Download The female-killing chromosome of the silkworm, Bombyx mori, was

Genetica (2006) 127:253–265
DOI 10.1007/s10709-005-4147-8
Springer 2006
The female-killing chromosome of the silkworm, Bombyx mori, was generated
by translocation between the Z and W chromosomes
T. Fujii1, N. Tanaka1, T. Yokoyama1, O. Ninaki1, T. Oshiki1, A. Ohnuma2, Y. Tazima3,
Y. Banno4, M. Ajimura5, K. Mita5, M. Seki6, F. Ohbayashi6, T. Shimada6 & H. Abe1,*
1
Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, 183-8509, Fuchu, Tokyo, Japan; 2Institute of Sericulture, Iikura 1053, 300-0324,
Ami-machi, Ibaraki, Japan; 3Nippon Silk Center, Gunma-cho 888-1, 370-3511, Gunma , Japan; 4Sinstitute of
Genetic resources, Kyushu University Graduate School of Bioresource and Bioenvironmental Science, Hakozaki 6-10-1, 812-8581, Higashi-ku, Fukuoka, Japan; 5National Institute of Agrobiological Science, Owashi
1-2, 305-8634, Tsukuba, Ibaraki, Japan; 6Department of Agricultural and Environmental Biology, Graduate
school of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, 113-8657, Bunkyo-ku, Tokyo,
Japan; *Author for correspondence: (Phone: +81-42-367-5848, Fax: +81-42-367-5848, E-mail:
[email protected])
Received 19 April 2005; Accepted 17 October 2005
Key words: Bombyx mori, deletion, female-killing, RAPD, sex chromosome, silkworm, translocation, W
chromosome, Z chromosome
Abstract
Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative
feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome,
Df(pSa+pW+od)Fem, derived from the translocation-carrying W chromosome (pSa+pW+od), is inert as
femaleness determinant. Moreover, this Df(pSa+pW+od)Fem chromosome has been thought to have a
female-killing factor because no female larvae having the Df(pSa+pW+od)Fem chromosome are produced.
Initially, to investigate whether the Df(pSa+pW+od)Fem chromosome contains any region of the W
chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The
Df(pSa+pW+od)Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly
indicate that the Df(pSa+pW+od)Fem chromosome contains the region of the W chromosome. Moreover,
by using phenotypic and molecular markers, we confirmed that the Df(pSa+pW+od)Fem chromosome is
connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the
Df(pSa+pW+od)Fem chromosome is viable. Therefore, we concluded that the Df(pSa+pW+od)Fem
chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the
death of the ZW-type diploid female having the Df(pSa+pW+od)Fem chromosome. Additionally, our
results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the
Df(pSa+pW+od)Fem chromosome and deleted Z chromosome was generated by translocation between the
Z chromosome and the translocation-carrying W chromosome, pSa+pW+od.
Introduction
In Bombyx mori, females are heterogametic (ZW),
and males are homogametic (ZZ) in sex chromosome constitution (Tanaka, 1916). The W chro-
mosome of Bombyx mori, is recombinationally
isolated from the Z chromosome because crossing
over is restricted to males in Bombyx mori
(Tanaka, 1913a, b; Sturtevant, 1915). Although
more than 200 visible mutations have been placed
254
on 28 linkage groups in Bombyx mori (Fujii et al.,
1998), no gene for a morphological character has
so far been found on a normal W chromosome.
The normal W chromosome of Bombyx mori does
not have any morphological characteristics
detectable under light-microscopic observation
(Traut, 1976). Therefore, the W chromosome is
difficult to analyze by conventional genetic and
cytologic methods.
To analyze the W chromosome at the molecular biological level, it is necessary to obtain the Wspecific nucleotide sequence. So far, 12 W-specific
RAPD markers have been identified in the silkworm strains maintained in Japan (Abe et al.,
1998, 2005). Abe et al. (2005) analyzed the presence or absence of the W-specific RAPD markers
on the aberrant W chromosomes, T(W;3)Ze and
T(W;10)+w-2. They revealed that these aberrant
W chromosomes are shorter than the normal W
chromosome. The deletion patterns of the W-specific RAPD markers on the aberrant W chromosomes showed the approximate position of several
W-specific RAPD markers on the W chromosome.
The function of the W chromosome of Bombyx
mori was first revealed by analyzing the sex ratios
and segregation of the marker gene located on the Z
chromosome in the filial triploid from the ZZWW
tetraploid female (Hasimoto, 1933). Hasimoto
presumed that W has a strong positive femaledetermining gene. Using a sex-limited strain which
have translocation-carrying W chromosome,
pSa+pW, Tazima (1944) confirmed that femaleness
in Bombyx mori is controlled by the presence of the
W chromosome irrespective of the number of Z
chromosomes. Through dissociation experiments
on the translocation-carrying W chromosome,
pSa+pW+od, Tazima (1954, 1964) presumed that
the putative feminizing gene (Fem) is located on a
restricted region of the W chromosome.
In the course of dissociation experiments on the
pSa+pW+od chromosome, Tazima isolated the
‘Z101 strain’; this strain has a paternally transmitted mutant W chromosome, Df(pSa+pW+od)Fem.
Based on genetic analysis, Tazima (1952) thought
that the Df(pSa+pW+od)Fem chromosome was
generated by the deletion of the Fem-containing
region of the pSa+pW+od chromosome (Figure 1)
and that a female having the Df(pSa+pW+od)Fem
chromosome would die during embryogenesis,
while a male having the Df(pSa+pW+od)Fem
chromosome would be viable. The Df(pSa+
X-ray
(a)
pSa +p
+od
Fem
psa+pW+od
(b)
pSa +p
+od
Fem
pSa +p
+od
(c)
Df(psa+pW+od)Fem
Figure 1. One possible model of the generation process of the
Df(pSa+pW+od)Fem chromosome based on Tazima (1952).
(a): The pSa+p W+od chromosome. (b): Generation of the
pSa+p fragment and the +od fragment, which lack the putative Fem gene, by X-ray irradiation. (c): Fusion of the pSa+p
fragment and the +od fragment.
p
W +od)Fem chromosome has been maintained
paternally since 1944. However, the difficulty entailed in conducting conventional genetic and
cytologic analyses of the W chromosome has retarded further analysis of the Df(pSa+pW+od)Fem
chromosome. Four questions are apparent about
the Df(pSa+pW+od)Fem chromosome, namely, (1)
why
female
embryos
having
the
Df(pSa+pW+od)Fem chromosome die during
embryogenesis; (2) how the Df(pSa+pW+od)Fem
chromosome was generated from the pSa+pW+od
chromosome; (3) whether the Df(pSa+pW+od)Fem
chromosome contains any region of the W chromosome derived from pSa+pW+od chromosome
besides the +p, pSa, and +od genes; and (4) whether
the Df(pSa+pW+od)Fem chromosome is attached
to other chromosomes.
In this report, we analyze the genetic features of
the Df(pSa+pW+od)Fem chromosome using phenotypic markers and molecular markers. We reveal that the Df(pSa+pW+od)Fem chromosome is
connected with the partially deleted Z chromosome and that this fused chromosome behaves as a
Z chromosome during male meiosis. Moreover, we
255
demonstrate that the Df(pSa+pW+od)Fem chromosome does not have a female-killing factor but
that partial deletion of the Z chromosome causes
the death of the ZW-type diploid female having
the Df(pSa+pW+od)Fem chromosome. Furthermore, we reveal the generation process of the
Df(pSa+pW+od)Fem chromosome. Additionally,
we discussed the chromosomal structure of the
pSa+pW+od chromosome, which is the ancestral
to the Df(pSa+pW+od)Fem chromosome.
Materials and methods
Genetic markers
For the phenotypic markers of the Z chromosome,
os (sex-linked translucent, 1-0.0), sch (sex-linked
chocolate, 1-21.5), and od (distinct translucent, 149.6) were used. For the phenotypic markers of
chromosome 2, the alleles of the p locus concerning body marking, p (plain, 2-0.0), +p (normal
marking, 2-0.0), and pSa (sable, 2-0.0) were used.
The dominance relation is pSa>+p>p. For the
phenotypic marker of the chromosome 3, Ze (zebra, 3-20.8) was used. For the molecular markers
of the W chromosome, we used 12 W-specific
RAPD markers (Abe et al., 1998, 2005). The sequences of the primers designed to amplify 12 Wspecific RAPD markers are described in Abe et al.
(1998, 2005). For the molecular markers of the Z
chromosome, N20.70b (Promboon et al., 1995),
Rcf96 (Shi, Heckel & Goldsmith, 1995), Bmkettin
(1-40.0) (Suzuki, Shimada & Kobayashi, 1999),
and T15.180a (Suzuki, Shimada & Kobayashi,
1998) were used. N20.70b, Rcf96, and T15.180a
are located around the os locus, the sch locus, and
the od locus, respectively (Shimada, unpublished
data). The sequences of the primers designed to
amplify the molecular markers on the Z chromosome are shown in Table 1.
Translocation-carrying W chromosomes
In this study, we used three translocation-carrying
W chromosomes, pSa+pW+od, +pW, and WZe,
which have a putative Fem gene.
Tazima (1944, 1964) discovered a strain in
which an attached chromosome composed of
two chromosomes 2, one having pSa and the
other having +p, was translocated onto one end
of the W chromosome. Hereafter, the translocated W chromosome is designated as pSa+pW
and an attached chromosome translocated onto
the W chromosome is designated as IIpSaII+p.
In this report, one side of the W chromosome
marked by the attached chromosome, IIpSaII+p,
is designated as ‘left’ following Tazima (1954).
By applying X-ray irradiation to the pSa+pW
chromosome, Tazima (1944) obtained a derivative of the pSa+pW chromosome that had lost
the pSa gene. Hereafter, this translocation-carrying
W chromosome is designated as +pW. Moreover,
Tazima (1948, 1964) connected the right end of the
pSa+pW chromosome with a short distal fragment
of the Z chromosome having the +od gene using a
high-temperature treatment. Hereafter, this translocated W chromosome is designated as pSa+
p
W+od (Figure 1(a)).
Using X-ray irradiation, Hasimoto (1948)
connected the W chromosome with a fragment of
chromosome 3 having Ze gene. Hereafter, this
translocated W chromosome is designated as WZe.
Table 1. Primer sequence for amplification of molecular markers on the Z chromosome
Molecular marker
N20.80b
Rcf96
Primer pair
Sequences
Aka- 2A
5¢-AAATACTCCTGCCCATATAGTGTCA-3¢
Aka- 2B
5¢-CTATTCACGAACGAAATGTCTTCGA-3¢
Rcf96- F1
5¢-AGTAATGATCATTGAAGCGG-3¢
Rcf96- R1
5¢-GGTCACCAGACGACAGAAAG-3¢
Bmkettin
ZD43
5¢-TCTAGCTTCCTCAGCTTGTTC-3¢
T15.180a
ZD44
T15- 1A
5¢-TGGGTGAAGCTGTATCAACTG-3¢
5¢-GATGCCACTACCCTAGCTTTGAC-3¢
T15- 1BREV
5¢-GACCGCTGCCGAGTCTCTCCAG-3¢
See Materials and methods for the location of the four molecular markers.
Product size (bp)
527
846
523
1012
256
It was elucidated that WZe lacks 2 of 12 W-specific
RAPD markers (Abe et al., 2005).
Inbred strains, marker strains, and sex-limited
strains
p50 and C108 strains are highly inbred strains and
that have been maintained by sister–brother mating as described by Promboon et al. (1995). POS
(p/p, os/W), PSCH (p/p, sch/W), POD (p/p, od/W),
and PSCHOD (p/p, sch od/W) are marker strains
having a normal W chromosome. The C137 strain
is a highly inbred sex-limited strain having the
+pW chromosome. Plain male (Z/Z, p/p) and
normal marking female (Z/+pW, p/p) are segregated in the C137 strain. The ZWII strain is a
sex-limited strain having the pSa+pW+od chromosome. Translucent skin plain male (od/od, p/p)
and normal skin sable female (od/pSa+pW+od,
p/p) are segregated in the ZWII strain. For convenience, body marking of the female having the
pSa+pW+od chromosome is designated as sable in
this report because pSa is epistatic to +p.
Z101 strain
Among the F1 descending from the X-ray irradiated female of the sex-limited strain having the
pSa+pW+od chromosome, Tazima (1948) found
an exceptional male having the +p, pSa, and +od
genes. This male was the founder of the Z101
strain having a paternally transmitted mutant W
chromosome designated as pSa+p( )+od (Tazima,
1952). For convenience, we designated the pSa+
p
( )+od chromosome as Df(pSa+pW+od)Fem in
this paper. The Df(pSa+pW+od)Fem chromosome
contains the +p, pSa, and +od genes. It has been
assumed that the Df(pSa+pW+od)Fem chromosome was generated by the deletion of Fem-containing region of the pSa+pW+od chromosome, as
shown in Figure 1 (Tazima, 1952). However, the
genetic behavior of the Df(pSa+pW+od)Fem
chromosome has not been elucidated yet. While, in
the Z101 strain, male larvae having the
Df(pSa+pW+od)Fem chromosome are observed,
female larvae having the Df(pSa+pW+od)Fem are
never observed (Tazima, 1952). Therefore, we have
maintained the Z101 strain by sister–brother
mating between translucent skin sex-limited zebra
plain female (od/WZe, p/p) and normal skin sable
male (od/od, p/p, pSa+p+od) (Figure 2). For con-
venience, the body marking of the male having the
Df(pSa+pW+od)Fem is designated as sable in this
report because pSa is epistatic to +p. In this cross,
translucent skin sex-limited zebra plain females
(od/WZe, p/p), translucent skin plain males (od/od,
p/p), normal skin sable males (od/od, p/p, pSa+
p
+od), and embryonic lethal eggs are segregated in
an almost equal ratio (Figure 2). It has been presumed that embryonic lethal eggs are females
having the Df(pSa+pW+od)Fem chromosome
(Tazima, 1952).
Hot water treatment
The ZZW triploid female silkworms were obtained
from eggs treated with hot water as described in
Yokoyama et al. (1990). The eggs were treated
with hot water (46C, 18 min) immediately after
oviposition (010 min). This hot-water treatment
causes the fusion of an ameiotic female pronucleus, 2n(ZW), with a male pronucleus, n(Z). More
than 90% of the individuals produced by this
method were ZZW triploid female (Yokoyama
et al., 1990).
DNA extraction from eggs and larvae
Genomic DNA was extracted from six embryonic
lethal eggs of the Z101 strain. Initially, to remove
any surface contamination of eggs, the eggs on the
egg-laying paper were carefully rinsed with water.
Then, each egg was separated from the egg-laying
paper carefully to prevent smashing them and
transferred into a 1.5 ml microtube. By adding
25 l‘ of a TE buffer (1 mM Tris–HCl, pH 8.0,
0.1 mM EDTA), eggs were then smashed and
homogenized by thrusting and twisting the pestle.
Then, genomic DNA was recovered from the 1 l‘
of the homogenate and diagnosed the PCR
markers using the GeneReleaser Kit (Bioventures,
Inc.) according to manufacture’s instructions.
Genomic DNA was also extracted from larval
posterior silk glands as described previously (Abe
et al., 1998). We used six individuals, including
both sexes for each strain.
PCR
PCR using 10-mer primers was carried out as
previously described (Abe et al., 1998). Briefly, 45
257
Figure 2. Fifth-instars larvae of the Z101 strain. (a): Translucent skin sex-limited zebra plain female (od/WZe, p/p). (b): Normal
skin sable male (od/od, p/p, pSa+p+od). (c): Translucent skin plain male (od/od, p/p ).
cycles of PCR were performed on a Zymoreactor
II Thermal cycler (ATTO Co.) as follows: 94C for
1 min, 37C for 1 min, and 72C for 3 min
followed by a final extension of 10 min at 72C.
PCR products were analyzed by electrophoresis on
2% agarose gels and stained with ethidium bromide. PCR using primers for W-Specific markers
(except Samu-ORF-1A and Samu-ORF-2A) was
performed as previously described (Abe et al.,
2005). Briefly, 40 cycles of PCR were performed as
follows: 94C for 1 min, 55C for 2 min, 72C for
3 min, and final extension at 72C for 10 min. For
the primer pair, Samu-ORF-1A and Samu-ORF2A, LA PCR using LA Taq polymerase (TaKaRa)
was performed on a Thermal Cycler TP2000 (TaKaRa) for 14 cycles of 98C for 20 s and 68C for
20 min following the initial denaturation at 94C
for 1 min; this was followed by 16 cycles of 98C
for 20 s, and 68C for 20 min+15 s/cycle, and a
final extension at 72C for 10 min. PCR using
primers for molecular markers of the Z chromosome was performed as follows: 40 cycle of 94C
for 1 min, 50C for 2 min and 72C for 3 min and
a final extension at 72C for 10 min.
GenBank accession number
The nucleotide sequence of the Maji RAPD
marker was deposited in the DDBJ, EMBL and
GenBank nucleotide databases under accession
number AB206653.
Results
Presence or absence of the W-specific RAPD
markers on the pSa+pW+od chromosome
and the Df(pSa+pW+od)Fem chromosome
We investigated the presence or absence of Wspecific RAPD markers on the pSa+pW+od and
Df(pSa+pW+od)Fem chromosomes. All 12 Wspecific RAPD markers were amplified from the
female of the ZWII strain having the pSa+pW+od
258
chromosome, while no W-specific RAPD markers
were amplified from the male of that strain (data
not shown). In the Z101 strain, the W-Mikan, WSamurai, and W-Bonsai RAPD markers were
amplified from the male (od/od, p/p, pSa+p+od)
having the Df(pSa+pW+od)Fem chromosome,
while no W-specific RAPD markers were amplified
from the male (od/od, p/p) without the Df(pSa+
p
W+od)Fem chromosome (data not shown).
These results strongly indicate that the Df(pSa+
p
W+od)Fem chromosome possesses three W-specific RAPD markers (W-Mikan, W-Samurai, and
W-Bonsai) derived from the pSa+pW+od chromosome.
Maji-2A (5’-ACAACATAACACGCACAACCA
GACA-3’), based on the sequencing result. We
compared the amplification pattern of the Maji
RAPD marker using a primer set, Maji-1B and
Maji-2A, on the genomic DNA of both sexes of the
C137, p50, and ZWII strains. The Maji RAPD
marker was amplified from the female of the
C137(+pW) and ZWII(pSa+pW+od) strains but
not from the female of the p50(normal W chromosome) strain or from males of these three strains
(Figure 3). These results strongly indicate that the
Maji RAPD marker is amplified from the fragment
of chromosome 2 translocated onto the W chromosome.
Identification of an RAPD maker on the fragment
of chromosome 2, IIpSaII+p, translocated
onto the W chromosome
Presence or absence of Maji
on the Df(pSa+pW+od)Fem chromosome
Previously, we screened a total of 3648 arbitrary
10-mer primers on the p50 and C137 strains, and
nine W-specific RAPD markers were identified
(Abe et al., 2005). In this study, we conducted a
more thorough screening and found one RAPD
marker, which is specific only to the female of C137
strain having the +pW chromosome. We named
this RAPD marker ‘Maji.’ The Maji RAPD marker is 1226 bp long. To convert the Maji RAPD
marker into a SCAR marker (1181 bp), we designed a new longer PCR primer pair, Maji-1B (5’ATACTTCGTCATTGTGGCTAGTTCT-3’) and
We investigated the presence or absence of the Maji
RAPD marker on the Df(pSa+pW+od)Fem chromosome in the Z101 strain. Maji was not amplified
from the female (od/WZe, p/p) and the male (od/od,
p/p) not having the Df(pSa+pW+od)Fem chromosome. On the other hand, the Maji RAPD marker
was amplified from the male (od/od, p/p, pSa+
p
+od) having the Df(pSa+pW+od)Fem chromosome (Figure 3). These results demonstrate that the
Df(pSa+pW+od)Fem chromosome possesses the
Maji RAPD marker. Thus, the Maji RAPD
marker is useful for the detection of the
Df(pSa+pW+od)Fem chromosome.
Figure 3. Amplification patterns of genomic DNA from females and males of the C137, p50, ZWII, and Z101 strains by using one
set of primers, Maji-1B + Maji-2A. Lane M, molecular-size markers (100 bp ladder; Invitrogen). The number at the left indicate
base pairs. The arrowhead indicates the Maji RAPD markers.
259
Diagnosing sex and genotype of the eggs
in the Z101 strain
It has been thought that females having the
Df(pSa+pW+od)Fem chromosome die during
embryogenesis in the Z101 strain (Tazima, 1952).
However, this assumption has not been confirmed
because it was impossible to analyze whether dead
eggs have the Df(pSa+pW+od)Fem chromosome
using conventional genetic method. Therefore, we
identified the sex and genotype of the embryonic
lethal eggs in the Z101 strain. We analyzed the
presence or absence of two molecular markers (WBMC1-Kabuki and Maji) in six embryonic lethal
eggs. The Maji RAPD marker was used as
molecular markers of the Df(pSa+pW+od)Fem
chromosome and the W-BMC1-Kabuki RAPD
marker was used as molecular marker of the Wze
chromosome. As a result, all six embryonic lethal
eggs were determined to be females having the
Df(pSa+pW+od)Fem chromosome because they
had two molecular markers (W-BMC1-Kabuki
and Maji) (data not shown). These results strongly
indicate
that
females
having
the
Df(pSa+pW+od)Fem chromosome die during
embryogenesis in the Z101 strain.
Presence or absence of molecular markers on the Z
chromosome in the embryonic lethal female
having the Df(pSa+pW+od)Fem chromosome
In a preliminary experiment, 28 chromosomes
were observed and extra chromosome were not
recognized in germ cells of males having the
Df(pSa+pW+od)Fem chromosome (data not
shown). Therefore, we hypothesized that the
Df(pSa+pW+od)Fem chromosome is connected
with a partially deleted Z chromosome. Based on
this hypothesis, it can be explained that the ZWtype diploid female having a deleted Z chromosome connected with the Df(pSa+pW+od)Fem
chromosome dies due to the deletion of the Z
chromosome, while the ZZ-type diploid male
having a deleted Z chromosome connected with
the Df(pSa+pW+od)Fem chromosome is viable
because it has a normal Z chromosome inherited
from mother which can compensate for the partial
deletion of the Z chromosome transmitted from
father.
To confirm the validity of this hypothesis, we
analyzed the presence or absence of the four
molecular markers on the Z chromosome (Table 1) in one of the six embryonic lethal eggs which
were determined to be female having the
Df(pSa+pW+od)Fem chromosome in the experiments mentioned above. As a result, N20.70b
marker was not amplified from the embryonic lethal egg, while three molecular markers (Rcf96,
Bmkettin, 15.180a) were amplified from it (data
not shown). These results suggest that the
Df(pSa+pW+od)Fem chromosome is connected to
the deleted Z chromosome, which lacks the left
end of the Z chromosome, where N20.70b
marker is located and that female having the
Df(pSa+pW+od)Fem chromosome die during
embryogenesis due to the deletion of the Z chromosome.
Connection of the Df(pSa+pW+od)Fem
chromosome with the deleted Z chromosome
We analyzed the Z chromosome of the males
having the Df(pSa+pW+od)Fem chromosome
using phenotypic markers, os (1-0.0), which is located left end of the Z chromosome. POS females
(p/p, os/W) were crossed to Z101 males having the
Df(pSa+pW+od)Fem chromosome (Figure 4(a)).
As a result, p+os female, p+os male and pSa+pos
male appeared almost 1:1:1 ratio (data not shown).
This
segregation
indicates
that
the
Df(pSa+pW+od)Fem chromosome is connected
with the deleted Z chromosome, which lacks the
+os locus, because plain (p) individuals were the
+os phenotype, while sable (pSa+p) individuals
were the os phenotype. Moreover, the absence of
the p os phenotype or the pSa+p+os phenotype,
which would result from the recombination between a normal Z chromosome and a deleted Z
chromosome, suggests that the Df(pSa+pW+od)
Fem chromosome is connected with the breakpoint
of the deletion (Figure 4(a)).
Locus of the breakpoint of the deletion
In preliminary experiments, we confirmed that the
deleted Z chromosome has the sch locus (1-21.5)
and the od locus (1-49.6) and there is recombination between a deleted Z chromosome and a normal Z chromosome (data not shown). Therefore,
we performed a crossover experiment to localize
the breakpoint of the deletion. We crossed
PSCHOD females (p/p, sch od/W) with males
260
Figure 4. Schematic illustration of the mating scheme. (a): POS Z101. (b): PSCHOD (PSCH Z101). (c): POD Z101. Arrows indicate the breakpoint of the deletion. See Materials and methods for the maker strains, POS, PSCH, and PSCHOD.
obtained by the cross between a PSCH female (p/p,
sch/W) and a Z101 male having the Df(pSa+
p
W+od)Fem chromosome (Figure 4(b); Table 2).
A1 (p, sch, od female), A2 (p, sch, od male), and A3
(pSa+p+od, +sch, +od male) resulted from the
recombination between the sch locus and the od
locus. However, the recombinant A3 (pSa+p+od,
+sch, +od male) was indistinguishable from the
non-recombinant male (pSa+p+od, +sch, od).
Therefore, the score of the A3 was estimated using
A1 (p, sch, od female) and A2 (p, sch, od male). We
estimated the score of the A3 to be {(A1)+(A2)}/
2. B1 (p, +sch, +od female), B2 (p, +sch, +od
male), and B3 (pSa+p+od, sch, od male) would
result from the double recombination. However,
B3 (pSa+p+od, sch, od male) could not be distinguished from the recombinant male (pSa+p+od,
sch, +od) that would result from the recombination
between the sch locus and the breakpoint of the
deletion. On the other hand, we could not detect
B1 (p, +sch, +od female) or B2 (p, +sch, +od male)
in these crosses. Therefore, we presumed that there
was no double recombination in these crosses between the breakpoint of the deletion, the sch locus,
and the od locus. The recombination value was
estimated to be 8.2% between the breakpoint of
deletion and sch and 33.6% between sch and od.
Because the distance between sch and od has been
determined to be 28.1 cM (Fujii et al., 1998), we
calculated the locus of the breakpoint of the
deletion to be 21.5-(8.2(28.1/33.6))=14.6 cM.
However, it is possible that the deletion and the
connection of the Df(pSa+pW+od)Fem chromosome to the deleted Z chromosome might affect
the recombination ratio between the normal Z
chromosome and the deleted Z chromosome.
Table 2. Crossover experiment between od, sch and the breakpoint of the deletion
Mating scheme
PSCHOD No. of crosses
3
(PSCHZ101)
See Figure 4(b) for mating scheme.
Sex
p+od
pod
psa +p +
od
sch
+sch
sch
+sch
$
112
34
195
0
0
0
#
113
27
198
0
21
313
sch
+sch
261
Therefore, further study is required to precisely
localize the breakpoint of the deletion.
Viability of the ZZW-type triploid female having
the Df(pSa+pW+od)Fem chromosome
We investigated whether the ZZW-type triploid
female having the Df(pSa+pW+od)Fem chromosome dies during embryogenesis or not. In order to
induce the ZZW-type triploid females, eggs from
the POD females (od/W, p/p) mated with the Z101
males were subjected to hot-water treatment
immediately after oviposition. A schematic illustration of the mating scheme is shown in Figure 4(c). As shown in Table 3, the sex ratio (female
: male) of larvae from untreated eggs is almost 1:2,
and no females having the Df(pSa+pW+od)Fem
chromosome were observed. On the other hand,
almost all of the larvae from treated eggs were female, including normal skin sable individuals having the Df(pSa+pW+od)Fem chromosome. Both
translucent skin plain female moths and normal
skin sable female moths from treated eggs showed
the typical features of the ZZW triploid female,
namely, they deposited a mixture of abnormal- and
normal-shapes eggs which died before hatching
(figure not shown). These results demonstrate that
the ZZW-type triploid female having the
Df(pSa+pW+od)Fem chromosome is viable.
Discussion
Structure of the Df(pSa+pW+od)Fem chromosome
and the pSa+pW+od chromosome
We clarified that the Df(pSa+pW+od)Fem chromosome has a part of the W chromosome which
contained 3 of 12 W-specific RAPD markers
(W-Mikan, W-Samurai, and W-Bonsai). In the
Z101 strain, neither the viability nor the sex phenotype of the male was affected by the presence of
the Df(pSa+pW+od)Fem chromosome in the Z101
strain. It seems likely that the segment of the W
chromosome contained in the Df(pSa+pW
+od)Fem chromosome (1) does not have a deleterious effect on male viability and (2) does not have
the putative Fem gene. As discussed earlier (Tazima, 1964; Abe et al., 2005), the W chromosome
may be devoid of functional genes, except the
putative Fem gene, which is located at a limited
portion of the W chromosome.
We verified that the Df(pSa+pW+od)Fem
chromosome is connected with the deleted Z
chromosome using molecular markers and phenotypic markers of the Z chromosome. Now, we
will try to determine how this complex chromosomal structure is constructed.
The pSa+pW chromosome was generated by
translocation of a fragment of chromosome 2,
II+pIIpSa, to the left end of the W chromosome
(Tazima, 1944, 1964). Subsequently, the
pSa+pW+od chromosome was produced by connecting the short fragment of the Z chromosome
having the +od gene with the pSa+pW chromosome
(Tazima, 1948, 1964). Through a dissociation
experiment on the pSa+pW+od chromosome, Tazima (1948, 1964) reached the conclusion that the
+od gene was translocated to the right end of the
pSa+pW chromosome as a result of exceptional
crossover between the Z chromosome and the
pSa+pW chromosome. However, he did not exclude
the possibility that the +od gene could be translocated to the end of the fragment of chromosome 2,
II+pIIpSa . Hereafter, we denote the former as the
right-end model and the latter as the left-end
model for the structure of the pSa+pW+od
chromosome.
Table 3. Segregation of larval characters in larvae hatched from the eggs treated with hot water
Mating scheme
Sex
Treated
pSa+p
p
+od
od
+od
Od
$
#
0
0
71
1
82
1
0
0
$
0
72
0
0
#
0
75
82
0
POD Z101
Control
See Figure 4(c) for mating scheme.
262
Figure 5. Schematic illustration of the Df(+odpSa+pW)Fem chromosome connected with the deleted Z chromosome and newly
designed structural model of the +odpSa+pW chromosome. (a): The pSa+pW chromosome. (b): The +odpSa+pW chromosome.
(c): DfZ–DfW chromosome composed of the Df(+odpSa+pW)Fem chromosome and the deleted Z chromosome. (d): A normal Z
chromosome.
Based on the right-end model, unusual chromosomal rearrangement is required in order to
explain the generation of the Df(pSa+pW+od)Fem
chromosome connected with the deleted Z chromosome as follows: (1) generation of the +od
fragment and the pSa+p fragment dissociated from
the pSa+pW+od chromosome; (2) generation of
the deleted Z chromosome; and (3) fusion of the
+od fragment, the pSa+p fragment, and the deleted Z chromosome. On the other hand, based on
the left-end model, the generation of the
Df(pSa+pW+od)Fem chromosome connected with
the deleted Z chromosome can easily be explained
by the translocation between the pSa+pW+od
chromosome and the Z chromosome. In fact, the
Z101 strain was derived from a female X-rayed
during the pupal stage, when the Z and W chromosomes were still in the same nucleus of the
oocyte and translocation between Z chromosome
and W chromosome was possible. Moreover, the
left-end model gains further support from the
following. First, though Tazima (1948, 1964)
thought that the pSa+pW+od chromosome was
generated through the exceptional crossover between the Z chromosome and the pSa+pW chromosome, the deficiency of a part of W
chromosome was not detected in the pSa+pW+od
chromosome; namely, the pSa+pW+od chromosome has 12 W-specific RAPD markers (data not
shown). Second, Sahara et al. (2003) could not
recognize the translocated chromosome fragment
at one end of the pSa+pW+od chromosome, but a
relatively long translocated chromosome fragment
could be recognized at the opposite end by means
of GISH or BAC-FISH. Based on these results, we
support the left-end model over the right-end
model. Hereafter, we designate the pSa+pW+od
chromosome and the Df(pSa+pW+od)Fem chromosome
as
+odpSa+pW
and
od Sa
p
Df(+ p + W)Fem, respectively.
Abe et al. (2005) concluded that the W-specific
RAPD markers are arranged in the order of WMikan and W-Samurai from one end of the W
chromosome. Therefore, we think that (1) Wspecific RAPD markers are arranged in the order
of W-Mikan, W-Samurai, and W-Bonsai from the
left end of the W chromosome, where the fragment
of chromosome 2, IIpSaII+p, is translocated
(Figure 5(a)); (2) the putative Fem gene is located
between the W-Bonsai RAPD marker and the
right end of the W chromosome (Figure 7 (a)); (3)
the +od fragment is not translocated to the W
region of the pSa+pW chromosome but it translocated to the end of the fragment of chromosome
263
2, IIpSaII+p (Figure 5(b)); (4) the left part of the Z
chromosome was replaced by the left part of
+odpSa+pW chromosome by the translocation
between the Z chromosome and the +odpSa+pW
chromosome, namely, the Df(+odpSa+pW)Fem
chromosome corresponds to the left part of the
+odpSa+pW chromosome (Figure 5(c)).
We think that the female-killing chromosome
in the Z101 strain is composed of five chromosome
fragments, as shown in Figure 5(c). It is very difficult to describe this complicated chromosomal
structure using the formal nomenclature. Therefore, we would like to propose the designation of
the female-killing chromosome as DfZ–DfW. The
structural model of the DfZ–DfW chromosome
shown in Figure 5(c) explains the Df(+odpSa
+pW)Fem chromosome in detail (see Introduction). However, we have not observed the
DfZ–DfW chromosome cytogenetically. Therefore, FISH analysis (Sahara et al., 2003; Yoshido
et al., 2005) is necessary to confirm the structure of
the DfZ–DfW chromosome.
Stable transmission and non-recombination
of the Df(+odpSa+pW)Fem chromosome
In the silkworm, it is presumed that sporadic loss
of the unstable chromosomal fragment carrying
the phenotypic marker gene during larval development causes the mosaic phenotype (Fujiwara
et al., 1991). On the other hand, we have never
observed a mosaic phenotype concerned with the
+p, pSa, and +od genes, which are located on the
Df(+odpSa+pW)Fem chromosome, during maintenance of the Z101 strain. We think that the
Df(+odpSa+pW)Fem chromosome is stably
transmitted from cell to cell and from father to son
because it is connected with partially deleted Z
chromosome (Figure 5(c)).
In the silkworm, crossing over is restricted to
males. Hence, the paternally transmitted
Df(+odpSa+pW)Fem chromosome, gains the
opportunity to undergo a crossover. The
Df(+odpSa+pW)Fem chromosome is composed of
four parts, that is, chromosome 2 having the +p
gene, chromosome 2 having the pSa gene, small
section of the Z chromosome having the +od gene,
and a segment of W chromosome containing the WMikan, W-Samurai, and W-Bonsai RAPD markers
(Figure 5(c)). Therefore, the Df(+odpSa+ pW)Fem
chromosome may undergo a crossover with a
normal chromosome 2 or Z chromosome. In the
mating experiments, we observed the recombination between the normal Z chromosome and
the deleted Z chromosome connected to the
Df(+odpSa+pW)Fem chromosome (Table 2).
However, in the course of maintenance of the Z101
strain, we never observed the recombination between (1) the pSa or +p genes on the
Df(+odpSa+pW)Fem chromosome and the p gene
on the normal chromosome 2; or (2) the +od gene on
the Df(+odpSa+pW)Fem chromosome and the od
gene on the normal Z chromosome (data not
shown). Moreover, Maji RAPD markers or 3 Wspecific markers have been identified to be located
on the Df(+odpSa+pW)Fem chromosome for more
than 60 years (Figure 3). These results suggest that
the Df(+odpSa+pW)Fem chromosome is recombinationally isolated from the homologous part of the
normal chromosome 2 or Z chromosome in the
Z101 strain.
Partial deletion of the Z chromosome connected
with the Df(+odpSa+pW)Fem chromosome
Tazima (1944) induced several kinds of deleted Z
chromosomes using X-rays. He confirmed that
female having these deleted Z chromosomes were
inviable. Therefore, he concluded that even a small
portion of the Z chromosome is necessary to
maintain the normal physiological function of the
female.
In the Z101 strain, we revealed that (1) a female
having the Df(+odpSa+pW)Fem chromosome is
bound to die during embryogenesis; (2) the
Df(+odpSa+pW)Fem chromosome is connected to
the deleted Z chromosome (Figure 5(c)); (3) the
ZZW-type triploid female, which has one normal
Z chromosome and one deleted Z chromosome
connected to the Df(+odpSa+pW)Fem chromosome, can survive (Table 3). Therefore, we concluded that the ZW-type diploid female having the
Df(+odpSa+pW)Fem chromosome dies during
embryogenesis due to the partial deletion of the Z
chromosome (Figure 5(c)). However, in a female
having this deleted Z chromosome, the exact point
at which development stops during the embryonic
stages has never been precisely identified. Further
study is required to elucidate the effect of this
deletion of the Z chromosome on embryogenesis.
If non-disjunction occurs between a normal Z
chromosome and a deleted Z chromosome
264
connected with the Df(pSa+pW+od)Fem chromosome during male meiosis in the Z101 strain, a
ZZW+
2A-type
female
having
the
Df(+odpSa+pW)Fem chromosome will result.
However, we have never obtained a female having
the Df(+odpSa+pW)Fem chromosome in the
course of maintenance of the Z101 strain. This fact
suggests that the deleted Z chromosome connected
with the Df(+odpSa+pW)Fem behaves as a Z
chromosome properly during male meiosis in the
Z101 strain.
In the silkworm, it is suggested that sex-linked
genes are not dosage-compensated (Suzuki, Shimada & Kobayashi, 1998, 1999). Koike et al.
(2003) revealed that most of 13 Z-chromosomelinked genes are expressed in a male-biased manner. These results suggest that the products of
genes on the Z chromosome are required at higher
levels in males than in females. In fact, Tanaka
(1939) showed that ZO diploid males exhibited
very low viability. However, in the Z101 strain, the
viability of a male having the deleted Z chromosome with the Df(+odpSa+pW)Fem chromosome
is normal. We speculate that one copy of the genes
on the segment of the Z chromosome deleted in the
Z101 strain may be sufficient for the normal viability of the male, though some genes on the other
part of the Z chromosome may require two copies.
Acknowledgements
This work was supported by PROBRAIN (to
K.M. and T.S.), the NIAS/MAFF Insect Technology Program (to K.M. and T.S.), and Grantsin-Aid for Scientific Research, JSPS/MEXT
(Nos. 16208006, 16011209 and 16011263). We
thank the Genetic Resource team, Institute of
Sericulture for providing Z101 strain.
References
Abe, H., M. Kanehara, T. Terada, F. Ohbayashi, T. Shimada,
S. Kawai, M. Suzuki, T. Sugasaki & T. Oshiki, 1998.
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: 243–254.
Abe, H., M. Seki, F. Ohbayashi, N. Tanaka, J. Yamashita, T.
Fujii, T. Yokoyama, M. Takahashi, Y. Banno, K. Sahara,
A. Yoshido, J. Ihara, Y. Yasukochi, K. Mita, M. Ajimura,
M.G. Suzuki, T. Oshiki & T. Shimada, 2005. Partial deletions of the W chromosome due to reciprocal translocation
in the silkworm Bombyx mori. Insect Mol. Biol. 14(4): 339–
352.
Fujii, H., Y. Banno, H. Doira, H. Kihara, Y. Kawaguchi, 1998.
Genetical stocks and mutations of Bombyx mori. Important
genetic resources, 2nd edition. Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka (in Japanese).
Fujiwara, H., O. Ninaki, M. Kobayashi, J. Kusuda & H.
Maekawa, 1991. Chromosomal fragment responsible for
genetic mosaicism in larval body marking of the silkworm,
Bombyx mori. Genet. Res. 57: 11–16.
Hasimoto, H., 1933. The role of the W-chromosome in the sex
determination of Bombyx mori . Jpn. J. Genet. 8: 245–
247(in Japanese).
Hasimoto, H., 1948. Sex-limited zebra, an X-ray mutation in
the silkworm. J. Seric. Sci. Jpn. 16: 62–64(in Japanese with
English summary).
Koike, Y., K. Mita, M.G. Suzuki, S. Maeda, H. Abe, K.
Osoegawa, P.J. deJong & T. Shimada, 2003. Genomic
sequence of a 320-kb segment of the Z chromosome of
Bombyx mori containing a kettin ortholog. Mol. Gen.
Genomics 269: 137–149.
Promboon, A., T. Shimada, H. Fujiwara & M. Kobayashi,
1995. Linkage map of random amplified polymorphic
DNAs (RAPDs) in the silkworm, Bombyx mori. Genet.
Res. Camb. 66: 1–7.
Sahara, K., A. Yoshido, N. Kawamura, A. Ohnuma, H. Abe,
K. Mita, T. Oshiki, T. Shimada, S-I. Asano, H. Bando & Y.
Yasukochi, 2003. W-derived BAC probes as a new tool for
identification of the W chromosome and its aberrations in
Bombyx mori. Chromosoma 112: 48–55.
Shi, J., D.G. Heckel & M.R. Goldsmith, 1995. A genetic
linkage map for the domesticated silkworm, Bombyx mori,
based on restriction fragment length polymorphisms.
Genet. Res. 66: 109–126.
Sturtevant, A.H., 1915. No crossing over in the female of the
silkworm moth. Amer. Nat. 49: 42–44.
Suzuki, M.G., T. Shimada & M. Kobayashi, 1998. Absence of
dosage compensation at the transcription level of a sexlinked gene in a female heterogametic insect, Bombyx mori.
Heredity 81: 275–283.
Suzuki, M.G., T. Shimada & M. Kobayashi, 1999. Bm kettin,
homologue of the Dorosophila kettin gene, is located on the
Z chromosome in Bombyx mori and is not dosage
compensated. Heredity 82: 170–179.
Tanaka, Y., 1913a. A study of mendelian factors in the
silkworm, Bombyx mori. J. Coll. Agri. Sapporo 5: 61–113.
Tanaka, Y., 1913b. Gametic coupling and repulsion in
silkworms. J. Coll. Agric. Sapporo 5: 115–148.
Tanaka, Y., 1916. Genetic studies in the silkworm. J. Coll.
Agric. Sapporo 6: 1–33.
Tanaka, Y., 1939. Non-disjunction of the sex chromosome in
the silkworm. Jap. J. Genet. 15: 359–361(in Japanese).
Tazima, Y., 1944. Studies on chromosome aberrations in the
silkworm. II. Translocation involving second and
265
W chromosomes. Bull. Seric. Exp. Stn. 12: 109–181(in
Japanese).
Tazima Y., 1948. Translocation of the Z chromosome to the W
chromosome of the silkworm, Bombyx mori. In Oguma
Commemoration Volume on Cytology and Genetics. 88–
91(in Japanese).
Tazima, Y., 1954. Mechanisms of the sex determination in the
silkworm, Bombyx mori. Proc. 9th Internat. Cong. Genet.
1953. Caryologia 6(suppl): 958–960.
Tazima, Y., 1964. The genetics of the silkworm. Academic
press, London.
Tazima Y., 1952. Inheritance of sex Silkworm Genetics, ed. Y.
Tanaka. 351–372 Shokabo, Tokyo (in Japanese).
Traut, W., 1976. Pachytene mapping in the female silkworm,
Bombyx mori L. (Lepidoptera). Chromosoma 58: 272–284.
Yokoyama, T., E. Sugai, T. Oshiki & Q. Pan, 1990. Induction
of the triploid silkworm, Bombyx mori, by the hot-water
treatment to the inseminated eggs immediately after oviposition. J. Seric. Sci. Jap. 59: 281–224 (in Japanese).
Yoshido, A., H. Bando, Y. Yasukochi & K. Sahara, 2005. The
Bombyx mori Karyotype and the Assignment of Linkage
Groups. Genetics 170(2): 675–685.