Hereditas 138: (2003)

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1 Hereditas 138: (2003) Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, Female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited p B silkworm (Bombyx mori ) strain TAKESHI YOKOYAMA 1, HIROAKI ABE 1, YASUO IROBE 1, KATSUJI SAITO 1, NOBUHIKO TANAKA 1, SHINYA KAWAI 1, FUMI OHBAYASHI 2, TORU SHIMADA 2 and TOSHIKAZU OSHIKI 1 1 Department of Biological Production, Faculty of Agriculture, Tokyo Uni ersity of Agriculture and Technology, Fuchu, Tokyo, Japan 2 Graduate School of Agriculture and Life sciences, The Uni ersity of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan Yokoyama, T., Abe, H., Irobe, Y., Saito, K., Tanaka, N., Kawai, S., Ohbayashi, F., Shimada, T. and Oshiki, T Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, Female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited p B silkworm (Bombyx mori ) strain. Hereditas 138: Lund, Sweden. ISSN Received March 11, Accepted April 28, 2003 The sex chromosomes of the silkworm, Bombyx mori, are designated ZW for the female and ZZ for the male. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD) marker, designated Female- 218, from the translocation-bearing W chromosomes. These W chromosomes contain a region of the second chromosome, which carries visible larval markers of the p loci. We used strain TWPB in which female larvae have black skin due to the p B gene (T(W;2)p B, + p /+ p ) while male larvae have whitish skin (+ p /+ p ). To determine whether the Female-218 RAPD marker is derived from the W region or a second chromosome fragment, we induced a detachment of the translocated W chromosome, T(W;2)p B, by treating the eggs with hot water at an early developmental stage. After hot water treatment, we obtained 27 white female larvae out of 4850 female larvae. The Female-218 RAPD marker was not amplified in 26 out of 27 white female larvae, and was amplified from one white female larva. Moreover, we obtained 11 black male larvae out of 5377 male larvae. Eight out of 11 black male larvae became adult moths, and the Female-218 RAPD marker was amplified from all eight male moths. Examination of the genetic relationship between the Female-218 RAPD marker and the second chromosome fragment of the translocated W chromosome strongly indicates that the Female-218 RAPD marker is amplified from the region of second chromosome fragment of the T(W;2)p B chromosome. Hiroaki Abe, Department of Biological Production, Faculty of Agriculture, Tokyo Uni ersity of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu, JP Tokyo, Japan. wfem@cc.tuat.ac.jp The sex chromosomes of the silkworm, Bombyx mori, are designated ZW (equivalent to XY) for the female and ZZ (XX) for the male (TANAKA 1916). Although 400 or more visible mutations have been placed on linkage maps in B. mori (GOLDSMITH 1995), no gene for morphological characters has been found on the normal W chromosome so far. However, several W chromosomes containing autosomal fragments were artificially constructed by X-ray irradiation (TAZIMA 1941; HASIMOTO 1948; TAZIMA et al. 1951; KIMURA et al. 1971). In strains with these W chromosomes, the dominant genes for egg color, blood color, and larval marking on the autosomal fragments are expressed in a sex-limited fashion. Therefore, all marked larvae are female, while all non-marked larvae are male. We previously identified four randomly amplified polymorphic DNA (RAPD) markers that are femalespecific. One of the four, designated Female-218 RAPD marker, is specific to females with translocation-bearing W chromosomes of B. mori (ABE et al. 1995, 1996; OHBAYASHI et al. 1996); the other three are designated W-Kabuki, W-Samurai, and W- Kamikaze RAPD markers, and are specific to females with normal W chromosomes containing no translocations (ABE et al. 1998). The Female-218 RAPD marker is 815 bp in length and is specific to translocation-carrying W chromosomes in females (ABE et al. 1995, 1996; OHBAYASHI et al. 1996). Although the Female-218 RAPD marker was among the first identified nucleotide sequences of the W chromosomes, it is not known whether the Female-218 RAPD marker is derived from the W region or the second chromosome fragment since these W chromosomes are artificially constructed by X-ray irradiation and contain regions of a second chromosome, which

2 Hereditas 138 (2003) carries visible larval markers of the p loci. Moreover, although mapping this Female-218 RAPD marker on the W chromosome is desirable, it is impossible to estimate the genetic distance between the four RAPD marker loci based on the recombination frequency, since crossing over is restricted to males in B. mori. These translocated W chromosomes are very stable under general rearing conditions. However, detachment of the attached second chromosome is often induced by external agents, such as X-rays and high temperature shocks (TAZIMA 1944). Therefore, in this study, we induced a detachment of the translocated W chromosome by hot water treatments and examined the genetic relation between the Female-218 RAPD marker and the second chromosome fragment of the translocated W chromosome. MATERIAL AND METHODS Silkworm strains We used Bombyx mori strains J137 (+ p /+ p ) and TWPB (Dp(2;W)p B, + p /+ p ). + p indicates the normal larval marking (whitish skin) gene, and p B is the black larval marking (black skin) gene, which is the dominant allele. Strain J137 has a normal W chromosome, while strain TWPB has the modified T(W;2)p B chromosome. Recently, TANAKA et al. (2000) found that one more chromosome, the fifth chromosome (linkage group), is attached to this T(W;2)p B chromosome. Therefore, the translocated W chromosome of the TWPB strain is made up of the W chromosome, the second chromosome fragment (p B fragment), and the fifth chromosome (linkage group). Strain TWPB, which is congenic to strain J137 with respect to the W chromosome, was constructed by crossing a female of the sex-limited p B strain from SASAKI (1955) with a male of the J137 strain more than 15 generations (OHBAYASHI et al. 1996). Hot water treatment After emergence, female moths of strain TWPB were crossed with J137 male moths for 3 hours. Since the female moths that are cooled lay their eggs promptly after being returned to room temperature, we refrigerated these female moths at 5 C for two days. After refrigeration, these female moths were allowed to oviposit at 25 C in a group and eggs were collected at 30 minutes interval until 270 minutes. These eggs were treated with hot water at 40 C for 10, 60, 90, 150 and 180 minutes. Control eggs were not treated with hot water. Then, these eggs were treated with HCl solution and incubated at 25 C to make them hatch without diapause. The larvae hatched from Detachment of the translocated W chromosome 149 treated eggs were reared by the usual method and segregation of larval characters and sexes was examined at the first day of the 5th instar. DNA and Female-218 RAPD marker Genomic DNA of larva and moth were extracted from posterior silk glands and legs, respectively, by the method of BENDER et al. (1983). DNA was further purified by phenol/chloroform extraction, ethanol precipitated, and resuspended in 0.1 TE buffer (1 mm Tris-HCl, ph 8.0, 0.1 mm EDTA). The Female-218 RAPD was amplified by PCR as described previously (ABE et al. 1996) using the W-1 and W-2 primers: W-1: 5 -CAGCCCAGAACT- CAAAATCG-3 and W-2: 5 -CTCAGCCCAGAG- ACGTGAGGTCT-3. RESULTS To examine the genetic relation between the Female- 218 RAPD marker and the second chromosome fragment of the translocated W chromosome, we carried out two dissociation experiments using hot water treatment. In the eggs treated at 30 min and 90 min after oviposition, the percentage of normally pigmented eggs decreased considerably regardless of the duration of the hot water incubation (data not shown). In particular, no larvae hatched from eggs treated for 180 min with hot water at 90 min after oviposition. However, in the eggs treated at times other than 30 and 90 minutes after oviposition, there was not a great difference in the percentage of normally pigmented eggs (from 50 to 80%), or hatchability of normally pigmented eggs (from 50 to 60%) (data not shown). Considering the very low frequency of detachment of the translocated W chromosome, it was necessary to raise as many individuals as possible. Therefore, we bred the hatched larvae as one group to reduce labor. In Exp. 1, we detected 6 white female larvae out of 2695 female larvae (Table 1). Two of six white larvae died during the 5th instar. Immediately after death, these two larvae were dissected and genomic DNA was extracted from the posterior silk glands of each individual; the Female-218 RAPD markers were not amplified from neither of these white females. The remaining four white larvae grew into adult moths, were crossed to J137 males, and produced eggs. After oviposition, genomic DNA extracted from the legs of each of these four female moths was used as templates for PCR; the Female-218 RAPD marker was not amplified from three of four female moths, and the Female-218 RAPD marker was amplified from only one female moth. In the female progenies of these four female moths, the body color of larvae was

3 150 T. Yokoyama et al. Hereditas 138 (2003) Table 1. Segregation of body color and sex at the fifth instar in lar ae hatched from the eggs treated with hot water. + p (White) p B (Black) + p (White) p B (Black) Mosaic Total Exp Cont Exp Cont white and the absence or presence of Female-218 RAPD marker was identical to their mothers. In Exp. 2, we detected 21 white female larvae out of 2155 female larvae (Table 1). All 21 white female larvae were dissected and genomic DNA was extracted from the posterior silk glands of the individuals. The Female-218 RAPD marker was not amplified from any of the 21 white female larvae. We also detected 11 black male larvae out of 2408 male larvae. The eleven black male larvae reached the pupal stage, but three of them died during pupal stage, and we could not extract DNA from these three pupae. However, eight male pupae became adult moths, and genomic DNA was individually extracted from the legs of these eight male moths, and was used as templates for PCR. The Female-218 RAPD marker was amplified from all eight male moths. Then, we crossed seven of these eight male moths to J137 females and obtained eggs. Among the progenies of these crosses, the ratio of black female: white female: black male: white male was 1:1:1:1 in each batch of eggs. Thus, in almost all of the larvae indicating the detachment of the second chromosome fragment of the translocated W chromosome, except only one female, the presence of the Female-218 RAPD marker was in accord with the expression of black larval skin color gene, p B. Additionally, we obtained many mosaic larvae characterized by black and white skin through treatment of hot water. Various degrees of mosaicism ranging from black and white mottled skin to bilateral mosaicism were recognized (data not shown). In the non-treated control, we detected only one mosaic larva and no white female or black male larvae out of 1250 larvae. DISCUSSION The egg nucleus just after deposition is in metaphase of the first maturation division, which is soon terminated by elimination of the first polar body. The second maturation division takes place about 60 min after deposition, and maturation of the egg nucleus is completed after the polar body is expelled (SAK- AGUCHI 1978). During these early developmental stages, we induced detachment of translocated W chromosome by hot water treatment. Since the translocated W chromosome is composed of three chromosomes, it seems likely that the breakage of the chromosome by hot water treatment would not occur equally over the entire chromosome. It is thought that the attachment sites between chromosomes are fragile. In fact, TAZIMA (1944) showed that the partial detachment or separation of the second chromosome fragment from the T(W;2)p chromosome is often induced by external agents such as X-rays and high temperature treatments. Therefore in this study, three cases of detachment in this translocated W chromosome could be expected. If a detachment occurs between the second chromosome fragment and the fifth chromosome, black females and white males would appear among the larvae hatched from eggs containing this type of pronucleus (Fig. 1b). These larvae could not be discriminated from normal black females and white males. If a detachment occurs between the W chromosome and the second chromosome fragment, black males and white females appear in the larvae hatched from the eggs containing these pronuclei (Fig. 1c). Therefore, these detachments are confirmed by dual characters of body color and sex. As mentioned above, the amplification of the Female- 218 RAPD markers were in accord with the expression of black larval skin color gene p B in all except one individual. These results strongly indicate that the Female-218 RAPD marker is amplified from a region in the second chromosome fragment on the T(W;2)p B. However, we did obtain one female individual with white skin and the Female-218 RAPD marker. One possible explanation for this discrepancy is that the Female-218 RAPD marker locus is located closer to the W chromosome than to the p B locus, and the translocated second chromosome fragment was cut off between the Female-218 RAPD marker and p B locus (Fig. 2). However, we could not exclude the

4 Hereditas 138 (2003) Detachment of the translocated W chromosome 151 Fig. 1. Schematic illustration of detachment patterns of the translocated W chromosome and presumed chromosome construction from the metaphase of first maturation division to early developmental stage and the expected larval skin colors. (a) Detachment does not occur. (b) Detachment occurs between the second chromosome fragment and the fifth chromosome. (c) Detachment occurs between the W chromosome and second chromosome fragment. (d) Two detachments occur: between the W chromosome and the second chromosome fragment and between the second chromosome fragment and the fifth chromosome. W: W chromosome, Z: Z chromosome, V: fifth chromosome, (II): second chromosome fragment, p B : black larval marking gene.

5 152 T. Yokoyama et al. Hereditas 138 (2003) Fig. 2. Presumed loci of the Female-218 RAPD marker and p B gene, and cutting pattern by hot water treatment of the white skin female larvae having Female-218 RAPD marker. possibility that the DNA of the p B locus itself was damaged. In addition to the T(W;2)p B chromosome, we maintain two other translocated W chromosomes, T(W;2)+ p and T(W;2)p Sa (p Sa codes for sable brownish skin). The origin of all three chromosomes is the T(W;2)p Sa chromosome (TAZIMA 1944). The T(W;2)p B -fifth chromosome used in this study can be observed under the microscope as an unusually large chromosome (TANAKA et al. 2000). On the other hand, no unusually large chromosomes are observed when strains with the T(W;2)+ p or T(W;2)p Sa chromosomes are observed (N. Tanaka, unpubl. data). The Female-218 RAPD marker is common to all three translocated W chromosomes. Therefore, it is thought that the Female-218 RAPD marker is not within the region of the attached fifth chromosome to T(W;2)p B. We hypothesize that the mechanism producing mosaic individuals in this study involves elimination of the second chromosome fragment detached from the translocated W chromosome in somatogenesis. It is assumed that two detachments occur in one cell: one between the second chromosome fragment and the fifth chromosome and one between the second chromosome fragment and the W chromosome (Fig. 1d). When the second chromosome fragment carrying p B gene is deleted in certain cells of epidermis in early developmental stages, these cells become white patches in the black skin. The frequencies of detachment and elimination of the second chromosome fragment is probably different in each individual. Therefore, various mosaic patterns will appear. In Diptera, the relationship between the transposable elements and evolution (degeneration) of the non-recombining Y chromosome has been extensively studied (CHARLESWORTH et al. 1994; CHARLESWORTH 1996, 2001). In Drosophila miranda, the Y chromosome degeneration is driven by the accumulation of transposable elements (STEINEMANN and STEINEMANN 1992, 1997; STEINEMANN et al. 1993). In B. mori, the second chromosome fragment translocated to W chromosome lost the opportunity of chromosomal recombination. Additionally, many retrotransposable elements have accumulated on the W chromosome in Bombyx (ABE et al. 2000, 2002). Therefore, this second chromosome fragment is probably degenerating due to the insertion of retrotransposable elements at present. The Female-218 RAPD marker will not be useful for further molecular analyses of this region of the W chromosome. However, the Female-218 RAPD marker will be useful for molecular analyses of the degenerating second chromosome fragment as a starting point for DNA sequencing. ACKNOWLEDGEMENTS This work was supported by the Research for the Future Program, the Japan Society for the Promotion of Science (to T. S.). REFERENCES Abe, H., Shimada, T., Yokoyama, T. et al Identification of a random amplified polymorphic DNA on the W chromosome of the Chinese 137 strain of the silkworm, Bombyx mori. J. Seric Sci. Jpn. 64: (In Japanese with English summary). Abe, H., Shimada, T., Kawai, S. et al Nucleotide sequence of the random amplified polymorphic DNA (RAPD) on the W chromosome of the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl. Entomol. Zool. 31: Abe, H., Kanehara, M., Terada, T. et al Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences. Genes Genet. Syst. 73: Abe, H., Ohbayashi, F., Shimada, T. et al Molecular structure of a novel gypsy-ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on the W chromosome of the silkworm Bombyx mori. Mol. Gen. Genet. 263: Abe, H., Sugasaki, T., Terada, T. et al Nested retrotransposons on the W chromosome of the wild silkworm Bombyx mandarina. Insect Mol. Biol. 11: Bender, W., Spierer, P. and Hogness, D. S Chromosome walking and jumping to isolate DNA from the Ace and rosy and bithorax complex in Drosophila melanogaster. J. Mol. Biol. 168: Charlesworth, B The evolution of chromosome sex determination and dosage compensation. Curr. Biol. 6: Charlesworth, B Genome analysis: more Drosophila Y chromosome genes. Curr. Biol. 11: R182 R184. Charlesworth, B., Sniegowski, P. and Stephan, W The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:

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