGTCTTGd

20 32.6 5K2D; Fluorescein; GTCTGCCTATCTCTGTTTAT CTTACCTACCTTACCCATTT6L7E Cy3

21 10.1 3H8E Cy3 TAGTGTAGGGAGCCATAGGG GATAGAGTGATTGAAGAGGA21 84 5C12G Fluorescein GTTCTGGTTGTTCGCTCATC CTGGTGTTTATCGTCCCTAC21 115.2 4B8B Fluorescein TAACGAGATTTGGGCATTCb GCTGGCTAGAGACATTTCAb

22 80.8 5B8H Cy3 GACTCCTGCGATTTAGTTTCf CAAAGAAAGTTTATACAGTG f

22 114.2 7E11E AAAGTGCGGGd/GGAGAGACTCd GTGACGTAGGd/AACGGCGACAd

23 83.2 6G3E Fluorescein GCCTTGGATGAGATAGAAT CGTGTGGTGCTTGGAATAG23 128.6 8G6G Cy3 CTGTAATCGTTTTGGCTGTG AAGTTAGTAATGCGTGTTCT

24 127.2 4L10D ACAGAATCAGCAAATACTCG CATAACACCCTCATAAAACT24 172.4 4A7H AACTATCGCTTCAATCAAAC TAAACCATAAACCGCACAAT

25 0 4C10D Cy3 AATCCAGTCAGGTTTTCA TTCCGTTTCTTCTCGTGTA AF28726725 1.3 6D4G Fluorescein AGTTTTTATCGCTTCTCC GATGTTCCAGTCGTCGTT

26 24.9 16J12H AAAGAAATAGGCAGGGTAGT AAAGAAATAGGCAGGGTAGT26 135.5 7H8A Cy3 GCTAAGTGGTATGTGAATCCb TTTGCTTTTGCCAGGGTGTCb AB010825

P 31.3 6L2A Cy3 GACCAATGCCd/GGTCCCTGACd GTTGCCGATCCd/TTCCGAACCCd

P 107.1 2B9A Fluorescein TAAACCGAAACTCAAGATT CTATGGCACAAAAAGAAAA

U 20.3 4D2A Cy3 AATCCATACTCTCGCTCTCAe ATAGTTTTCCAATCCACCAAe

U 45.1 7L2G Cy3 ACAGTAGGATGAAATGGAAT TTATGTGTCTCGGAATGA

a Yasukochi (1999).b Yasukochi et al. (2004).c Yasukochi et al. (2005).d Yasukochi (1998).e Wu et al. (1999).f Shimada et al. (1994).


Figure 1.—Detection of theWZ bivalent (Z is linkage group1) using Z-BACs 9A5H, 14I7D,5H3E, and a female-derivedwhole-genomic probe for the Wchromosome (A). Red signalsare from Z-BACs and green sig-nals are from the whole-genomicprobe. The chromosomes arecounterstained with DAPI (lightblue). The arrow points to theheterochromatic block of an au-tosomal bivalent (see text). N,nucleolus. Bar, 10 �m. (B andC) Linkage map with RAPD loci(in black) from Yasukochi(1998) and the map positions ofBACs (in red) corresponding tothe signals on the Z chromosome.

well-spread pachytene nuclei could be discriminated BAC-FISH had previously been applied to fine map-ping in extended chromatin fibers (Weier 2001). The(Figure 3A). We arranged the bivalents of one cell (Fig-

ure 3B, cell 4 in Table 2) according to their average method commonly used to identify chromosomes byFISH is either chromosome painting (Cremer et al.length calculated from 12 karyotyped complements.

The probe cocktail did not include markers for the 1988; Lichter et al. 1988; Pinkel et al. 1988) or by usingcentromeric chromosome-specific repetitive sequencechromosomes corresponding to linkage groups 1(Z),

11, and 24. Those corresponding to linkage groups 11 probes (Hizume et al. 2002; Vischi et al. 2003), whichare not available in B. mori. Karyotyping of the wholeand 24 could be recognized from the DAPI pattern

alone. And, Z-specific probes proved unnecessary as the chromosome complement has been achieved in the hu-man and in the mouse with sophisticated color schemesprobe cocktail reliably produced extra label on the W

chromosome to which the Z chromosome was synapsed and probe mixes of five and seven different fluoro-in the pachytene stage. phores. M-FISH (Speicher et al. 1996; Jentsch et al.

Length measurements of 12 karyotyped complements 2001) and spectral karyotyping (Liyanage et al. 1996;are listed in Table 2. It is obvious that the various chro- Schrock et al. 1996) are two of these methods. Wemosomes occupy similar but not constant positions in had decided against chromosome painting. Probes forthe order of length. Systematic individual length changes chromosome painting are usually generated from sortedduring pachytene development are not apparent but chromosomes (Van Dilla et al. 1986; Collins et al.cannot be excluded. Considering the rather shallow 1991; Vooijs et al. 1993), from microdissected chromo-length gradient especially in the middle part of the B. somes (Guan et al. 1994), or from a densely spacedmori complement, however, variation in position may chromosome-specific BAC array (Lysak et al. 2001).be merely due to measurement error (the median of Sorting chromosomes is probably not possible in Bom-standard error is 0.101 with the range from 0.019 to byx due to the similarity of chromosome sizes (see Table0.340) and/or variation in individual chromosome 2). Microdissection of chromosomes is confronted withspreading. the same problem if one does not wish to rely on a

single microdissected anonymous chromosome. A col-lection of 28 sufficiently dense chromosome-specific

DISCUSSION BAC arrays would have been a feasible alternative forprobe generation. But this would have been a ratherThe high number and small size of the chromosomesexpensive alternative with respect to costs and experi-and the absence of suitable cytogenetic markers likemental work and it would have given less information.bands and localized centromeres have hitherto inhib-The obvious advantage of using only a few BACs andited chromosome identification in B. mori. We circum-two different fluorophores per chromosome is the gen-vented the problem by (1) hybridizing selected fluores-eration of anchor points that relate to the geneticalcence-labeled BACs to the chromosomes (BAC-FISH)map. Besides mere identification of the chromosome,and (2) using pachytene instead of mitotic chromo-they provide a framework for physical mapping of othersomes. Pachytene chromosomes have the advantage ofBACs and allow us to distinguish between the two chro-an extended length (4.3 times longer than mitotic chro-mosome ends.mosomes on average) and a reduced number (28 biva-

The two B. mori whole-genome shotgun sequencinglents instead of 56 single mitotic chromosomes). In thisprojects, Mita et al. (2004) and Xia et al. (2004), pres-way, we were able to identify all 28 chromosomes of the

B. mori complement individually. ently under way have already achieved 3� and 6� cover-



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mack et al., 1996 Multicolor spectral karyotyping of mouse chor-age, respectively, of the genome. The karyotype and theomosomes. Nat. Genet. 14: 312–315.anchor points provided in this article afford a basis to Lysak, M. A., P. F. Fransz, H. B. Ali and I. Schubert, 2001 Chromo-

map contigs directly or with little experimental effort some painting in Arabidopsis thaliana. Plant J. 28: 689–697.Marec, F., and W. Traut, 1993 Synaptonemal complexes in femaleto the respective chromosomes and linkage groups. Our

and male meiotic prophase of Ephestia kuehniella (Lepdoptera).results already disclose that the map of 28 linkage Heredity 71: 394–404.groups mainly based on RAPD markers (Yasukochi Mita, K., M. Kasahara, S. Sasaki, Y. Nagayasu, T. Yamada et al.,

2004 The genome sequence of silkworm, Bombyx mori. DNA Res.1998) truly reflects the chromosome status in B. mori.11: 27–35.Since the BACs have been proven to give chromo-

Pinkel, D., J. Landegent, C. Collins, J. Fuscoe, R. Segraves et al.,some-specific signals, identification of individual mitotic 1988 Fluorescence in situ hybridization with human chromo-

some specific libraries: detection of trisomy 21 and translocation(as opposed to meiotic pachytene) chromosomes byof chromosome 4. Proc. Natl. Acad. Sci. USA 85: 9138–9142.BAC-FISH will probably also be possible in B. mori. BAC-

Promboon, A., T. Shimada, H. Fujiwara and M. Kobayashi, 1995FISH karyotyping of mitotic chromosomes or mapping Linkage map of random amplified polymorphic DNAs (RAPDs)other probes relative to our anchor points, however, in the silkworm, Bombyx mori. Genet. Res. 66: 1–7.

Rasmussen, S. W., 1976 The meiotic prophase in Bombyx mori fe-will be difficult or impossible due to the small size ofmales analyzed by three-dimensional reconstructions of synapto-the mitotic chromosomes. On the other hand, the BAC- nemal complexes. Chromosoma 54: 245–293.

FISH karyotyping method is probably easily adaptable Sahara, K., F. Marec and W. Traut, 1999 TTAGG telomeric re-peats in chromosomes of some insects and other arthropods.to other species with similar problems of chromosomeChromosome Res. 7: 449–460.recognition. Sahara, K., F. Marec, U. Eickhoff and W. Traut, 2003a Moth sexchromatin probed by comparative genomic hybridization (CGH).We thank Walther Traut (Luebeck, Germany) for critical discus-Genome 46: 339–342.sions and Yasuhiro Yamada for technical assistance. This study was

Sahara, K., A. Yoshido, N. Kawamura, A. Ohnuma, H. Abe et al.,partially supported by Grant-in-Aid for Scientific Research no.2003b W-derived BAC probes as a new tool for identification15380227 from the Japan Society for the Promotion of Science (K.S.of the W chromosome and its aberrations in Bombyx mori. Chro-and Y.Y.) and by the Insect Technology Project from the Ministry ofmosoma 112: 48–55.

Agriculture, Forestry and Fisheries in Japan, no. 2102 (Y.Y.) and no. Schrock, E., S. Du Manoir, T. Veldman, B. Schoell, J. Wienberg2013 (K.S.). et al., 1996 Multicolor spectral karyotyping of human chromo-

somes. Science 273: 494–497.Shi, J., D. G. Heckel and M. R. Goldsmith, 1995 A genetic linkage

map for the domesticated silkworm, Bombyx mori, based on restric-LITERATURE CITED tion fragment length polymorphisms. Genet. Res. 66: 109–126.Shimada, T., T. Hasegawa, K. Matsumoto, N. Agui and M. Koba-Collins, C. C., W. L. Kuo, R. Segraves, J. C. Fuscoe, D. Pinkel et

yashi, 1994 Polymorphism and linkage analysis of the protho-al., 1991 Construction and characterization of plasmid librariesracicotropic hormone gene in the silkworm, Bombyx mori. Genet.enriched in sequences from single human chromosomes. Geno-Res. 63: 189–195.mics 11: 997–1006.

Speicher, M. R., S. G. Ballard and D. C. Ward, 1996 KaryotypingCremer, T., P. Lichter, J. Borden, D. C. Ward and L. Manuelidis,human chromosomes by combinatorial multi-fluor FISH. Nat.1988 Detection of chromosome aberrations in metaphase andGenet. 12: 368–375.interphase tumor cells by in situ hybridization using chromosome

specific library probes. Hum. Genet. 80: 235–246. Tan, Y., C. Wan, Y. Zhu, C. Lu, Z. Xiang et al., 2001 An amplifiedDoira, H., 1983 Linkage maps of Bombyx mori status quo in 1983. fragment length polymorphisms map of the silkworm. Genetics

Sericologia 23: 245–269. 157: 1277–1284.Fujii, H., Y. Banno, H. Doira, H. Kihara and Y. Kawaguchi, 1998 Traut, W., 1976 Pachytene mapping in the female silkworm, Bombyx

Genetical Stocks and Mutations of Bombyx mori: Important Genetic mori L. (Lepidoptera). Chromosoma 58: 275–284.Resources, Ed. 2. Institute of Genetic Resources, Faculty of Agricul- Traut, W., K. Sahara, T. D. Otto and F. Marec, 1999 Molecularture, Kyushu University, Fukuoka, Japan. differentiation of sex chromosome probed by comparative geno-

Glaser, R. W., 1917 ‘Ringer’ solutions and some notes on the physio- mic hybridization. Chromosoma 108: 173–180.logical basis of their ionic composition. Comp. Biochem. Physiol. Van Dilla, M. A., L. L. Deaven, K. L. Albroght, N. A. Allen,2: 241–289. M. R. Aubuchon et al., 1986 Human chromosome specific DNA

Guan, X. Y., P. S. Meltzer and J. M. Trent, 1994 Rapid generation libraries: construction and availability. Biotechnology 4: 537–552.of whole chromosome painting probes (WCPs) by chromosome Vischi, M., I. Jurman, G. Bianchi and M. Morgante, 2003 Karyo-microdissection. Genomics 22: 101–107. type of Norway spruce by multicolor FISH. Theor. Appl. Genet.

Hizume, M., F. Shibata, Y. Matsusaki and Z. Garajova, 2002 Chro- 107: 591–597.mosome identification and comparative karyotypic analyses of Vooijs, M., L. C. Yu, D. Tkachuk, D. Pinkel, D. Johnson et al., 1993four Pinus species. Theor. Appl. Genet. 105: 491–497. Libraries for each human chromosome, constructed from sorter-Jentsch, I., I. D. Adler, N. P. Carter and M. R. Speicher, 2001

enriched chromosomes by using linker-adaptor PCR. Am. J. Hum.Karyotyping mouse chromosomes by multiple-FISH (M-FISH).Genet. 52: 586–597.Chromosome Res. 9: 211–214.

Weier, H. U. G., 2001 DNA fiber mapping techniques for the assem-Kawaguchi, E., 1928 Zytologische Untersuchungen am Seidenspin-bly of high-resolution physical maps. J. Histochem. Cytochem.ner und seinen Verwandten. I. Gametogenesese von Bombyx mori49: 939–948.L. und Bombyx mandarina M. und ihres Bastarde. Z. Zellforsch.

Wu, C., S. Asakawa, N. Shimizu, S. Kawasaki and Y. Yasukochi,7: 519–552.1999 Construction and characterization of bacterial artificialKawamura, N., 1979 Cytological studies on the mosaic silkwormchromosome libraries from the silkworm, Bombyx mori. Mol. Gen.induced by low temperature treatment. Chromosoma 74: 179–Genet. 261: 698–706.188.

Xia, Q., Z. Zhou, C. Lu, D. Cheng, F. Dai et al., 2004 A draftLichter, P., T. Cremer, J. Borden, L. Manuelidis and D. C. Ward,sequence for the genome of the domesticated silkworm (Bombyx1988 Delineation of individual human chromosomes in meta-mori). Science 306: 1937–1940.phase and interphase cells by in situ suppression hybridization

Yamamoto, T., 2000 Silkworm strains, pp. 45–49 in Strain Mainte-using chromosome specific library probes. Hum. Genet. 80: 224–nance and Databank for Life Science, edited by N. Nakatsuji. Kyori-234.

Liyanage, M., A. Coleman, S. Du Manoir, T. Veldman, S. McCor- tsu Shuppan, Tokyo [in Japanese].


Yasukochi, Y., 1998 A dense genetic map of the silkworm, Bombyx Yasukochi, Y., L. A. Ashakumary, C. Wu, A. Yoshido, J. Nohata etmori, covering all chromosomes based on 1018 molecular mark- al., 2004 Organization of the Hox gene cluster of the silkworm,ers. Genetics 150: 1513–1525. Bombyx mori : a split of the Hox cluster in a non-Drosophila insect.

Yasukochi, Y., 1999 A simple and accurate method for generating Dev. Genes Evol. 214: 606–614.co-dominant markers: an application of conformation-sensitive Yasukochi, Y., Y. Banno, K. Yamamoto, M. R. Goldsmith and H.gel electrophoresis to linkage analysis in the silkworm. Mol. Gen. Fujii, 2005 Integration of molecular and classical linkageGenet. 261: 796–802. groups of the silkworm, Bombyx mori (n � 28). Genome (in press).

Yasukochi, Y., 2002 PCR-based screening for bacterial artificialchromosome libraries. Methods Mol. Biol. 192: 401–410. Communicating editor: J. Birchler

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The Bombyx mori Karyotype and the Assignment of Linkage ...2 22.3 1I12H Fluorescein CACAGGGCTTTTTGGTTCTA GCTTTTATGTTATTCACTCG 2 60.3 5J8G Cy3 AAAACGGCTAACTAACGAAG TGAGAAACAGGAGACTACT