In situ hybridization identifies the diploid progenitor species of Coffea arabica (Rubiaceae)

 The most important commercial coffee species, Coffea arabica, which is cultivated in about 70% of the plantations world-wide, is the only tetraploid (2n=4x=44) species known in the genus. Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) were used to study the genome organization and evolution of this species. Labelled total genomic DNA from diploid species (C. eugenioides, C. congensis, C. canephora, C. liberica) closely related to C. arabica was separately used as a probe in combination with or without blocking DNA to the chromosome spreads of C. arabica. GISH discriminated between chromosomes of C. arabica only in the presence of an excess of unlabelled block DNA from the species not used as a probe. Among the range of different species combinations used, DNA from C. eugenioides strongly and preferentially labelled 22 chromosomes of the tetraploid C. arabica, while the remaining 22 chromosomes were labelled with C. congensis DNA. The similarity of observations between C. arabica and the two diploid species using two ribosomal genes with FISH with respect to metaphase chromosomes provided additional support to the GISH results. These results confirm the allopolyploid nature of C. arabica and show that C. congensis and C. eugenioides are the diploid progenitors of C. arabica. Received: 2 February 1998 / Accepted: 12 May 1998     

Characterization of Aegilops uniaristata chromosomes by comparative DNA marker analysis and repetitive DNA sequence in situ hybridization

RFLP analyses were performed on wheat-Aegilops uniaristata Vis. addition and translocation lines to confirm the identity of added N-genome chromosomes. Complete 1N, 3N, 4N, 5N and 7N chromosome additions were identified, while the complete long arm and only part of the short arm was identified for chromosome 2N. There were no wheat-like 4/5 and 4/7 translocations in the Ae. uniaristata chromosomes. Chromosome 3N carried an asymmetric pericentric inversion, and the translocation line was a product of centric fusion between the long arms of chromosomes 3B and 3N. Chromosome-specific RAPD and microsatellite markers were also identified for all the added Ae. uniaristata chromosomes available in this set of addition lines. A new genomic in situ hybridization protocol combining pre-annealing of probe and blocking DNA and prehybridization with blocking DNA was developed to differentiate the very closely related genomes of Ae. uniaristata and wheat. Hybridization sites for the repetitive DNA sequences pAs1, pSc119.2 and pTa71 were identified on the N-genome chromosomes of Ae. uniaristata using the fluorescent in situ hybridization technique. Results showed deviation from the previously published ideogram of this species. A new ideogram, which shows the hybridization sites for the above sequences, was produced in which the chromosomes are arranged according to their homoeologous group. Received: 23 April 1999 / Accepted: 6 August 1999     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt     

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Genome characterization and relationships between two species of the genusLobelia (Campanulaceae) determined by repeated DNA sequences

Three repeated DNA sequences (rDNA 5S, 18S-5.8S-26S and telomeric repeats) were localised in the genomes ofLobelia brasiliensis andL. imperialis var.kanitzii (subg.Tupa), both with 2n = 28, by fluorescence in situ hybridization (FISH). The results were used to analyse the genomic relationship between the species. With probe pTa71, the karyotypes of these species showed only one NOR site. Probe pTa794, which contains 5S rDNA, demonstrated differences between the species. Telomeric sequences, studied with probe pLT11, were not detected in ectopic sites, but different telomeres showed signals of varying intensity. Based on the results obtained, considerations are made on karyotype evolution inLobelia.     

The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18s-5.8s-25s rRNA genes, 5s rRNA genes,telomere-like sequences,and a family of centromeric repetitive DNA sequences

A knowledge of genome organization is important for understanding how genomes function and evolve, and provide information likely to be useful in plant breeding programmes involving hybridization and genetic manipulation. Molecular techniques, including in situ hybridization, molecular cloning and DNA sequencing, are proving valuable tools to investigate the structure, organization, and diversity of chromosomes in agricultural crops. Heterologous labelled 18 s-5.8 s-25 s (pTa71) and 5 s rDNAs (pTa794) were used for in situ hybridization on Vigna unguiculata (L.) Walp. chromosomes. Hybridization with 18 s-5.8 s-25 s rRNA gene probes occurred at the same chromosomal sites which were positive to the CMA fluorochrome. Silver staining of nucleolar-organizing regions indicated that all the rDNA sites detected using the 18 s-5.8 s-25 s rRNA gene probe possessed active genes. Degenerate telomeric repeats gave hybridization signals at the telomeres of most chromosomes and no intercalary sites were detected at metaphase; the sequences appear to have no preferential distribution in interphase nuclei. A repetitive DraI family from V. unguiculata was cloned (pVuKB1) and characterized. The DraI repeat is 488 nucleotides long, AT rich (74%), and hybridized on all chromosomes in the centromeric areas. The presence of this sequence family was investigated by Southern hybridization in different Vigna species and other Leguminoseae. It was only detected in V. unguiculata, and hence represents a species-specific DNA sequence.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Cytological localization and organization of dispersed middle repetitive DNA sequences of Drosophila subobscura

To study the middle repetitive fraction of the Drosophila subobscura genome, 26 phage clones containing repetitive sequences were examined by Southern DNA blot analysis and by in situ hybridization to polytene chromosomes. These results led to a classification of the clones according to five different types of hybridization patterns. Two types, each containing seven clones, are characterized by hybridization at 100 to 300 sites dispersed over the euchromatic parts of the chromosomes, and in addition by one prominently labelled chromosome band. One of these two classes also showed strong labelling of the chromocentre. The remaining types of hybridization pattern lacked a prominent band but showed hybridization either to the euchromatic regions or to the chromocentre or both. Chromosome A (=X) was the preferred location of prominently labelled bands and it also showed an excess of labelling by some clones. Some of the cloned dispersed sequences were localized cytologically on chromosomes of larvae from crosses between different strains of D. subobscura and between two closely related species, in order to detect heterozygosity at hybridization sites. Comparisons of the chromosomal distribution of labelling sites showed differences in number and location, indicating the possibility of transposition events.     

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome

Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome. Received: 30 May 1997; in revised form: 20 August 1997 / Accepted: 20 August 1997     

In situ hybridization in Actinidia using repeat DNA and genomic probes

 In situ hybridization has been used to probe chromosome spreads of hexaploid Actinidia deliciosa (kiwifruit; 2n=6x=174) and tetraploid A. chinensis (2n=4x=116). When a species-specific repeat sequence, pKIWI516, was used, six hybridization sites were observed in some accessions of tetraploid A. chinensis and all of A. deliciosa. Southern analysis with the pKIWI516 probe revealed that there are two types of tetraploid A. chinensis. Genomic probes from diploid A. chinensis (2n=2x=58) did not differentiate the genomes of hexaploid A. deliciosa and tetraploid A. chinensis, irrespective of the presence or absence of blocking DNA. The results indicate that the genomes of polyploid Actinidia species are similar but not identical. The origin of A. deliciosa is discussed. Received: 29 June 1996 / Accepted: 5 July 1996     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa, representing the four basic genomes of the genus, by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on the chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Hybridization of the pSc119.2 sequence to the chromosomes and Southern blots of digested DNA detected signals in Hordeum bulbosum, Hordeum chilense, Hordeum marinum and Hordeum murinum 4x, but not in Hordeum murinum 2x and Hordeum vulgare ssp. spontaneum. A maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. The species carrying the same I-genome differed in the presence (Hordeum bulbosum) or absence (Hordeum spontaneum) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of Hordeum murinum, but its absence in the diploid cytotype, suggests that the tetraploid is not likely to be a simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences give information about both the interrelationships of the species and the evolution of the repetitive sequences. Received: 17 March 1999 / Accepted: 16 June 1999     

Probing the W chromosome of the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis>, with sequences from microdissected sex chromatin

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.     

Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization

Summary A species-specific, dispersed repetitive DNA sequence was cloned from Nicotiana plumbaginifolia and used in dot blots and in situ hybridizations to analyze asymmetric somatic hybrids of N. tabacum(+)kanamycin-resistant N. plumbaginifolia. Dot blot hybridization data, using the cloned, species-specific repetitive DNA as a probe, showed that some of the hybrids contain only 1%–5% N. plumbaginifolia DNA, whereas others contain 15%–25%. In situ hybridization of the probe to chromosome spreads showed that the extremely asymmetric hybrids retain a single N. plumbaginifolia chromosome; the hybrids with higher dot blot values were found to have 8 to 12 N. plumbaginifolia chromosomes and chromosome fragments. In situ hybridization also revealed translocations between N. plumbaginifolia and N. tabacum chromosomes in 3 of 8 hybrids studied. RFLP analysis using a 5S gene probe showed the presence of N. plumbaginifolia-specific 5S banding patterns in most hybrids examined, including those that retain only a single N. plumbaginifolia chromosome.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

<Emphasis Type="Italic">Agropyron elongatum</Emphasis> chromatin localization on the wheat chromosomes in an introgression line

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F₅ line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1–3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid II-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-03₁₂₉₆ from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels