Heredity (2010) 104, 393–400 & 2010 Macmillan Publishers Limited All rights reserved 0018-067X/10 $32.00 www.nature.com/hdy ORIGINAL ARTICLE Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement DC Cabral-de-Mello1, RC Moura2 and C Martins1 1Departamento de Morfologia, Instituto de Biocieˆncias, UNESP-Universidade Estadual Paulista, Botucatu, Sa˜o Paulo, Brazil and 2Departamento de Biologia, Instituto de Cieˆncias Biolo´gicas, UPE-Universidade de Pernambuco, Recife, Pernambuco, Brazil Chromosomal banding techniques and repetitive DNA genes, we showed that ribosomal 18S rDNAs are located in mapping are useful tools in comparative analysis and in the chromosomes 3 and 4, whereas 5S rRNA and histone H3 elucidation of genome organization of several groups of genes are colocalized in chromosomal pair 2 and show eukaryotes. In this study, we contributed to the knowledge of an apparently interspersed organization. Moreover, these Coleoptera genomes by reporting the chromosomal organi- genes are not present in the B chromosome, suggesting that zation of repetitive DNA sequences, as well as the presence the B chromosome did not originate from chromosomal pairs and characteristics of a B chromosome in two natural 2, 3 or 4. On the other hand, mapping of the C0t-1 DNA populations of Dichotomius geminatus (Coleoptera; Scar- fraction showed that the B chromosome is enriched in abaeidae) using classical, chromosomal banding and mole- repetitive DNA elements, also present in the standard cular cytogenetic techniques. As in other coleopteran complement, indicating an intraspecific origin of this element species, the heterochromatin was mainly concentrated in in D. geminatus. These results will contribute to our under- pericentromeric regions and the B chromosome was standing of genome organization and evolution of repetitive composed almost entirely of heterochromatin. Physical elements in Coleoptera and other insects regarding both mapping using double fluorescent in situ hybridization was A and B chromosomes. performed for the first time in Coleoptera; using DNA probes Heredity (2010) 104, 393–400; doi:10.1038/hdy.2009.126; for 5S and 18S ribosomal RNA (rRNA) and histone H3 published online 16 September 2009 Keywords: cytogenetics; C0t-1 DNA; FISH; genome evolution; heterochromatin; supernumerary chromosome Introduction retrotransposons, are responsible for the variations in the sizes of eukaryotic genomes (Cavalier-Smith, 1985; Elder Repetitive DNA elements make up a large portion of and Turner, 1995). In particular, repetitive DNAs are of eukaryotic genomes and include tandem arrays and great importance to molecular cytogenetics and represent dispersed repeats. Tandem repeats comprise microsatel- excellent chromosomal markers that are very useful in lite, minisatellite and satellite DNAs (satDNA) (Charles- studies of species evolution, supernumerary chromo- worth et al., 1994) and multigenic families, such as somes, sex chromosomes and for the identification of histones and ribosomal RNAs (rRNAs) (Maxon et al., chromosomal rearrangements; these repetitive sequences 1983; Hadjiolov, 1985). Dispersed repeats are represented are even used in applied genetics. Probes of repeated by transposons and retrotransposons (Charlesworth DNA elements, such as satDNA, rDNA, and to lesser et al., 1994). satDNA has been characterized as highly extent, histones, have been used extensively for tracking abundant and ubiquitous in eukaryotic genomes and is historical and ongoing karyotype repatterning in fishes, located in heterochromatic chromosomal compartments. mammals, mollusks, insects, plants and other groups. These sequences are more variable than the sequences of Repeated DNA elements have found an application in multigenic families and, together with transposons and studies involving supernumerary B chromosomes, which occur in addition to standard karyotypes and are found in B15% of eukaryotic species. These elements are Correspondence: Professor C Martins, Departamento de Morfologia, generally heterochromatic and are composed of repeti- Instituto de Biocieˆncias, UNESP—Universidade Estadual Paulista, tive DNA, mainly satDNA. However, B chromosomes Botucatu, Sa˜o Paulo, 1861 8-000, Brazil. E-mail: [email protected] can also harbor functional genes, such as rRNA genes Received 15 June 2009; revised 5 August 2009; accepted 17 August (Camacho, 2005; Jones et al., 2008). Among coleopterans, 2009; published online 16 September 2009 the presence of B chromosomes has been observed in Mapping of repetitive DNAs in D. geminatus DC Cabral-de-Mello et al 394 several families, such as Buprestidae (Moura et al., 2008), Biosystems, Foster City, CA, USA) with a Dynamic Cantharidae (James and Angus, 2007), Cicindelidae Terminator Cycle Sequencing Kit (Applied Biosystems), (Proenc¸a et al., 2002) and Scarabaeidae (Angus et al., as per the manufacturers’ instructions. Nucleic acid 2007). In Scarabaeidae, analysis of B chromosomes has sequences were subjected to BLAST (Altschul et al., 1990) been restricted to polymorphism characterization using searches at the NCBI website (http://www.ncbi.nlm.nih. conventional staining, and there is little information gov/blast) to check for similarities to other previously about the genomic content of these elements. deposited sequences. The sequences were deposited in the Cytogenetic studies in Coleoptera that focus on NCBI database under the following accession numbers: repetitive sequences are scarce and are frequently GQ443313 (18S rRNA gene), GQ443312 (5S rRNA gene) restricted to chromosomal banding (C-banding), base- and GQ443311 (histone H3 gene). specific fluorochromes and, to a lesser extent, fluorescent An enriched library with repetitive sequences of a 0B in situ hybridization (FISH) using ribosomal DNA individual of D. geminatus was constructed on the basis of (rDNA) or satDNA as probes (Roz˙ek et al., 2004; Bione renaturation kinetics of C0t-1 DNA (DNA enriched for et al., 2005a; Palomeque et al., 2005). With the aim of highly and moderately repetitive DNA sequences) (Zwick contributing to the knowledge of coleopteran genomes, et al., 1997, Ferreira and Martins, 2008). DNA samples we investigated the organization of repeated DNA (200 ml of 100–500 ng mlÀ1 genomic DNA in 0.3 M NaCl) elements in the karyotype of Dichotomius geminatus and were autoclaved for 30 min at 1.4 atmospheres of pressure described the association of 5S rRNA and histone H3 and 120 1C and the fragmented DNA was separated by genes, as well as the characteristics of a newly detected B electrophoresis with a 1% agarose gel. Expected DNA chromosome. fragments should have ranged in size from 100 to 1000 bp. Samples of 50 ml DNA fragments were denatured at 95 1C for 10 min, placed on ice for 10 s and transferred into a Materials and methods 65 1C water bath for reannealing. After 1 min of reanneal- ing, the samples were incubated at 37 1C for 8 min with Animals, DNA samples and chromosome preparation 1 U S1 nuclease to permit digestion of single-stranded Samples of D. geminatus (Arrow 1913) individuals were DNA. The samples were immediately frozen in liquid o 0 00 o 0 00 collected in Igarassu (7 50 03 S:34 54 23 W) (43 males) nitrogen and DNA was extracted with phenol–chloro- o 0 00 o 0 00 and in Maracaı´pe (8 31 47 S:35 01 71 W) (23 males), form. The DNA fragments obtained were used as probes Pernambuco State, Brazil, using pitfall traps. The for FISH in 0B and 1B individuals. genomic DNA of individuals with 0B chromosomes was extracted using the phenol–chloroform procedure Fluorescence in situ hybridization described by Sambrook and Russel (2001). Meiotic chromosomes were obtained from testicular The plasmids containing the 18S rRNA and histone H3 cells. All individuals from Igarassu provided usable genes, and the C0t-1 DNA fraction were labeled by nick preparations, although only eight animals from Mara- translation, using biotin-14-dATP (Invitrogen, San Diego, caı´pe were useful to this study. The rest of the Maracaı´pe CA, USA). For simultaneous hybridization (double sample was not used because the individuals were not at FISH), the 5S rRNA gene clone was labeled using the appropriate stage of sexual maturity. Slides for digoxigenin-11-dUTP (Roche, Mannheim, Germany). conventional chromosome analysis were stained with Meiotic chromosome slides were incubated with RNase (100 mgmlÀ1) for 1.0 h and with pepsin (10 mgmlÀ1) for 2% Lacto-acetic orcein. Slides used for C-banding, silver 1 nitrate staining and FISH analysis were prepared in 45% 20 min in a moist chamber at 37 C. The slides were fixed acetic acid and coverslips were removed after freezing at room temperature using 37% formaldehyde in the preparations by immersing in liquid nitrogen for a phosphate buffer detergent solution and dehydrated in few seconds. C-banding was performed according to the 70 and 100% ethanol for 5 min. The hybridization mixture (100 ng denatured probe, 50% formamide, method described by Sumner (1972) and silver nitrate À1 staining was conducted according to Rufas et al. (1987). 10 mg ml dextran sulfate, 2 Â SSC), in a final volume of 15 ml, was dropped onto slides that were previously denatured using 70% formamide, 2 Â SSC for 40–60 s at Isolation of repetitive DNA 70 1C. In double FISH experiments, 15 ml hybridization Partial sequences of 18S rRNA, 5S rRNA and histone H3 mixture of each