ISSN 1022 7954, Russian Journal of Genetics, 2015, Vol. 51, No. 12, pp. 1233–1242. © Pleiades Publishing, Inc., 2015. Original Russian Text © N.B. Rubtsov, T.V. Karamysheva, A.S. Bogdanov, I.V. Kartavtseva, M.N. Bochkarev, M. Iwasa, 2015, published in Genetika, 2015, Vol. 51, No. 12, pp. 1423–1432. ANIMAL GENETICS
Comparative Analysis of DNA Homology in Pericentric Regions of Chromosomes of Wood Mice from Genera Apodemus and Sylvaemus N. B. Rubtsova, b, T. V. Karamyshevaa, A. S. Bogdanovc, I. V. Kartavtsevad, M. N. Bochkareva, and M. Iwasae aInstitute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences (SB RAS), Novosibirsk, 630090 Russia bNovosibirsk State University, Faculty of Science, Department of Cytology and Genetics, Novosibirsk, 630090 Russia cKoltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, 119334 Russia dInstitute of Biology and Soil Science, Far Eastern Branch, Russian Academy of Sciences (FB RAS), Vladivostok, 690022 Russia eNihon University, Kameino 1866, Fujisawa, Kanagawa 252 8510, Japan e mail: [email protected]@; [email protected]@; iwasa.masahiro@nihon u.ac.jp Received April 2, 2015. In final form June 13, 2015.
1 Abstract—In the present study, an analysis of the DNA homology of the pericentric chromosomal regions 1 2 and pericentric heterochromatin in distantly related species of wood mice (species from the Apodemus genus, as well as from the Apodemus and Sylvaemus genera) was conducted by fluorescent in situ hybridization (FISH) with microdissected DNA probes obtained from the corresponding chromosomal regions of these 1 species. Cross hybridization of microdissected DNA probes obtained from pericentric C positive blocks of chromosomes of Sylvaemus species with chromosomes of Apodemus species, as well as DNA probes from 1 pericentric C positive blocks of chromosomes of Apodemus species with chromosomes of Apodemus and Syl 1 vaemus species, showed that DNA repeats homologous to the pericentric regions in other species represented 1 dispersed repeats in C negative chromosomal regions, as well as in several regions bordering pericentric C positive and C negative regions in heterochromosomes and autosomes and in distal regions in the long 1 arms of several autosomes. The results indicate that the level of DNA homology in pericentric chromosomal regions decreases with an increase in the differentiation level and a decrease in the kinship between the com pared forms and species of wood mice. Most likely, degeneration of the DNA repeats is accompanied by a gradual destruction of repeat clusters and their replacement by new, nonhomologous repeats in almost all 1 pericentric regions (some old repetitive sequences might be “extruded” into interstitial or telomeric regions of chromosomes). These processes, which are observed in some species from Sylvaemus genus in distantly related species of Sylvaemus and Apodemus genera, have almost achieved the final stages.
2 1 Keywords: fluorescent in situ hybridization (FISH), heterochromatin, DNA repeats, evolution, pericentric chromosomal regions DOI: 10.1134/S1022795415120091
INTRODUCTION alterations in the heterochromatin size and content 2 might affect both the spatial organization of the cell Species formation, and the subsequent indepen nucleus and probably the successful performance of dent evolution of the formed species, results in the several stages of cellular meiosis. Estimation of the sig accumulation of differences in their genomes. Analy nificance of the divergence of C positive pericentric 1 sis of these differences makes it possible to estimate the chromosomal regions for the formation of reproduc divergence level and time. The initial stages of new species formation appear to be the most complex for tive isolation and species’ formation might be con comparative analysis and determination of the taxo ducted as a result of their comparative analysis, both in nomic state. Polymorphism detection in several genes different races from one species and in different spe might provide reasonable conclusions, but not in all cies. Wood mice represent the optimal model for such cases. In these cases it becomes more appropriate to studies. Comparative analysis of pericentric hetero 1 2 explore highly variable genomic elements, which chromatin between wood mice from con specific pop 1 include C positive pericentric chromosomal regions ulations of several species of Sylvaemus genus, repre predominantly consisting of DNA repeats. Moreover, sentatives of the chromosomal forms and races of the
1233 1234 RUBTSOV et al.
small wood mouse Sylvaemus uralensis, as well as spe The genus of the Eastern Palearctic wood mice cies from different Sylvaemus genera, was conducted Apodemus s. str., being more ancient, includes species [1, 2]. Comparative analysis of the differences in DNA significantly differing in various genetic parameters; it 1 2 pericentric heterochromatin in wood mice was per should be mentioned that the genetic distances formed via the obtainment of microdissected DNA between the Apodemus s. str. species (except, probably, probes from the corresponding chromosomal regions for the distances between the groups of A. agrarius– and subsequent cross hybridization of the obtained A. chevrieri and A. semotus–A. draco–A. latronum), DNA probes with the chromosomes of all of the stud based on several markers, were comparable to the dif ied species. ferentiation between the Western Palearctic wood mice and the Eastern Palearctic, or even the house Differences in the number of DNA repeats in mouse, i.e. they actually achieved the genus level [6, 7, 1 C positive pericentric regions were observed between the Asian and European races of the small wood mice 12. 13–17]. A. argenteus and A. gurkha appear to be S. uralensis, as well as between chromosomal forms of maximally distant from other Apodemus and Sylvae European race [1, 3]. The comparison of chromo mus species [10, 12, 16, 18 – 20]. somes from different species of Sylvaemus genus Our goal was to estimate the degree of homology of resulted in qualitative differences in the DNA content DNA sequences of pericentric chromosomal regions 1 1 2 of the pericentric heterochromatin. As a result of het and in phylogenetically more distant species of wood erologous FISH, several species demonstrated an mice (between species of the Apodemus genus and absence of homologous sequences in C positive compared to species of the Sylvaemus genus). regions, while the others reported a rapid decrease in their quantity and/or homology level. For instance, 3 such differences were detected in the related allopatric MATERIALS AND METHODS species S. flavicollis and S. ponticus. Heterologous in In total, 72 individuals from five Sylvaemus species situ cross hybridization conducted in different Syl and four Apodemus species were studied. The animals’ vaemus species also revealed clusters of homologous species, collection site, sample size, and a brief char repeats in interstitial and telomeric regions of the long acterization of the karyotype (2n, NF) are described 1 arms in the absence of FISH signal in pericentric below. Data on the C block location in mice karyo regions. Representatives of several species were even characterized by the absence of clusters of homolo types of Sylvaemus species and intraspecific forms of gous repeats in any chromosomal region as a result of A. uralensis, S. sylvaticus have been published previ heterologous FISH. The homology of dispersed ously [2]. Diagnostics of species and intraspecific repeats distributed in C negative chromosomal forms was preliminary conducted via karyologic, regions was observed in all species on the basis of the allozyme, and molecular genetic analysis. 2 rather rapid evolution of repeats in heterochromatin regions [2]. S. uralensis (2n = NF = 48) Our previous research was performed on wood European race, eastern European chromosomal mice of the same subgenus. Therefore, the question of form. whether the detected evolutionary DNA mechanisms 1 2 of pericentric heterochromatin fit larger taxonomic Russia: Moscow oblast, Zaraiskii region, Zaraisk categories (subgenera and genera), as well as the char neighborhoods (1 ); Samara oblast, Borskii region, 1 2 acter of the differences in pericentric heterochromatin Gerasimovka and Gostevka neighborhoods (1, 1); in the species comprising these taxa, remains open. This question concerning wood and field mice might Saratov oblast, Ozinskii region, Modin neighborhoods 2 be solved via comparative analysis of heterochromatin (1 ); Orenburg oblast, Kuvandykskii region, Kan regions in representatives from the Sylvaemus and cherovo Railroad Station neighborhoods (1, 1); 1 Apodemus genera. Sverdlovsk oblast, Visimskii Reserve (1 ); Kurgan oblast, Polovinskii region, neighborhoods of Zaural’e 1 I.Ya. Pavlinov et al. [4, 5] considered two subgenera of wood and field mice: @Apodemus@ and @Sylvaemus@ comprised Railroad Station and Polovinnoe village (1, 1); one genus. S.V. Mezhzherin [6, 7] proposed three subgenera: Omsk oblast, Tavricheskii region, Amre Railroad Sta @Apodemus@, @Sylvaemus@, and @Alsomys@. Many taxono mists share the previous proposals for unity of the @Apode tion neighborhoods (1, 1). mus@ sensu lato genus with division into subgenera [8, 9] or European race, southern European chromosomal species groups [10–12]. Our point of view is congruent with I.Ya. Pavlinov et al., dividing @Apodemus@ senso lato genus form. Russia: Krasnodar krai, Sochi neighborhoods: into two: Western Palearctic wood mice of the @Sylvaemus@ Hosta outskirts (1 ); 5 km northeast of Krasnaya genus (including @Sylvaemus@ and @Karstomys@ subgenera) and Eastern Palearctic wood and field mice of @Apodemus@ Polyana, Caucasian Reserve (1 ). Russia, Kabar sensu str. genus (including @Apodemus@ and @Alsomys@ sub dino Balkaria, Nalchik outskirts (1 ). Armenia, Lori genera) on the basis of geographical principle with assumption of the morphological and genetic differences. oblast, Lermontovo neighborhoods (2 ).
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 COMPARATIVE ANALYSIS OF DNA HOMOLOGY 1235
Asian race. Russia, Novosibirsk outskirts in several Russia, Chita oblast, Borzinskii region, Ust’ Ozernoe km from Berdsk (1 ). Kazakhstan, Pavlodar oblast, neighborhoods (1 ). Russia, Primorskii krai: Khan Pavlodarskii region, Rozovka neighborhoods (1 ). kaiskii region, Novo Nikolaevka neighborhoods Kazakhstan, eastern Kazakhstan oblast, Tarbagataiskii (2 ); Pogranichnii region, Pad’ Karantinnaya region, 30 km southwest of Aksuat village, Tarbagatai (1 ); Shkotovskii region, Novonezhino neighbor ridge, flood lands of Karabuga River (2 ). Kaza hoods (1 ); Nadezhdinskii region, Kiparisovo neigh khstan, Almaty oblast: Aksuiskii region, 33 km from Matai village toward Kuraksu village, floodlands of borhoods (1 ). Aksu River (1 ); Alakol’skii region, Bibakan village neighborhoods, floodlands of Orta Tentek River (1 ); Kerbulakskii region, 10 km northeast of Rud Apodemus (Alsomys) peninsulae nichnyi village, Dzhungarskii Alatau (1 ); Kerbulak Russia, Novosibirsk oblast: Novosibirsk outskirts skii region, Altynemel’ ridge, Altynemel’ pass (1 ). several km from Berdsk and Akademgorodok territory Turkmenistan, Lebapskii velayat, Kugitang ridge, (1 – 2n = NF = 48 + 7 additional chromosomes; 1 Airybaba mountain and neighborhoods: Darai Dara canyon, Svintsovii rudnik neighborhoods; Kyrkkyz – 2n = NF = 48 + 6 additional chromosomes); Iskit canyon, Khodzhaipil’ neighborhoods (1, 2 ). imskii region, Morozovo neighborhoods (1 – 2n = NF = 48+ 6 additional chromosomes). Russia, Altai Republic: Shebalinskii region, Cherga neighborhoods S. flavicollis (1 – 2n = NF = 48 + 8 additional chromosomes); Russia: St. Petersburg neighborhoods (3, 2 Turochakskii region, Artybash neighborhoods (1 – – 2n = NF = 48; 1, 1 – 2n = NF = 48 + 1 addi 2n = NF = 48+ 8 additional chromosomes). Japan, tional chromosome); Penza oblast, Belinskii region, Hokkaido Island, Tomakomai neighborhoods (1 – Shiryaevo neighborhoods (2 – 2n = NF = 48); 2n = NF = 48 + 8 additional chromosomes). Samara oblast, Stavropolskii region, Bakhilova Poly ana neighborhoods, Zhigulevskii Reserve (1 – 2n = NF = 48). Apodemus (Alsomys) speciosus (2n = 48, NF = 56)
S. ponticus (2n = NF = 48) Japan: Hokkaido Island, Tomakomai neighbor Russia, Krasnodar krai, Sochi neighborhoods: hoods (1 ); Honshu Island (2 ). Russia, Hosta outskirts (2 ); 5 km northeast of Krasnaya Kunashir Island (1 ). Polyana, Caucasian Reserve (2 ). Apodemus (Alsomys) argenteus (2n = 46, NF = 52) S. sylvaticus, vohlynensis form (2n = NF = 48) Russia: Belgorod oblast, Borisovskii region, Boris Japan, Hokkaido Island, Tomakomai neighbor ovka neighborhoods, Belogor’e Reserve (1 ); Ros hoods (1 ). tov on Don, floodlands of Temernik River (1 ). The preparation of metaphase chromosomes, their C and Q staining for heterochromatin segments 2 S. fulvipectus (2n = NF =48) detection, microdissection, obtainment of DNA probes from dissected C positive pericentric regions, 1 Russia, Rostov oblast, Sal’skii region, Mayak in situ hybridization and signals registration were con neighborhoods, Salsk steppe (1 ). Turkmenistan, ducted according to previously described methods [1, Balkanskii velayat, Kopetdag ridge, Ioldere canyon, 2]. Syunt Khasardagskii Reserve (1 ). The results of differential staining and FISH were analyzed with an AXIOSKOP 2 Plus microscope Apodemus (Apodemus) agrarius (2n = 48, NF = 56) (Zeiss); microimage registration and adjustment was Russia, Yekaterinburg neighborhoods (2 ). performed with a CCD camera with corresponding Russia, Novosibirsk oblast: Novosibirsk neighbor filters (Chroma, Zeiss) and ISIS3 software (META Systems Gmbh, Germany). The study was also con hoods (2 ); Kochkovskii region, Kochki neighbor ducted on the equipment of the Center of Collective hoods (2, 2). Russia, Altai Republic, Ust’ Use for microscopy analysis of biological objects of SB Kanskii region, Chernii Anui neighborhoods (2 ). RAS and FEB RAS.
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 1236 RUBTSOV et al.
RESULTS of a signal in C blocks of the one pair of acrocentric and metacentric chromosomes with the intensive Microdissected DNA probes and analysis staining of pericentric heterochromatin of other auto 1 2 of their specificity somes and the X chromosome of A. argenteus confirms Microdissected DNA probes were obtained from the hypothesis of significant heterochromatin hetero 2 1 the C positive pericentric region of one of the longest geneity in this species’ karyotype [30]. autosomes of A. peninsulae, A. speciosus, and A. argen DNA probe Asp intensively stained only the teus (one for each species). They were designated as C positive pericentric regions of sex chromosomes in 1 the combination of the first letter of Latin name of the A. speciosus karyotype; signals of weak or medium genus and two first letters of species (i.e. Ape, Asp, and intensity have been detected in pericentric regions 1 Aar, respectively). Moreover, for the comparison of corresponding to the heterochromatin size in approx 2 Apodemus and Sylvaemus species, we included DNA imately half of autosomes differing in size (Fig. 1, c; probes Spo, Sfl, Sfu, Ssy and Sur5, which were previ Table 1). No specificity of fluorescent signal in the ously obtained from S. ponticus, S. flavicollis, S. fulvi telomeric chromosomal regions was observed. pectus, S. sylvaticus, and the southeuropean chromo somal form of S. uralensis into the analysis; their char No significant intraspecific variance in the charac acteristics have been represented previously [1, 2]. ter of FISH signals were detected in any Apodemus Only metaphase chromosomes were used from the species via in situ hybridization with the obtained wood mice A. agrarius for heterologous FISH with the microdissected DNA probe with chromosomes of probes mentioned above. conspecific samples from several populations, except for the B chromosomes of the eastern Asian mouse; The specificity of each DNA probe was tested via in their wide variability in the presence and location of situ hybridization with chromosomes in the same DNA repeats homologous to autosomal pericentric het 1 2 example from which it was obtained. DNA probes Ape erochromatin sequences was revealed earlier [36, 37]. and Aar demonstrated precise signals in C positive 1 pericentric regions of almost all the chromosomes, together with an intensive signal in the dissection FISH signals obtained via cross hybridization region (Fig. 1). However, A. peninsulae was character of DNA probes with the chromosomes of various ized by Ape probe hybridization on the border of Apodemus species, as well as via comparison | 1 C positive and C negative pericentric regions of X of Apodemus and Sylvaemus species chromosome, in the C positive telomeric segment of the Y chromosome, and in various regions of the B The results of in situ hybridization of DNA probes chromosomes (Fig 1, a), which is evidence of the pres Sur5, Sfl, Sfu, Spo and Ssy with the chromosomes of ence of sequences homologous to those characteristic Apodemus species are shown in Table 2 and Fig. 2. The 1 2 for pericentric heterochromatin. Analogous cases of FISH patterns demonstrated no large diversity and FISH signal detection in telomeric segments of dis frequently demonstrated an intensive staining of telo tinct chromosomes during hybridization with probes meric regions of distinct autosomes or specific signals 1 obtained from C positive pericentric regions have at the border of the pericentric C positive and C neg 1 been described above for the yellowthroat mouse ative chromosomal regions, as well as in the pericen 1 S. flavicollis and the vohlynensis form of the wood tric C blocks (in the whole segment or in some part) of mouse S. sylvaticus [2]. heterochromosomes. As a result of in situ hybridiza Interesting results were revealed via FISH with tion of the Spo probe with A. speciosus chromosomes, DNA probe Aar and metaphase chromosomes of the an intensive signal was detected in the pericentric C 1 A. argenteus sample from which it was obtained. block of the large autosomal pair. In many cases the Intensive signals of this probe were detected, both in compact signals have not even been detected: the 1 2 all C blocks of pericentric heterochromatin in almost chromosomal staining reported a diffusive character all acrocentric autosomes and in the centromeric C almost along the whole chromosomal length except block of one pair of medium metacentrics, as well as in for their C positive pericentric regions (see, for 1 the large C positive block of X chromosome (Fig. 1, instance, FISH results with DNA probe Sur5, b). FISH of the DNA probe Aar demonstrated no spe Table 2). 1 cific DNA staining in the pericentric region of the Simultaneous in situ hybridization with A. specio smallest acrocentric pair and the pair of small meta sus and A. agrarius chromosomes of three DNA probes 1 centrics. The absence of a FISH signal in the pericen tric region of the one pair of small metacentrics and (Ssy, Sfl, and Spo) revealed their detection in coincid intensive staining of this region in the one pair of ing, overlapping, or neighboring regions (Fig. 3). medium metacentric autosomes is probably caused by Cross hybridization of DNA probes Ape, Asp and a difference in the content of the repeated sequences Aar with the chromosomes of representatives of Apo 1 2 forming pericentric heterochromatin of these auto demus and Sylvaemus genera (Fig. 4) resulted in the 1 somes. The pericentric region probably consists of detection of a diffusive signal on almost the whole other sequences and could not be detected by DNA chromosomal length, except for their pericentric het 1 2 probe Aar in the small metacentrics pair. The absence erochromatin segments.
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 COMPARATIVE ANALYSIS OF DNA HOMOLOGY 1237
X
X Y
X Y
X
(a) (b) (c) Fig. 1. FISH of microdissected DNA probes with metaphase chromosomes of samples from the same species (FISH signals are green): a DNA probes Ape with the chromosomes of A. peninsulae sample from Novosibirsk, ; b DNA probes Aar with the chromosomes of A. argenteus sample from Hokkaido Island, Tomakomai neighborhoods, ; c DNA probes Asp obtained from males from Hokkaido Island with the chromosomes of A. speciosus from Honshu Island, . The arrows stand for the signals detected on B chromosomes of A. peninsulae (a). The arrows → point to the small acrocentrics and metacentrics of A. argenteus absent of signals to DNA probe Aar; arrows point to the signals detected by DNA probe Aar on the one pair of medium metacentrics of A. argenteus (b). X, Y—sex chromosomes. Total staining of the chromosomes was performed with DAPI (red sig nal).
X Y B X B B B B Y
B X
Y (a) (b) (c)
Fig. 2. FISH signals obtained during in situ hybridization of DNA probes Sfl (red signal) and Spo (green signal): a with chromo some of A. peninsulae sample from Novosibirsk, ; b with the chromosomes of A. agrarius sample from the neighborhoods of Kochki village of Novosibirsk oblast, ; c with the chromosomes of A. speciosus sample from Hokkaido Island and Tomakomai neighborhoods, . The short and long arrows point to the signal of DNA probe Sfl in B chromosomes (B) and in telomeric regions of A. peninsulae autosomes, respectively. X, Y—sex chromosomes. Complete staining of chromosomes was performed with DAPI (blue signal).
DISCUSSION metaphase chromosome of the Apodemus genus resulted in the absence of precise signals in half of the 1 The level of DNA homology in pericentric chromosomal combinations. In the cases of detected FISH signals, regions in genetically distant species they were single and their location was limited to the of wood and field mice pericentric regions of sex chromosomes and autoso 1 mal large pairs (in almost all cases, fluorescence was According to the results of heterologous FISH, the observed at the border of the C positive and C nega most genetically distant species of wood and field tive regions), as well as to distal regions of the long mice, which belongs to the Apodemus genus or to Apo arms of several chromosomes, as was reported for Syl demus and Sylvaemus genera, were characterized by vaemus species. the smallest resemblance in DNA sequences in the 1 1 2 pericentric regions and pericentric heterochromatin. All of the reciprocal variants of heterologous FISH The comparison of different Sylvaemus species in the case of comparison of eastern Palearctic and resulted in the absence of even weak compact signals western Palearctic wood mice (i.e. hybridization of and the presence of diffusive fluorescence in three of DNA probes obtained from Apodemus species with 20 cases (15%) with the use of the same DNA probe metaphase chromosomes of Sylvaemus species) dem (Sfu) [2]. The hybridization of the same DNA probes onstrated the absence of clusters of homologous (Sur5, Sfl, Spo, Sfu, and Ssy) with the murine repeats with a diffusive fluorescence character only.
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 1238 RUBTSOV et al.
their significant genetic differentiation and older age relative to Sylvaemus species.
Evolution of DNA sequences in the pericentric regions 1 and pericentric heterochromatin of wood a 1 2 nd field mice at different differentiation stages Y Accordingly, the summary of the obtained findings allows the conclusion that wood mice from different X populations of the same species or belonging to various intraspecific forms possess DNA sequences compris ing the pericentric chromosomal regions and pericen 1 1 tric heterochromatin and are characterized by high 2 resemblance and number of DNA repeats, which Fig. 3. Simultaneous in situ hybridization of three DNA probably determines the observed differences in FISH probes, Ssy (green signal), Sfl (yellow signal), and Spo (red patterns [1, 2]. However, the following differentiation signal), with the chromosomes of A. agrarius sample from level is characterized by a rapid decrease in the DNA Yekaterinburg neighborhoods, . X, Y—sex chromo somes. Complete staining of the chromosomes was per homology of pericentric chromosomal regions and 1 formed with DAPI (blue signal). The image was obtained pericentric C heterochromatin in the genetically 1 2 with specific METASYSTEMS Gmbh software for multi close, allopatric S. flavicollis and S. ponticus species, 3 color FISH. Only intensive signals significantly exceeding which appears more obvious during the comparison of the background level are shown, while signals of the diffu “good” and even genetically distant species of wood sive type are not shown. and field mice up to the complete absence of precise signals. This might be caused by two processes: the Accordingly, in light of the complete set of DNA degeneration of homologous DNA sequences (due to probes used to compare wood mice belonging to vari signal weakening with the maintenance of the sizes of the fluorescent zones) and/or rupture of their clusters ous genera, this FISH pattern appears to be predomi and the formation of all or the majority of pericentric 1 nant (75% of combinations). It probably represents a chromosomal regions and pericentric C heterochro 1 2 complete or almost complete renewal of DNA content matin from absolutely different, nonhomologous 1 in pericentric chromosomal regions, i.e. the replace DNA sequences that extrude the residues of the ment of the previous DNA sequences by novel nonho former DNA repeats into interstitial or telomeric mologous DNA repeats, and the previous sequences regions of the chromosomes. Comparison of Sylvae are either eliminated or extruded into other chromo mus species demonstrating diverse variants of hybrid somal regions and are maintaind as distinct degener ization of microdissected DNA probes with the chro mosomes resulted in the observation of both trends, ated single copies or small clusters incapable of stable including the degeneration of homologous sequences hybridization with probes. Notably, the same observa with maintenance of the location of their clusters (see tion was shown during a comparison of different spe FISH of DNA probe Sfl with S. ponticus chromo cies of eastern Palearctic wood mice, thus confirming somes) [2] and the replacement of homologous
X X
X
Y (a) (b)
Fig. 4. FISH signals obtained via in situ hybridization: a DNA probes Ape with the chromosomes of A. agrarius sample from Yeka terinburg neighborhoods, ; b DNA probes Aar with the chromosomes of A. peninsulae sample from Novosibirsk, . X, Y—sex chromosomes. FISH signals are red. Complete staining of the chromosomes was conducted with DAPI (blue signal).
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 2 1 COMPARATIVE ANALYSIS2 OF1 2 DNA2 HOMOLOGY2 2 1 1 12391 References [19, 26–30, our data] our data] [31, 32, our data] [26, 33–35, our data] C heterochromatin occupies the whole C heterochromatin short arm and a third part of the long (in its proximal part) of the X chromosome. results in a C staining of the Y chromosome than that darkness that is weaker complete in the pericentric regions of autosomes. af of the X chromosome Heterochromatin ter quinacrine staining is characterized by fluorescence. Heterochromatin “delayed” is divided into two of X and Y chromosomes separate types, including X (X type) and heterochroma Y (Y type) chromosomal tin, according to the character of staining with fluorochromes Rather large and bright pericentric C blocks [21–26, The X chromosome possesses interstitial The X chromosome together with inserts of heterochromatin pericentric C block. The Y chromosome stained via appears to be almost completely C staining possesses large a peri The X chromosome centric C segment and bright interstitial signal in the distal regions of arms; Y is stained in dark but has more chromosome dense pericentric and interstitial blocks species Apodemus differs in its unique A. argenteus Character of C heterochromatin distribution Character of C heterochromatin “delayed” fluorescence in the majority of peri “delayed” C blocks after treatment with centric autosome as in the heterogeneity of het quinacrine, as well chromosomes segments of various erochromatic Three fluorochromes. upon staining with several isolat were autosomes types of heterochromatic of the first (M1 ed, including heterochromatin type) and second (M2 type) pairs of metacen of the pericentric as heterochromatin trics, as well (acrocentric regions of acrocentric chromosomes type) Rather large and bright pericentric C blocks are of the main set; present in all chromosomes and interstitial C telomeric presence of the weak pairs of autosomes segments is possible in several ed only in several submeta and acrocentric pairs. ed only in several One of the medium acrocentric pairs possesses ir C segments regular staining of telomeric is exclu location in autosomes Heterochromatin pericentric; C blocks are present in the ma sively jority of pairs Rather large pericentric C blocks are detected in all autosomes. tion in the karyotypes of investigated of investigated tion in the karyotypes in B chromosomes in autosomes in X–Y chromosomes This species is characterized by wide polymorphism by the number of B chro point to (0–30), their size (from mosomes as their lo as well large), and morphology, cation, size, and precision of heterochro bright C segments (in matin blocks: from interstitial state) to diffu the cetromeric, (in interstitial regions) sive B chromosomes were unobserved were B chromosomes detect large pericentric C blocks were Relatively are rare (0–1), possess B chromosomes acrocentric morphology and a weak C staining B chromosomes are rarely detected B chromosomes (0–1), possess a metacentric morphology, a point to the middle and are sized from The character of C heterochromatin distribu The character of C heterochromatin /NF n 48/48 2 Species, 46–48/56 48/54–56 46/52–54 A. agrarius A. speciosus A. argenteus A. peninsulae Table 1. Table 1 1 1 1 1 1 2 2 2 2 2
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 1240 2 RUBTSOV2 et al. 1 1 2 1 1 1 1 1 1 1 Apodemus tive and C negative pericentric and C negative tive Diffusive signal at background level signal at background level Diffusive part of auto only in euchromatic as in the prolonged as well somes, (C+) region of X Signals of medium intensity in (C–) region neighboring pericentric het in the pair of large au erochromatin in the tel(C–) region of an tosomes; (the other pair of large autosomes sequences ho same with observed mologous to Sfl probe); in (periC+) signal in the part region of X. Diffusive of B chromosomes Signal of medium intensity in (C–) region neighboring pericentric het in the pair of large au erochromatin signals in (periC–) intensive tosomes; region of X and Y at slight distance (see Fig. 3) centromere from signals in the (C–) region Weak neighboring the pericentric hetero in the pair of large auto chromatin signals in the tel(C+) intensive somes, regions of a pair small autosomes and in the middle part of Y (distal pericentric region staining with from Asp probe) Diffusive signal at the Diffusive in background level region of euchromatic of all chromosomes the main set and in B outside chromosomes (periC+) blocks signal at the Diffusive in background level region of euchromatic out all chromosomes side (periC+) blocks signal at the Diffusive of background level part of all euchromatic outside chromosomes (periC+) blocks signal at the border of C posi ⎯ d Ssy with metaphase chromosomes of wood mice of the of wood d Ssy with metaphase chromosomes hybridization of DNA probes in situ Diffusive signal at the back Diffusive in the euchromatic ground level as as well part of all autosomes, in prolonged (C+) region of X Signal of medium intensity in (C–) region neighboring peri centric C block in the pair of large autosomes. Diffusive sig nals along the arms outside peri centric C blocks were observed in heterochromosomes, B chro mosomes, and other autosomes (Fig. 2, a) signal in (C–) region Intensive neighboring pericentric C block in the pair of large autosomes. signals along the arms Diffusive outside pericentric C blocks in other auto observed were and heterochromosomes somes (Fig. 2, b; 3) Intensive signal in peri(C+) re gion of the one pair large au tosomes. Diffusive signal along the arms outside pericentric C blocks was detected in other au tosomes and heterochromo somes mes, as well in tel(C–) regions of peri(C+) regions of C negative chromosomal segments; (C+/C–) chromosomal C negative ⎯ Character of signals obtained during distal regions of long arms in acrocentrics. Intensive signals in tel(C–) regions of the Intensive Diffusive one pair of large autosomes. in euchromat signal at background level in other chromosomes ic part is observed Intensive signals in one pair of large autoso peri(C+/C–) regions of X and Y. Signals of different intensity and location in the arms of B chromosomes (see Fig. 2, a) signals in (C–)regions slightly Intensive region in X the centromeric distant from and Y (see Fig. 2, b; 3) Intensive signals in X, Y, as well irregular signals in two small pairs of metacentric autosomes (fig. 2, c) ⎯ hybridization of DNA probes Sur5, Sfl, Spo, Sfu, an C positive, (C–) C positive, ⎯ in situ Sur5 Sfl Spo Sfu Ssy sex chromosomes; tel sex chromosomes; ⎯ Diffusive signal at the back Diffusive in the euchromatic ground level out region in all chromosomes in side (periC+) blocks; some observed crease in the signal was in G segments signal at the back Diffusive re in euchromatic ground level outside gion of all chromosomes in (periC+) blocks; several creases of the signal in G seg observed ments was signal at background Diffusive part of all in euchromatic level outside (periC+) chromosomes signal increase in blocks; some G segments is observed Location of signals detected via pericentric chromosomal regions; (C+) pericentric chromosomal ⎯ A. agrarius of species A. speciosus A. argenteus Metaphase A. peninsulae chromosomes periC chromosomal regions; X, Y chromosomal Table 2. Table genus 1 RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 COMPARATIVE ANALYSIS OF DNA HOMOLOGY 1241
sequences with nonhomologous ones. Finally, evolu Scientific Center of RAS) and N.E. Kolcheva (Insti tion in the wood and field mice probably results in the tute of Plant and Animal Ecology Ural Branch of 1 replacement of sequences in all pericentric chromo RAS). somal regions, while a comparison of genetically dis This study was supported by the Russian Founda tant species reported the final stage of this process. tion for Basic Research (project no. 14 04 00086a, The examples of prolonged preservation of clusters 14 04 00785a, 15 04 03871a), as well as by the budget of ancient DNA sequences that are homologous in dif project VI.53.1.4. 1 ferent species in interstitial, telomeric, and even peri centric regions of distinct chromosomes appear to be interesting. In this case the preservation of clusters of REFERENCES such DNA repeats probably have some functional sig 1. Karamysheva, T.V., Bogdanov, A.S., Kartavtseva, I.V., nificance. For instance, a significantly high level of et al., Comparative FISH analysis of C positive blocks resemblance of genes encoding 5S, 18S, and 28S of centromeric chromosomal regions of pygmy wood rRNA and the constant location of one part of their mice Sylvaemus uralensis (Rodentia, Muridae), Russ. J. clusters on homeologous chromosomes were demon Genet., 2010, vol. 46, no. 6, pp. 712–724. strated in the wood mice of Apodemus and Sylvaemus 2. Rubtsov, N.B., Karamysheva, T.V., Bogdanov, A.S., genera [38]. et al., Comparative FISH analysis of C positive regions of chromosomes of wood mice (Rodentia, Muridae, In conclusion, the series of studies [1–3] for the Sylvaemus), Russ. J. Genet., 2011, vol. 47, no. 9, pp. first time included an analysis of the homology of a 1096–1110. large set of anonymous DNA sequences comprising 3. Bogdanov, A.S. and Rozanov, Yu.M., Variability in size 1 1 the pericentric chromosomal regions and pericentric of the nuclear genome in pygmy wood mouse Sylvae C blocks in several conspecific populations, intraspe mus uralensis (Rodentia, Muridae), Russ. J. Genet., cific forms, and Apodemus and Sylvaemus species by 2005, vol. 41, no. 10, pp. 1123–1129. preliminary assessment of their kindred. Unfortu 4. Pavlinov, I.Ya., Yakhontov, E.L., and Agadzhanyan, nately, no analogous research was conducted in other A.K., Mlekopitayushchie Evrazii (sistematiko animal groups except for the experiments with in situ geograficheskii spravochnik) (Mammals of Eurasia: hybridization of distinct DNA sequences with the A Systematic and Geographic Reference Book), vol. 1: chromosomes of several species of house mice of the Rodentia, Moscow: Mosk. Gos. Univ., 1995. Mus genus [39, 40] and wood mice of the Apodemus 5. Mlekopitayushchie Rossii: sistematiko geograficheskii and Sylvaemus genera [19, 38, 41]. Hence, the ques spravochnik (Mammals of Russia: Systematic and Geo graphic Reference Book), Pavlinov, I.Ya. and Lis tion of the universality of the evolutionary mecha ovskii, A.A., Eds., Moscow: KMK, 2012. 1 2 nisms of pericentric chromosomal regions and hetero 6. Mezhzherin, S.V., Genetic differentiation and phylo chromatin described above remains open. The solu genetic relationships among Palearctic mice tion to this problem requires a comparison of data (Rodentia, Muridae), Russ. J. Genet., 1997, vol. 33, obtained from several groups of animals with similar no. 1, pp. 65–72. characters of differentiation. 7. Mezhzherin, S.V., Revision of mice of the genus Apo demus (Rodentia, Muridae) in Northern Eurasia, Vestn. Zool., 1997, vol. 31, no. 4, pp. 29–41. ACKNOWLEDGEMENTS 8. Corbet, G.B., The Mammals of the Palaearctic Region: We are grateful to V.P. Vekhnik (Zhigulevskii a Taxonomic Review, London: Cornell Univ. Press, Reserve). E.I. Zholnerovskaya (Institute of Systemat 1978. ics and Ecology of Animals of SB RAS), V.I. Ermolaev 9. Corbet, G.B. and Hill, J.E., The Mammals of the (Institute of Cytology and Genetics of SB RAS), Indomalayan Region: a Systematic Review, Oxford: S.B. Pole (Kazakh Scientific Center of Quarantine Oxford Univ. Press, 1992. and Zoonotic Diseases), P.A. Til’ba, B.S. Tuniev 10. Musser, G.G., Brothers, E.M., Carleton, M.D., and (Sochi National Park), and R.I. Druzev (Kabardino Hutterer, R., Taxonomy and distributional records of Balkarian State University) for their assistance with oriental and European Apodemus, with a review of the animal collection and the opportunity for laboratory Apodemus–Sylvaemus problem, Bonner Zool. Beitr., processing of the material. We specifically thank 1996, vol. 46, nos. 1–4, pp. 143–190. O.V. Brandler (Koltzov Institute of Developmental 11. Musser, G.G. and Carleton, M.D., Superfamily Muroidea, Mammal Species of the World: a Taxonomic Biology of RAS) and A.Yu. Puzachenko (Institute of and Geographic Reference, Wilson, D.E. and Reeder, Geography of RAS) for direct help with the expedi D.M, Eds., Baltimore: Johns Hopkins Univ. Press, tions’ preparation and performance. Part of the mice 2005, pp. 894–1531. were donated by V.V. Marochkina (The Amudarya 12. Serizawa, K., Suzuki, H., and Tsuchiya, K., A phyloge State Nature Reserve), V.S. Lukarevskii, netic view on species radiation in Apodemus inferred Yu.M. Koval’skaya (A.N. Severtsov Institute of Ecol from variation of nuclear and mitochondrial genes, ogy and Evolution of RAS), I.Yu. Baklushinskaya Biochem. Genet., 2000, vol. 38, nos. 1–2, pp. 27–40. (A.N. Severtsov Institute of Ecology and Evolution), 13. Mezhzherin, S.V. and Zykov, A.E., Genetic divergence V.V. Stakheev (Institute of Arid Zones of the Southern and allozyme variation among mice of the genus Apo
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 1242 RUBTSOV et al.
demus s. lato (Muridae, Rodentia), Tsitol. Genet., 1991, 28. Yoshida, M.C., Sasaki, M., and Oshimura, M., Karyo vol. 25, no. 4, pp. 51–59. type and heterochromatin pattern of the field mouse, 2 14. Filippucci, M.G., Macholán, M., and Michaux, J.R., Apodemus argenteus Temminck, Genetics, 1975, vol. 45, Genetic variation and evolution in the genus Apodemus no. 3, pp. 397–403. (Muridae: Rodentia), Biol. J. Linn. Soc., 2002, vol. 75, 29. Fukuoka, H. and Udagawa, T., On the banding struc no. 3, pp. 395–419. tures of the chromosomes of the field mouse, Apodemus 15. Michaux, J.R., Chevret, P., Filippucci, M. G., and argenteus Temmink, with a note on the number varia Macholan, M., Phylogeny of the genus Apodemus with tion, Proc. Jpn. Acad., Ser. B, 1979, vol. 55, no. 10, a special emphasis on the subgenus Sylvaemus using the pp. 492–496. nuclear IRBP gene and two mitochondrial markers: 30. Obara, Y. and Sasaki, S., Fluorescent approaches on cytochrome b and 12S rRNA, Mol. Phylogenet. Evol., the origin of B chromosomes of Apodemus argenteus 2002, vol. 23, pp. 123–136. Hokkaido, Chromosome Sci., 1997, vol. 1, pp. 1–5. 16. Chelomina, G.N., Lesnye i polevye myshi: molekul yarno geneticheskie osnovy evolyutsii i sistematiki 31. Hirai, H., Moriwaki, K., and Uchida, T.A., Compara (Wood and Field Mice: Molecular Genetic Aspects of tive analyses of Japanese wood mice from the Oki Evolution and Systematics), Vladivostok: Dal’nauka, islands and the mainland of Japan based on biochemi 2005. cal genetics and cytogenetics, J. Fac. Agr., Kyushu Univ., 1980, vol. 25, no. 1, pp. 1–8. 17. Bogdanov, A.S., Stakheev, V.V., Zykov, A.E., et al., Genetic variation and differentiation of wood mice 32. Saitoh, M. and Obara, Y., Chromosome banding pat from the genus Sylvaemus inferred from sequencing of terns in five intraspecific taxa of the large Japanese field the cytochrome oxidase subunit 1 gene fragment, Russ. mouse, Apodemus speciosus, Zool. Sci., 1986, vol. 3,
no. 5, pp. 785–792. J. Genet., 2012, vol. 48, no. 2, pp. 186–198. ˆ
18. Saitoh, M., Matsuoka, N., and Obara, Y., Biochemical 33. Vujos evicˆ , M., Rimsa, D., and Zivkovic, S., Patterns of systematics of three species of the Japanese long tailed G and C bands distribution on chromosomes of three field mice, Apodemus speciosus, A. giliacus and A. argen Apodemus species, Z. Säugetierkd., 1984, vol. 49,
teus, Zool. Sci., 1989, vol. 6, no. 5, pp. 1005–1018. pp. 234–238.ˆ 19. Fukushi, D., Kuro o, M., Shichiri, M., et al., Molecu lar cytogenetic analysis of the highly repetitive DNA in 34. Murariua , D. and Stepan, C., The distribution of the constituent heterochromatin and the G banding pat 2 the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus , tern in the genome of Apodemus agrarius (Pallas, 1771) Apodemus (Mammalia, Muridae), Cytogenet. Cell Genet., 2001, vol. 92, nos. 3–4, pp. 254– Trav. Mus. Hist. Nat. “Grigore 263. Antipa”, 1986, vol. 28, pp. 267–270. 20. Suzuki, H., Sato, J.J., Tsuchiya, K., et al., Molecular 35. Kartavtseva, I.V. and Pavlenko, M.V., Chromosome phylogeny of wood mice (Apodemus, Muridae) in East variation in the striped field mouse Apodemus agrarius Asia, Biol. J. Linn. Soc., 2003, vol. 80, pp. 469–481. (Rodentia, Muridae), Russ. J. Genet., 2000, vol. 36, no. 2, pp. 162–174. 21. Radzhabli, S.I. and Borisov, Yu.M., Variants of B chro mosome system in continental forms of Apodemus pen 36. Rubtsov, N.B., Karamysheva, T.V., Kartavtseva, I.V., et insulae (Rodentia, Muridae), Dokl. Akad. Nauk SSSR, al., B chromosomes: DNA, origin, evolution, Biol. 1979, vol. 248, no. 4, pp. 979–981. Membr., 2005, vol. 22, no. 3, pp. 196–211. 22. Volobuev, V.T., B chromosome system of the Asian 37. Rubtsov, N.B., Kartavtseva, I.V., Roslik, G.V., et al., wood mouse Apodemus peninsulae (Rodentia, Features of the B chromosome in Korean field mouse Muridae): 1. The structure of the karyotype, G and C Apodemus peninsulae (Thomas, 1906) from Trans bands and the variation in chromosome number, Gene baikalia and the Far East identified by the FISH tika (Moscow), 1980, vol. 16, no. 7, pp. 1277–1284. method, Russ. J. Genet., 2015, vol. 51, no. 3, pp. 278– 23. Borisov, Yu.M., Geographical variation of the variants 288. of additional chromosomes system in continental forms 38. Matsubara, K., Nishida Umehara, Ch., Tsuchiya, K., of Apodemus peninsulae (Rodentia, Muridae), Izv. Sib. et al., Karyotypic evolution of Apodemus (Muridae, Otd. Akad. Nauk. SSSR, Ser. Biol., 1980, vol. 15, no. 3, Rodentia) inferred from comparative FISH analyses, pp. 61–69. Chromosome Res., 2004, vol. 12, pp. 383–395. 24. Borisov, Yu.M., Variation of cytogenetic structure of the 39. Kuznetsova, I., Podgornaya, O., and Ferguson Smith, Apodemus peninsulae (Rodentia, Muridae) populations M.A., High resolution organization of mouse centro in West Sayany, Genetika (Moscow), 1990, vol. 26, meric and pericentromeric DNA, Cytogenet. Genome no. 8, pp. 1484–1491. Res., 2006, vol. 112, nos. 3–4, pp. 248–255. 25. Abe, S., Han, S.H., Kojima, H., et al., Differential staining profiles of B chromosomes in the East Asiatic 40. Ostromyshenskii, D.I., Kuznetsova, I.S., Komissa wood mouse Apodemus peninsulae, Chromosome Sci., rov, A.S., Kartavtseva, I.V., and Podgornaya, O.I., Tan 1997, vol. 1, pp. 7–12. dem repeats in the rodent genome and their mapping, Cell Tissue Biol., 2015, vol. 9, no. 3, pp. 217–225. 26. Kartavtseva, I.V., Kariosistematika lesnykh i polevykh myshei (Rodentia, Muridae) (Karyosystematics of 41. Hirning, U., Schulz, W.A., Just, W., et al., A compara Wood and Field Mice), Vladivostok: Dal’nauka, 2002. tive study of the heterochromatin of Apodemus sylvati 2 27. Kobayashi, T. and Hayata, I., Revision of the genus cus and Apodemus flavicollis, Chromosoma, 1989, Apodemus in Hokkaido, Annot. Zool. Jpn., 1971, vol. 44, vol. 98, no. 6, pp. 450–455. no. 4, pp. 236–240. Translated by A. Kazantseva
RUSSIAN JOURNAL OF GENETICS Vol. 51 No. 12 2015 SPELL: 1. pericentric, 2. heterochromatin, 3. allopatric