Comparative FISH Analysis in Five Species of Eyprepocnemidine
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Heredity (2003) 90, 377–381 & 2003 Nature Publishing Group All rights reserved 0018-067X/03 $25.00 www.nature.com/hdy Comparative FISH analysis in five species of Eyprepocnemidine grasshoppers J Cabrero1, A Bugrov2,3, E Warchałowska-S´liwa4,MDLo´pez-Leo´n1, F Perfectti1 and JPM Camacho1 1Departamento de Gene´tica, Universidad de Granada, 18071 Granada, Spain; 2Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, 630091 Novosibirsk, Russia; 3Novosibirsk State University, 630090 Novosibirsk, Russia; 4Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, 31-016 Krako´w, Poland The chromosomal localization of ribosomal DNA, and a species, however, lacked the 180 bp tandem repeat, and 180 bp satellite DNA isolated from Spanish Eyprepocnemis showed rDNA clusters in one (S9 in Thisoicetrinus pteros- plorans specimens, has been analysed in five Eyprepocne- tichus), two (S9 and S10 in Eyprepocnemis unicolor;M8 and midinae species collected in Russia and Central Asia. S11 in Heteracris adspersa), or three (S9,S10, and S11 in Caucasian E. plorans individuals carried each of the two Shirakiacris shirakii) chromosome pairs. The implications of DNAs, but the rDNA was limited to only two chromosomes these findings for the evolution of these two chromosome (S9 and S11) in sharp contrast to Spanish specimens that markers in this group of species are discussed. show 4–8 rDNA clusters and to Moroccan specimens which Heredity (2003) 90, 377–381. doi:10.1038/sj.hdy.6800255 carry rDNA in almost all chromosomes. The four remaining Keywords: rDNA; satDNA; heterochromatin; grasshopper; nucleolus organizer region Introduction change (Maggini et al, 1991), and the amplification of minor rDNA loci (Dubcovsky and Dvo¨rak, 1995). As the genome era progresses, it is being more apparent The presence of satDNA is characteristic of most that eukaryote genomes contain a significant proportion eukaryote organisms. It is restricted to heterochromatic of repetitive DNA of various types. Two of the tandem chromosome regions and is typically noncoding. It repetitive DNA classes, that is, ribosomal (rDNA) and usually consists of a tandemly repeated unit of size satellite DNA (satDNA), are very different in genetic role varying, in most cases, from 10 to 100 bp (Charlesworth (rDNA is very important functionally, but satDNA seems et al, 1994). to be nonfunctional) but they share some evolutionary SatDNA constitutes a rapidly evolving marker, which properties (eg concerted evolution). makes it useful for phylogenetic studies. Its presence or Ribosomal DNA is arranged in tandem arrays (con- absence, and the divergence of sequences shared among taining transcriptional units coding for three of the four species, may be powerful tools, throwing light on the rRNA types) located at one or more chromosome evolutionary relationships among closely related species. regions, the so-called nucleolus organizer regions Such information has proven to be useful in a variety of (NORs). NORs are evolutionarily dynamic, which makes organisms, for example, insects (Bachmann and Sperlich, them excellent markers for phylogenetic studies (Cabrero 1993; Juan et al, 1993; Bachmann et al, 1994; Pons et al, and Camacho, 1986; Amemiya and Gold, 1988; Santos 1997), fishes (Garrido-Ramos et al, 1995; de la Herra´n et al, and Fox, 1988). Interspecies comparison of NOR number 2001; Lanfredi et al, 2001), birds (Madsen et al, 1992), and chromosome location may be a good tool for mammals (Hamilton et al, 1992; Wijers et al, 1993; macroevolutionary studies. Intraspecific variation, more- Volobouev et al, 1995; Lee et al, 1999), and plants (Galasso over, when detected, is indicative of the complex et al, 2001). microevolutionary patterns shown by rDNA. The most The grasshopper Eyprepocnemis plorans harbours a B remarkable conclusion of rDNA research at these two chromosome polymorphism which has become a para- levels is that NORs seem to be able to spread through the digm of the long-term evolution of parasitic B chromo- genome thus creating new rDNA loci (Castro et al, 2001). somes, since it has provided clear evidence for an arms Several spreading mechanisms have been suggested, for race between the coevolving A and B chromosomes example, transposition (Schubert and Wobus, 1985), (Camacho et al, 1997). These B chromosomes are mainly insertion of extrachromosomal rDNA amplified during made up of two repetitive DNAs, that is rDNA and a oogenesis (Phillips et al, 1988), the presence of repetitive 180 bp satDNA, which are also present in many A elements facilitating nonhomologous chromosome ex- chromosomes (Lo´pez-Leo´n et al, 1994, 1995b; Cabrero et al, 1999). To ascertain the origin of these B chromo- Correspondence: JPM Camacho, Departamento de Gene´tica, Universidad somes is, therefore, necessary to investigate the presence de Granada, 18071 Granada, Spain. E-mail: [email protected] and chromosome distribution of these two repetitive Received 31 July 2002; accepted 29 January 2003 DNAs in geographically distant E. plorans populations as Comparative FISH analysis J Cabrero et al 378 well as in some other closely related species, since such a Results study might uncover the ancestral patterns and the evolutionary pathways that could explain the present As described elsewhere (Bugrov et al, 1999), the five chromosome distribution. In this paper, we analyse the species show 2n ¼ 22+X0#/XX~ acrocentric chromo- presence and chromosome localization of these two somes, with two long (L1 and L2), six medium (M3–M8) repetitive DNAs in five Eyprepocnemidine species from and three short (S9–S11) autosomes, and the X chromo- Russia and Central Asia, including E. plorans. some showing a size similar to that of the longest M autosomes. Caucasian males of E. plorans show two rDNA clusters located on the S9 and S11 autosomes (Figure 1a). The Materials and methods 180 bp tandem DNA repeat, however, is located on A total of 24 adult male grasshoppers belonging to five paracentromeric regions of the X chromosome and eight Eyprepocnemidinae species, collected at several localities autosomal bivalents (excluding M8,S9, and S10) (Figure in Russia and Central Asia (see locations in Table 1 in 1a,b). Note that S11 is the only standard chromosome Bugrov et al, 1999), were analysed by fluorescence in situ carrying the two repetitive DNAs. None of the four other hybridization (FISH) and silver impregnation. Two of the Eyprepocnemidinae species carried the E. plorans repe- species analysed belonged to the genus Eyprepocnemis (E. titive DNA, suggesting that it is species specific. In plorans, five males collected at Daghestan, North Cauca- consistency with rDNA location, silver impregnation sus, and E. unicolor, six males collected at Tajikistan), the showed active NORs only in bivalents S9 and S11 three other species being Heteracris adspersa (five males (Figure 2a). from Daghestan), Thisoicetrinus pterostichus (five males In the remaining species, the rDNA was located on a from Daghestan), and Shirakiacris shirakii (three males variable number of bivalents: one in Thisoicetrinus from Primorskij kray). pterostichus (S9) (Figure 1e), two in Eyprepocnemis unicolor Chromosome preparations were made by squa- (S9 and S10) (Figure 1c) and Heteracris adspersa (M8 and shing two testis follicles in 50% acetic acid. After S11) (data not shown), and three in Shirakiacris shirakii (S9, 10 min, the coverslip was removed with a razor blade S10, and S11) (Figure 1d). Silver impregnation showed after freezing the preparation by immersion in liquid that all rDNA clusters were active in all four species nitrogen for a few seconds. Silver impregnation was (Figure 2; data not shown for T. pterostichus). performed following the technique described by Rufas et al (1982). Discussion For FISH, in order to eliminate cell cytoplasm and thus facilitate probe accessibility, each slide was incubated in Species-specific satDNA has been found in some cases, 150 ml of pepsin (50 mg/ml in 0.01N HCl) at 371Cina for example, the cave cricket Dolichopoda schiavazzii humid chamber for 10–30 min. After three washes in (Bachmann et al, 1994). In these cases, this marker distilled water, the slides were dehydrated in an ethanol provides very useful information for microevolutionary series at 70, 90, and 100% for 3, 3, and 5 min, respectively, studies. When the same satDNA is shared by other and then were air dried. Slides were stored at 601C species, however, it becomes a valuable marker for overnight before in situ hybridization. phylogenetic studies. For example, it may be shared by FISH was performed with two different DNA probes: part of a genus (eg in Tribolium flour-beetles, Juan et al, pTa71, which contains a 9-kb EcoRI repeat unit of rDNA 1993), a complete genus (eg Peromyscus rodents, Hamil- isolated from Triticum aestivum (Gerlach and Bedbrook, ton et al, 1992, Pimelia beetles, Pons et al, 1997, and Lens 1979), and pEpD15, which contains a 180 pb DraI legumes, Galasso et al, 2001), several genera in a same fragment of a tandemly repetitive DNA isolated from family (eg the RBMII sequences in the avian Anatidae E. plorans (Lo´pez-Leo´n et al, 1994, 1995b). The probe family, Madsen et al, 1992, and the DraI satDNA in the DNA was labelled by nick translation with Fluorogreen Sparidae fish family, de la Herra´n et al, 2001), and even a 11-dUTP or Fluorored 11-dUTP, using standard techni- whole family (eg the EcoRI satDNA in the Sparidae fish ques. FISH was performed following the technique family, de la Herra´n et al, 2001) or order (eg the human described in Lo´pez-Leo´n et al (1994). In brief, the two gamma-X centromeric satellite DNA, which seems to be DNA probes were mixed to a final concentration of 5 ng/ widespread among primates, Lee et al, 1999). ml in a solution containing 40% formamide, 10% dextran The 180 bp tandem DNA repeat isolated from Spanish sulfate, 0.1% sodium dodecyl sulphate (SDS) in 1 Â SSC, specimens of E.