Chromosome Research (2007) 15:315Y325
DOI: 10.1007/s10577-007-1128-z# Springer 2007

DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh.
(Orthoptera, Acrididae)
Alexander G. Bugrov1,2*, Tatiana V. Karamysheva3, Eugeny A. Perepelov1,3, Eugeny A. Elisaphenko3,
Denis N. Rubtsov1, Elżbieta Warchałowska-Śliwa4, Haruki Tatsuta5 & Nikolay B. Rubtsov1,31
Novosibirsk State University, 630090 Novosibirsk, Russia; Tel: +7(383)3397564; Fax: +7(383)3397564;
E-mail: bugrov@fen.nsu.ru; 2Institute for Systematics and Ecology of Animals, Siberian Branch of Russian
Academy of Sciences, 630091 Novosibirsk, Russia; 3Institute of Cytology and Genetics, Siberian Branch of
Russian Academy of Sciences, 630090 Novosibirsk, Russia; 4Department of Experimental Zoology, Institute of
Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland;5
National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan* Correspondence
Received 10 January 2007. Received in revised form and accepted for publication by Herbert Macgregor 5 February 2007

Key words: B-chromosomes, DNA sequences, FISH analysis

Abstract
A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome
microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty ofthem were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the
DDBJ/GenBank/EMBL database (http://www.ddbj.nig.ac.jp/) was performed. Different patterns of signals wereobserved after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted
either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A
chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes.
Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived
from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telometricregions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large
regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B
chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely
related Podisma species.

Introduction
Extra chromosomes (Bs) to the standard complement (A) have been reported in many species.
Extensive studies have been performed on Bs,
including analysis of B chromosome distribution
across species, the structure and origin of Bs,
effects of Bs on the standard complement, transmission of Bs, population dynamics and evolution,and integration of Bs into the A genome (Camacho

et al. 2000); but little is known about the DNAcontent of Bs. The B chromosomes in grasshoppers
are relatively well studied. High morphologicaldiversity in size and location of C-negative andC-positive regions in Bs, and regions enriched with
rDNA and satellite repeats, have been described for
Eyprepocnemis plorans (Camacho et al. 1997a,b,Henriques-Gil et al. 1984, Henriques-Gil & Arana
1990, Bakkali et al. 1999, Cabrero et al. 1999).
Despite similar DNA content, the origin of at leastA. G. Bugrov et al.

316
two types of Bs in this species appeared to be
independent (Cabrero et al. 2003). A partial sequenceof the B chromosome in E. plorans has been obtained(data presented by Professor J. P. Camacho at theSecond B Chromosome Conference, Bubion, Granada,
Spain, 26Y29 June, 2004).
An extensive study of Bs was performed inanother group of grasshopper species belonging to
the genus Podisma. In P. sapporensis Shir., cytological variation of B chromosomes has been found insome populations in Hokkaido, Sakhalin and Kuril
islands. The Bs of this species have been divided into
seven morphotypes according to their size, morphology and C-banding patterns (Warchałowska-Śliwa
et al. 2001). A DNA library derived from the B of
P. sapporensis Shir. (morphotype B1) was generated
by chromosome microdissection followed with DOPPCR and used in a study on B chromosome
organization and distribution of homologous DNA
in A and B chromosomes of Podisma species(Bugrov et al. 2003, 2004). The comparative FISH
study on the Bs of other morphotypes revealed
regions containing DNA showing no homology to
the B1 chromosome. In some Bs only small regions
have shown signals after FISH with a microdissected
DNA probe derived from the B1 chromosome(Bugrov et al. 2004). An analysis of repetitive DNA
distribution in A and B chromosomes suggested that
B chromosome evolution in P. sapporensis, at leastin some cases, was mainly associated with DNA
insertions into an original A chromosome followedby amplification of inserted DNA and deletion ofsome ancestral chromosome regions (Bugrov et al.
2004).
The diversity of molecular organization of the Bs
in P. sapporensis raised new questions on themechanisms of B chromosome origin and organization in closely related species belonging to the genus
Podisma. A comparative analysis of morphology andmolecular organization of B chromosomes in theP. sapporensis group distributed in Sakhalin, Kunashiri, Hokkaido and Honshu Islands would be ofgreat help to understand the further evolution of B
chromosomes and mechanisms that may be involvedin these processes. The present study is a detailed
analysis of the B chromosome in P. kanoi and acomparison of B chromosome organization in two
closely related species, P. kanoi and P. sapporensis.
An analysis of repetitive DNA distribution in A
chromosomes of these species was also performed.

Material and methods
SamplesDuring JulyYAugust 2004 and 2005 a total of 39
females and 77 males of P. kanoi Storozhenko, 1993
were collected from three sites on Honshu Island (MtShirane, Manza, and Mt Naeba). Females wereplaced in separate cages according to locality. Aftera few days the females laid egg pods in moist, coarse
sand. All males and 53 embryos were used for
cytogenetic analysis. The specimens of P. sapporensis sapporensis captured from populations in
Hokkaido Island have been described earlier (Bugrov
et al. 2003).
Chromosome analysis
Chromosomes for routine cytogenetic analysis and
FISH were prepared from embryos and testes of adultmales as described by Bugrov et al. (2000, 2004).
Males were injected with 0.1% colchicine for 1.5Y2.0h.
The testes were fixed in alcohol:glacial acetic acid(3:1) for 15min, and then kept in 70% ethanol. Airdried preparations were prepared by squashing thetesticles in 45% acetic acid and freezing in dry ice.
Each portion of the eggs was stored in a separate Petri
dish with moist sand and kept at room temperature.
After 15Y20 days of incubation the eggs were placedin a solution of 0.05% colchicine in insect saline andthe tops of the nonmicropylar end were removed.
They were incubated at 30 C0 for 1.5Y2h. The
embryos were dissected out of the eggs and placed
into a 0.9% sodium citrate solution at room temperature. After 20Y30 min they were fixed in 3:1
methanol:acetic acid. Air-dried preparations wereprepared on slides by macerating the embryos in adrop of 60% acetic acid.
Meiotic and mitotic chromosomes were stainedwith Giemsa, DAPI and a standard C-banding
technique (Sumner 1972).
DNA probes
Meiotic chromosome microdissection followed with
DOP-PCR was used for preparation of DNA probes
from the whole P. kanoi B chromosome (Bk) andfrom the central region of the B chromosome arm(Bm). Briefly, dissected regions of eight copies of Bs
were transferred into 40nl of buffer solution in a

DNA content of the B chromosomes in Podisma kanoi

317

Figure 1. C-banded and NOR karyotypes of Podisma kanoi. (a) Mitotic metaphase in embryo; (b) karyogram of mitotic metaphase; (c)metaphase II, indicates B chromosome; (d) AgNOR stained metaphase I, arrows indicate NORs. Scale bars = 10mm.

siliconized micropipette tip, treated with proteinase
K, and then amplified using DOP-PCR with theMW6 primer (Rubtsov et al. 2000, 2002).
The DNA libraries were labeled with biotin-16dUTP or digoxigenin-11-dUTP in 15 PCR cycles(Rubtsov et al. 2000). The DNA probes were used for
FISH analysis of meiotic and mitotic chromosomes of
P. sapporensis and P. kanoi. FISH was performed
according to a standard protocol (Rubtsov et al.
2002) with salmon sperm DNA as the DNA carrier.
Biotin- and digoxigenin-labeled DNA probes werevisualized with avidin-FITC and mouse antidigoxigenin antibodies conjugated to Cy3, respectively.
Microdissected DNA probes generated from the B
chromosome of P. sapporensis (B1) and the euchromatic part of the smallest A chromosome (EUR1)(Bugrov et al. 2004) were also used for FISH
analysis.
Biotin-, digoxigenin-, and dinitrophenyl-labeled,cloned DNA fragments derived from microdissected

DNA libraries were obtained by PCR with M13
direct and reverse primers in 25 cycles.
The rDNA-probe was a nick-translation labeled
fragment of human 18S rDNA cloned in pHr13(Malygin et al. 1992).
The chromosomes were counterstained with DAPI
and analyzed using an AXIOSKOP 2 Plus microscope (Zeiss) equipped with a CCD camera, filter
sets and the ISIS3 image-processing package ofMetasystems GmbH in the Microscopic Centre of
the Institute of Cytology and Genetics SB RAS,
Novosibirsk, Russia.
Sequencing of B chromosome DNA
DNA fragments of the B chromosome for dideoxy
sequencing were produced with two techniques:
1. DNA fragments from the microdissected P. kanoi
B chromosome DNA library were ligated to the318
XhoI site of the plasmid pBluescript II KS+ andthen used for transformation of competent Escherichia coli XLI-Blue cells. Colonies of recombinants were selected by their color.
2. Alternatively the pCR4Blunt-TOPO vector with
the TOPO Shotgun Subcloning Kits (Invitrogen)were used according to the manufacturer’s recommendation. Recombinant colonies were selected
on ampicillin plates.
The cloned DNA fragments were sequenced with
FM13 direct_ and reverse primers in both cases.
These primers were used to sequence cloned fragments on an Applied Biosystems 371A Automated
DNA Sequencer utilizing BigDye terminators (ABI
PRISM).
The BLAST and FASTA packages were used forcomputer analysis of obtained sequences. A homology search was done among the sequences of the
DDBJ (http://www.ddbj.nig.ac.jp/).

Results
C- and Ag-NOR-banding patterns of the A and B
chromosomes
The karyotype of P. kanoi showed a standard
chromosome complement typical for the vast majority of grasshoppers (White 1973, Hewitt 1979),
consisting of 22 acrocentric autosomes plus a single
acrocentric X chromosome in males (2n S=23, X0)
and two Xs in females (2n 9=24, XX) (Figure 1a,b).
Pericentromeric C-blocks were found in all chromosomes of the set. These blocks are large, andapproximately uniform in size. All A chromosomes
possessed extremely short arms. The X and two pairsof the medium-sized autosomes have C-positive
blocks in their telomeric regions (Figure 1a,b).
Active NORs were detected with Ag-NOR-banding.
A larger NOR was localized in the telomeric regionof the X, a smaller one was found in the telomericregion of a medium-sized autosome (Figure 1c).
Five out of 51 samples from the Mt Shirane
population were characterized by the presence of B
chromosomes. Three samples with Bs were foundamong 17 samples from Mt Naeba. Bs were notfound in the Manza population. All B chromosomes
showed a similar morphology, i.e. being acrocentrics
of medium size (Figure 1d) with C-positive staining.

A. G. Bugrov et al.
No C-negative region was revealed in P. kanoi Bs, incontrast to the Bs in P. sapporensis (Bugrov et al.
2003, 2004).
Chromosome polymorphism in the population of
P. s. sapporensis and P. s. krylonensis has been describedearlier (Bugrov et al. 2001, 2004, Warchałowska-Śliwa
et al. 2001) in detail.
FISH with microdissected DNA probes on A and B
chromosomes in P. kanoi and in P. sapporensis
FISH with the B1 probe derived from a P. sapporensis
B1 chromosome on the Bs in P. kanoi gave signals
only in pericentromeric and telomeric regions; bothof them were very weak. Some of the A chromosomes showed signals in pericentromeric regions(Figure 2a). The FISH signal was also detected in
telomeric regions of the X chromosome and in one pairof autosomes. No C-positive region of A and B
chromosomes in either species was painted with the
EUR1-probe derived from the euchromatic part of thesmallest A chromosome of P. sapporensis (Figure 2a),while all C-negative regions of A chromosomes
showed a strong signal in the studied species. Taken
together, this information encouraged us to prepare
two microdissected DNA probes from the P. kanoi B
chromosome. The first probe (Bk DNA probe) was
derived from the whole B chromosome found in themale from the Shirane population. The second probe(Bm DNA probe) was prepared from the middle
section of the same B. FISH with the Bk DNA probegave dot-like signals in short arms and probably inpericentric regions of all A chromosomes (Figure2b). It should be noted that the short arms ofchromosomes in P. kanoi are very small. They arealmost indiscernible in meiotic chromosomes. Basedon the results of FISH experiments with meiotic
chromosomes, it is impossible to discriminate thepericentric repeats and subtelomeric repeats of theshort arms because in both cases the FISH signal
covered the pericentric regions and short arms ofchromosomes. The bright painting of the B chromosomes masked the signal on A chromosomes. In
order to analyze the FISH signal on A chromosomes,
FISH was performed on samples without B chromosomes. These FISH experiments revealed that thesignal was more intense on three pairs of autosomes.
Furthermore, on these chromosomes FISH with the
Bk DNA probe produced signals on larger regions,
including the short arms and pericentric regions while

DNA content of the B chromosomes in Podisma kanoi

319

Figure 2. FISH of B1 (red signal) and EUR1 (green signal) probes on P. kanoi chromosomes. (a) Metaphase II; FISH of Bk- (green signal)probe in P. kanoi chromosomes; (b) spermatogonial metaphase plate with B chromosome; (c) metaphase II without B. X and B indicated.
Scale bars = 10 mm.

on other chromosomes only a slight, distant, dot-likesignal was registered (Figure 2c). FISH with the B1probe (DNA probe derived from the P. sapporensis
B1) gave intensive and widespread signals on pericentric regions of P. kanoi A chromosomes. In
pericentric regions, the FISH signal of the B1 probe
was close to the FISH signal of the Bk and Bm DNA
probes. In telomeric regions of long arms of the X andone middle-sized autosome the FISH signals of the B1probe were co-localized with the FISH signals of therDNA probe.
FISH with microdissected DNA probes derived
from the B chromosome of P. kanoi on chromosomesof P. sapporensis gave a background-like signal
along all chromosomes but no region-specific signal
was revealed.
FISH with the rDNA-probe on A and B chromosomesin P. sapporensis and P. kanoi
FISH with rDNA on chromosomes of P. kanoi gave
intense signals in telomeric regions of long arms of
the X and one middle-sized autosome (Figure 3a).
No signal was detected on P. kanoi B chromosomes
in any sample except for one collected from the Mt
Naeba population (Figure 3b). The intensity of the

Figure 3. FISH of rDNA probe on P. kanoi meiotic chromosomes in metaphase I; X and B indicated. (a) Plate with a B chromosome without
a NOR; (b) FISH of Bk (red signal) and rDNA (green signal) probes on P. kanoi meiotic chromosomes; (c) FISH of rDNA probe (green
signal) in P. sapporensis meiotic chromosomes. Scale bars = 10mm.

WB

#29

#85
#86
#87

14

15
16
17

55,13
66,89
57.88

PR
PR
ND

ER

ND
ND

PR

PR

PR
PR

PR
ND

PR

PR
PR
PR
ND

Signal on As

(6)

NSH
Clone showed 98% homology to H. sapiens
BAC clone RP11-804M4 (acc AC073284)
Clone showed 75% homology to Locusta migratoria
solitarious EST clone (ACC CO843638)
The tandem repeats with 38bp consensus, NSH
NSH
NSH

Clone showed 76% homology to Locusta migratoria
solitarious EST clone (ACC CO856777)
Ditto

NSH
NSH

Part of clone consist of tandem repeat; size of repeat
unit is 17 bp; clone showed 69% homology tosubtelomeric repeats of Bombyx mori
Ditto
Ditto

NSH
NSH
NSH
NSH

Comments(7)#93 Y 100%#118 Y 100%

#33 Y 91%
#10 Y 91%

#6Y 93%, #97% Y 76%#6 Y 79%, #43 Y 76%#43 Y 93%, #97 Y 76%#12 Y 77%, #19 Y 83%, #37 Y 80%#1 Y 77%, #19 Y 82%, #37 Y 77%#1 Y 83%, #12 Y 82%, #37 Y 82%#1 Y 80%, #12 Y 77%, #19 Y 82%

Homology to other clones(8)

WB, the whole B chromosomes; PR, pericentric regions; PR+DR, pericentric region and distal region of long arm of B chromosomes; ER, euchromatic regions of A chromosomes; ND,
no region with detectable specific signal; NSH, no significant homology with known DNA sequences.

234
296
406

328
WB
WB
WB

ND
ND

Clones showed no homology to each other
12
#23
137
63.50
13
#26
272
61,98
61.59

PR

66,80

11

244

PR

Group of clones showed homology to each other
10
#93
244
66,80

#118

PR+DR
PR+DR

50.62
46.18

Group of clones showed homology to each other
8
#10
365
58.63
9
#33
301
56,88

160
301

WB
WB

#43
#97

6
7

Signal on Bs

WB

AT (%)(5)

Group of clones showed homology to each other
5
#6
193
53.89

Size (b.p)

(4)

WB
PR
WB
WB

Clone number

N(3)Group of clones showed homology to each other
1
#1
233
60.94
2
#12
216
60.19
3
#19
291
58.08
4
#37
334
61.98

(2)

(1)

Table 1. Cloned DNA fragments derived from the B chromosome

320
A. G. Bugrov et al.

DNA content of the B chromosomes in Podisma kanoi

321

FISH signal on this B was similar to the signal in NORs
of A chromosomes. The distribution of rDNA in
P. sapporensis was different. In P. sapporensis FISH
with rDNA gave a signal in C-positive regions of theBs of different morphotypes and in additional Cpositive short arms of A chromosomes (Bugrov et al.
2004) (Figure 3c). No signal was detected in telomericregions of long arms of the A chromosomes.

regions of A and B chromosomes and subtelomericregions of the Bs; vi Y two clones gave only abackground-like signal on A and B chromosomes(Table 1). Results of FISH with DNA probes based on
five cloned DNA fragments are shown in Figure 4.
Sequences of the clones were submitted to DDBJ/
GenBank/EMBL (http://www.ddbj.nig.ac.jp/) (Accession Nos: EF053372YEF053387) and comparedwith known sequences. 16 of them were AT-enriched.
Only one clone (clone #97) was GC-enriched. This
clone showed 69% homology to a tandem repeat in
Bombyx mori (Kang et al. 2004). The size of therepeated element in clone #97 was 17bp (TGAA
ACCTGACCTGACC). In P. kanoi its copies were
distributed along the entire B; however, FISH withthe probe based on this clone revealed no specificsignal on A chromosomes. Nine clones could be
combined into three groups according to their homology to each other: i Y clones #1, #12, #19, and #37; ii
Y clones #6, #43, and #97; iii Y clones #10 and #33(Table 1). Clones #93 and 118 were identical. They
showed 76% homology to Locusta migratoria solitarious phase hind-leg cDNA (EST clone; ACC
CO856777). Clone #29 showed 75% homology to
Locusta migratoria solitarious phase EST clone(ACC CO843638). This clone painted the whole B
chromosome and pericentric regions of A chromosomes. DNA probes based on two clones (# 23 and#26) showed no FISH signal on A or B chromosomes. Furthermore, clone #26 showed 98% homology to human BAC RP11-804M4 (ACC AC073284).

Sequencing of cloned DNA fragments and their FISH
on A and B chromosomes in P. kanoi
In order to analyze the DNA content of the Bs in
P. kanoi, 153 DNA fragments derived from the Bk
microdissected library were cloned in pBluescript II
KS+ (60 clones) and in pCR4Blunt-TOPO vectors(93 clones). The size of cloned DNA fragments
ranged from 100 up to 500 bp. A total of 20 cloned
DNA fragments were sequenced and computer
analysis of their sequences was performed. Three ofthem showed homology to plasmid DNA and were
excluded from further analysis.
Patterns of FISH signal obtained with the remaining 17 clones were divided into six groups: i Y six
clones painted the whole B and gave a signal in A
chromosome pericentric regions; ii Y one clone
painted the whole B and gave a slight signal in A
chromosome arms; iii Y three clones painted the whole
B and gave no signal in A chromosomes; iv Y threeclones painted the pericentric regions of A and B
chromosomes; v Y two clones hybridized to pericentric

Figure 4. FISH of P. kanoi cloned DNA fragments on P. kanoi meiotic chromosomes: (a) #12 probe (red), #10 probe (green) (metaphase II);(b) #23 probe (red), #43 (green) probe (metaphase I); (c) Y #19 (green) (metaphase II). Counterstained with DAPI. Arrow indicates B
chromosome. Scale bars = 10mm.

322
There are probably only a few copies of DNA
elements homologous to these clones, although wecannot exclude contamination as their origin. However, most of the cloned DNA fragments were
obviously derived from the B chromosome. Fifteenof 17 DNA probes based on these clones painted thewhole B or some of its regions.

Discussion
FISH with DNA probes ranging in size from 100 to
500bp marks chromosome regions only if there are
numerous copies of homologous sequences present in
the chromosome. The patterns of FISH signal on theBs obtained with DNA probes based on cloned DNA
fragments indicate a wide distribution of numerous
copies of repeats in the B in P. kanoi. Repeats

A. G. Bugrov et al.
homologous to cloned DNA fragments were found in
the Bs and in pericentric regions of A chromosomes.
Through the analysis of the mechanisms of B
chromosome formation we considered the hypothetical evolutionary pathways of chromosome rearrangements and possible B chromosome organization basedon the presented data (Figure 5). We assumed that the
B chromosome at its initial stage was small, andsimilar to human small supernumerary marker chromosomes (SMCs), reviewed in detail in Liehr et al.
(2004). Generally, SMCs are formed as a result of
the deletion of chromosome arms followed bytelomerization of chromosome regions at the breakpoints or inverted duplication of short arm andpericentromeric regions of acrocentric chromosomes.
In contrast to human SMCs, the size of the initial
B chromosome was enlarged through DNA amplification. Indeed, six DNA probes based on cloned

Figure 5. Hypothetical mechanisms of B chromosome formation and evolution in P. kanoi. Chromosome regions are described in the figure.
Arrows indicate chromosome breaks, formation of inverted duplication, centromere inactivation, chromosome end telomerization, amplification of
DNA in chromosome regions. The first break led to small additional chromosome formation as result of deletion of most of the long arm
accompanied with inverted duplication or telomerization of the chromosome end. In case of inverted duplication the next step was one centromere
inactivation. Further evolution in both hypothetical mechanisms was associated with DNA amplification in different chromosome regions.

DNA content of the B chromosomes in Podisma kanoi

323

DNA fragments painted the whole B and gave asignal on pericentric regions or short arms of A
chromosomes. Two other DNA probes gave a signal
on pericentric regions including short arms of A and
B chromosomes and on subtelomeric regions of thelong arm of the B. Three DNA probes marked only
the pericentric regions of A and B chromosomes(Table 1). This means that most of the B chromosome DNA was derived from the original A
chromosome.
In Figure 5 we summarize the hypothetical pathways of B chromosome evolution by A chromosome rearrangements and DNA amplification indifferent regions of the initial B chromosome. The
formation of the initial B as a ring chromosome was
excluded from our analysis. This type of rearrangement requires two simultaneous breaks in both armsof the ancestral chromosome. Furthermore, in orderto form the modern morphology of the B, one more
break in the ring chromosome followed by telomerization of chromosome regions at the breakpoints
is required. This sequence of events is considered tooccur rarely, if ever. It is also unlikely that contemporary Bs are derived from the deletion of theancestral chromosome arm followed by telomerization of regions at the breakpoint and DNA amplification because of the existence of two DNA
probes (#10 and #33) that produced a signal on
pericentric regions, including the short arm, and on
subtelomeric regions of the long arm of B chromosomes (Figure 4a, Table 1). Therefore, the mostpromising hypothesis that agrees well with thepresent results is that the initial B chromosome isan inverted duplication of the short arm andpericentromeric region of an acrocentric chromosome followed by an inactivation of one centromere. A similar mechanism was shown forformation of human small SMCs from acrocentricchromosomes. Moreover, as shown by the locationof FISH signals, DNA fragments from different
regions have been amplified in the B. Different
regions of A and B chromosomes were marked byprobes based on cloned DNA fragments derived
from the B chromosome. In all cases FISH of theseprobes painted large regions on B chromosomes,
implying independent DNA amplification in different regions. Therefore, all regions of the initial B
were possibly amplified during B chromosome
evolution. Despite only 17 cloned DNA fragments
in FISH experiments, these probes painted various

regions of chromosomes, e.g. only the pericentromeric region, pericentromeric and subtelomericregions, the whole B chromosomes, and slightly
euchromatic regions of A chromosomes.
Unexpectedly, DNA clones that marked relatively
small B chromosome regions were overrepresented.
Three of 17 clones marked only the pericentric
region and two produced a signal in both pericentricand subtelomeric regions. These regions cover asmall part of the entire B, and the DNA clones
derived from these regions should comprise a small
portion of the B chromosome DNA library. B
chromosome probe #19 helps to resolve this contradiction (Figure 4c). This probe painted the entire B
chromosome; however, signal intensity was muchstronger in the pericentric region than in the longchromosome arm. Some copies of other pericentricspecific DNA sequences were probably also present
in the long chromosome arm as dispersed repeats.
It is uncertain whether all of the contemporary B
chromosome DNA is derived from the initial B or
partially from inserted DNA elements. In humans thephenomenon of duplicons associated with DNA
transposition has been described and molecularmechanisms involved in duplicon formation werediscussed by Sharp et al. (2005) and Bailey et al.
(2003). Insertion of DNA elements into the B
chromosome could have taken place by similar
mechanisms. DNA probe #29 painted the whole B
but gave only a very weak, dispersed signal on
euchromatic A chromosome regions. Other DNA
probes (#37, #87, #97) also painted the whole B but
gave no signal on the A chromosomes. Unfortunately
we could not ascertain the origin of these DNA
fragments.
Our present results strongly suggest that at least
some DNA transpositions into the B chromosomes
occurred after formation from the ancestral B. The
rDNA cluster found in one of the studied Bs may be
the result of rDNA transposition during the laterstage of B chromosome evolution. Regions enrichedwith rDNA were found in B chromosomes in manyother species (Green 1990). In general there are twoforms of rDNA distribution in B chromosomes
including large regions enriched with rDNA in the
B chromosomes of some species (Cabrero et al.
2003), and clusters of rDNA shown to be small
NORs in others (Rubtsov et al. 2004). It should be
noted that almost all B chromosomes in P. sapporensis contained large regions enriched with rDNA. In

A. G. Bugrov et al.

324
contrast to P. kanoi, rDNA in A chromosomes in
P. sapporensis is located in pericentric regions and in
additional short arms. The ancestor of the B
chromosomes in P. sapporensis probably had acluster of rDNA in its pericentric region or short
arm and rDNA was included into the initial B as aresult of chromosome rearrangement that led toinverted duplication of the short arm and proximal
part of the long arm. In P. kanoi, NORs were locatedin subtelomeric regions of long arms of the X and the
middle-sized acrocentric autosome. As suggested,
formation of the supernumerary chromosome in
P. kanoi would not lead to the inclusion of rDNA
in the initial B chromosome. Indeed, rDNA wasatypical for the Bs in P. kanoi. The rDNA cluster
found in the only B chromosome could haveoriginated from a standard NOR of an A chromosome.
Transposition of rDNA could have occurred by meansof alternate telomere lengthening, the mechanismsof which include non-homologous recombinationof subtelomeric regions (Londono- Vallejo et al.
2004, Laud et al. 2005). It is likely that thelarge difference in molecular composition between
P. kanoi and P. sapporensis Bs is due to the different patterns of rDNA distribution in A chromosomesin these. While B chromosomes in P. sapporensis
included spacious regions enriched with rDNA, asmall cluster of rDNA was found only in one B
chromosome of P. kanoi.
Generation of the B chromosome DNA library,cloning of B chromosome DNA fragments, andcomparative FISH analysis showed that the B
chromosomes in P. kanoi consist mainly of repetitive
DNA. A detailed study of DNA composition andevolution of the B chromosomes in P. kanoi throughcomparative analysis of DNA sequences revealed thatthe sequenced DNA fragments were mostly AT-richand some of them consisted of short repetitive units(Table 1).
Short repeated sequences were also found in shortcloned DNA fragments. Their size in clones #6, #43,
and #97 was equal to 17bp. In clone #97 this repeat
showed differences in two positions. In clones #6 and#43 repeat units were identical. The presence ofsimilar repeats in these clones led to the highhomology between them (Table 1). A high level ofhomology was revealed between these clones andDNA of subtelomeric regions in Bombix mori chromosomes. Substantial similarity between clones # 10
and #33 was also disclosed (91% homology), as well

as for the group of clones #1, #12, #19 and #37(Table 1). Another group (clones #29, #93, #118)
showed homology with different EST clones
extracted from L. migratoria. The identical clones#93 and #118 are probably derived from the samefragment that was amplified during DNA librarygeneration. The rest of the sequenced DNA fragments did not show significant homology with known
sequences.
FISH of different DNA probes gave different
painting patterns in pericentromeric regions and shortarms of A chromosomes. Some marked pericentromeric regions of all chromosomes, while others gavea signal in pericentromeric regions only in a few
chromosomes. These data show that there are DNA
sequences that are common to all pericentromericregions, while the distribution of other sequences inpericentromeric regions is limited to one or a few
pairs of A chromosomes. Some clusters of repeat
may generate a hot break spot in one of theautosomes, increasing the frequency of B chromosomes formation.
In conclusion we have to mention that P. sapporensis and P. kanoi are very closely related species.
Results of comparative molecular cytogenetic analysis of A and B chromosomes performed in this study
showed that relatively small chromosome reorganization such as relocation of NORs from pericentromeric regions into telomeric regions or, on thecontrary, could have a significant influence on further
karyotype evolution in these species. As for B
chromosomes in P. sapporensis they are enrichedwith rDNA that was included in the Bs, probably
since the moment of their formation. In P. kanoi theBs, had probably no rDNA in the beginning and theirevolution was associated mainly with amplificationof different repeated sequences. We suppose that inanimals the frequency of B chromosome formationand their further evolution strongly depends on sometraits of molecular and morphological chromosome
organization, for example on Fhot spot_ in pericentromeric regions of acrocentric chromosomes, different types of repeats distribution, and NOR location.

Acknowledgements
The authors thank Dr Shin-ichi Akimoto and Dr
Masahiro Ohara, for all-round help in Japan. Part ofthis study was realized in the Hokkaido University

DNA content of the B chromosomes in Podisma kanoi
Museum by A. Bugrov as Visiting Professor in 2005.
The study was supported by a grant from the
Presidium of the Russian Academy of Sciences
FBiodiversity and genome dynamics_ for A. Bugrov;
the Russian Fund of Basic Researches (RFBR) No.
05-04-48230a for T. Karamysheva; 05-04-48221a
and 06-04-08211 for N. Rubtsov; a grant by thePolish State Committee for Scientific Reasearch(KBN) no. 2P04C 08826 for E. Warchałowska-Śliwa
and A. Bugrov; and Grant-in-Aid from the Ministryof Education, Science, Sports and Culture of Japan(no. 16770069 to H. Tatsuta). Thanks are also due tothe Ministry of Environment of Japan for providing
approval to collect materials in Joeshin-etsu National
Park, Honshu, Japan.

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