Genome relationships in the genus Dasypyrum: evidence from molecular phylogenetic analysis and in situ hybridization

The genus Dasypyrum (or Haynaldia) consists of two species, D. villosum and D. breviaristatum. However, the genomic relationships between these two species remain unclear. The objective of this study was to provide molecular phylogenic and cytological evidence on the evolutionary relationships of the genus Dasypyrum. Sequences of Chloroplast DNA (cpDNA) and α-gliadin genes both support the hypothesis that diploid D. breviaristatum is the progenitor of tetraploid D. breviaristatum, and the diploid D. villosum and D. breviaristatum evolved parallel from an ancestral species. Genomic and fluorescence in situ hybridization using ribosomal DNA and rye repetitive DNA sequence as probes also indicated that tetraploid D. breviaristatum originated from diploid D. breviaristatum.     

Genomic relationships among diploid and polyploid species of the genus Eryngium L. using genomic in situ hybridization

Eryngium L. (Umbelliferae) is a large genus including more than 250 species worldwide. The large morphological variability in this genus makes it difficult to delimit the species or to establish phylogenetic relationships. The occurrence of different ploidy levels within the genus might indicate a hybrid origin of the polyploid species. In the present study, the chromosome number and karyotype of E. regnellii are reportedfor the first time and the ploidy level of a population of E. paniculatum is confirmed. We compare the genomes of the diploids E. horridum and E. eburneum, the tetraploids E. megapotamicum and E. regnellii, and the hexaploids E. pandanifolium (as a representative of the whole pandanifolium complex) and E. paniculatum using genomic in situ hybridization (GISH). Although it was not possible to identify the parental species of the polyploid taxa analyzed, the GISH technique allowed us to postulate some hypotheses about their origin. Eryngium horridum and E. eburneum do not seem to be the direct progenitors of the polyploids analyzed. On the other hand, it seems that other diploid species unrelated to E. horridum and E. eburneum are involved in their origin. Our results are consistent with morphological and phylogenetic studies, indicating a close relationship between the species of the series Latifolia.     

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     

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes.

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.     

Genomic origin and organization of the allopolyploid Primula egaliksensis investigated by in situ hybridization

Background and Aims: Earlier studies have suggested that the tetraploid Primula egaliksensis(2n = 40) originated from hybridization between the diploidsP. mistassinica (2n = 18) and P. nutans (2n = 22), which werehypothesized to be the maternal and paternal parent, respectively.The present paper is aimed at verifying the hybrid nature ofP. egaliksensis using cytogenetic tools, and to investigatethe extent to which the parental genomes have undergone genomicreorganization. Methods: Genomic in situ hybridization (GISH) and fluorescent in situhybridization (FISH) with ribosomal DNA (rDNA) probes, togetherwith sequencing of the internal transcribed spacer (ITS) regionof the rDNA, were used to identify the origin of P. egaliksensisand to explore its genomic organization, particularly at rDNAloci. Key Results: GISH showed that P. egaliksensis inherited all chromosomes fromP. mistassinica and P. nutans and did not reveal major intergenomicrearrangements between the parental genomes (e.g. interchromosomaltranslocations). However, karyological comparisons and FISHexperiments suggested small-scale rearrangements, particularlyat rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromaticknobs characteristic of the maternal parent P. mistassinicaand maintained only the rDNA loci of P. nutans. These resultscorroborated sequence data indicating that most ITS sequencesof P. egaliksensis were of the paternal repeat type. Conclusions: The lack of major rearrangements may be a consequence of theconsiderable genetic divergence between the putative parents,while the rapid elimination of the ITS repeats from the maternalprogenitor may be explained by the subterminal location of ITSloci or a potential role of nucleolar dominance in chromosomestabilization. These small-scale rearrangements may be indicativeof genome diploidization, but further investigations are neededto confirm this assumption.     

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.     

Screening of cloned recombinant DNA in bacteria by in situ colony hybridization.

We have developped in situ methods of colony hybridization in which there is no need to replicate colonies one by one prior to hybridization. The best method consists in promoting partial lysis of the colonies on the plates by means of a resident thermoinducible prophage. It appears that colonies are heterogeneous with respect to prophage induction, so that survivors remain in each colony. Blotting onto nitrocellulose filters and hybridization with a highly radioactive probe permits the screening of many thousands of colonies per plate for the presence of a DNA sequence carried by a plasmid and complementary to the probe. This procedure greatly facilitates the isolation of recombinant plasmids which carry a specific DNA sequence. We also describe a second, less efficient procedure which does not use prophage induced lysis, and is potentially usable with B2 or EK2 safety systems, without modification of the bacterial hosts.     

Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization

Summary We report a general procedure which allows the application of whole cosmid cloned genomic sequences for non-radioactive in situ hybridization. The presence of highly repetitive sequences, like Alu and Kpn fragments, is eliminated through competition hybridization with Cot-1 DNA. The method has been tested and optimized with several randomly chosen cosmids of the human thyroglobulin (Tg) gene (8q24). At present, the procedure can be performed with three of the four tested individual cosmids. In cases where a single clone does not result in a specific signal, a larger fragment may be required, which can be accomplished by using two (partially overlapping) cosmids of the same region. The advantages and further potentialities of such a hybridization approach are discussed.     

Restriction endonuclease recognition and Southern hybridization of bromodeoxyuridine-substituted genomic DNA

Abstract. Human glioma cell lines exposed to various concentrations of bromode-oxyuridine (BrdUrd) were studied to determine the effect of BrdUrd substitution on restriction endonuclease recognition and Southern hybridization of genomic DNA. BrdUrd substitution had no effect on the recognition of restriction endonucleases. When the exposure to BrdUrd was 2 h or less and the BrdUrd substitution rate was less than 40%, there was no difference in the density of hybridized bands after Southern hybridization using human non-recombinant complementary DNA as a probe. Hybridization was suppressed significantly by exposures longer than 24 h or BrdUrd substitution rates greater than 40%. These results suggest that the BrdUrd substitution rate and the exposure time to BrdUrd influence the hybridization reaction by a DNA probe. Brief exposure (up to 2 h) to BrdUrd does not influence restriction endonuclease recognition or Southern hybridization of genomic DNA.     

Intergenomic translocations of polyploid oats (genus Avena) revealed by genomic in situ hybridization

Wild and cultivated hexaploid oats share the same genomes (AACCDD) and display a considerable level of interspecific variation in both plant and chromosome morphology. The GISH was utilized to detect the interspecific genomic compositions in four hexaploid and two tetraploid oats using total genomic DNA of Avena eriantha (a C-genome diploid) as probe. Intergenomic translocations between A/D and C-genome chromosomes were frequently observed in hexaploid and tetraploid species. In the hexaploid, two pairs of A/D genome segments on C-genome chromosome (A/D-C) translocation and four to six pairs of C-genome segments on A/D genome chromosome (C-A/D) translocation were clearly identified whilst the number of A/D-C translocations was constant among species. In the tetraploid A. maroccana (AACC), a pair of A-C and four pairs of C-A translocations were observed. Moreover, the A/D translocation segments on chromosome 5C was detected only in A. byzantina and A. maroccana, whilst A/D-C translocations were observed on the 1C and 7C of A. sativa, A. fatua and A. sterilis. A. byzantina did however also carry the 1C rearrangement. This result shows that A. byzantina has retained a similar genomic constitution to the tetraploid ancestor of hexaploid oats, A. maroccana. Three pairs of A-C translocations were detected only in A. murphyi (AACC), and two pairs of those were the 1C and 7C as well as the three hexaploid species except A. byzantina.     

Limitations of in situ hybridization with total genomic DNA in routine screening for alien introgressions in wheat

In situ hybridization with total genomic DNA (GISH) has become a powerful tool in characterization of alien introgressions in wheat. With recent simplification it can now be used in large scale screening for new chromosome constructs. Its level of resolution in routine applications was tested on sets of recombined wheat-rye chromosomes with genetically determined positions of the translocation breakpoints. The resolution level of GISH visualized by an enzymatic color reaction was much lower than that of GISH with fluorescent probes but both techniques failed to reveal the presence of some distally located breakpoints. The limits of resolution for the two methods were at least 9.8 and 3.5 cM of the relative genetic lengths of chromosome arms, respectively, in configurations with proximal rye and terminal wheat segments when rye DNA was used as a probe. When wheat DNA was used as a probe, a terminal wheat segment estimated to be ca. 1.6 cM in length could not be visualized. An example of induced recombination between a chromosome of Agropyron elongatum and wheat illustrates that these resolution limits of GISH may hamper isolation of critical translocation breakpoints in a chromosome engineering effort.     

Genomic sequencing reveals gene content,genomic organization,and recombination relationships in barley

Barley (Hordeum vulgare L.) is one of the most important large-genome cereals with extensive genetic resources available in the public sector. Studies of genome organization in barley have been limited primarily to genetic markers and sparse sequence data. Here we report sequence analysis of 417.5 kb DNA from four BAC clones from different genomic locations. Sequences were analyzed with respect to gene content, the arrangement of repetitive sequences and the relationship of gene density to recombination frequencies. Gene densities ranged from 1 gene per 12 kb to 1 gene per 103 kb with an average of 1 gene per 21 kb. In general, genes were organized into islands separated by large blocks of nested retrotransposons. Single genes in apparent isolation were also found. Genes occupied 11% of the total sequence, LTR retrotransposons and other repeated elements accounted for 51.9% and the remaining 37.1% could not be annotated. Electronic Publication     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes

In situ DNA hybridization using biotin-labelled leptospiral DNA was performed on clinical specimens to investigate its usefulness as a technique for the identification of Leptospira interrogans. The applicability of this test in blood, urine and liver smears was demonstrated. In situ DNA hybridization can be completed in only 4 h and it combines the advantage of visualization of the leptospiral morphology with the specificity of the hybridization reaction. No cross-hybridization was observed with other bacteria. This study shows that hybridization in situ can be simple to perform and may contribute to a rapid diagnosis.     

Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization

 Three lines of the tetraploid wheat Aegilops ventricosa Tausch (2n=4x=28), which contains good resistance to eyespot, were analysed using fluorescent in situ hybridization. Probes used included rDNA, cloned repeated sequences from wheat and rye, simple-sequence repeats (SSRs) and total genomic DNA. The banding patterns produced could be used to distinguish most chromosome arms and will aid in the identification of Ae. ventricosa chromosomes or chromosome segments in breeding programmes. All lines had a single major 18S-25S rDNA site, the nucleolar organizing region (NOR) in chromosome 5N and several minor sites of 18S-25S rDNA and 5S rDNA. A 1NL.3DL, 1NS.3DS translocation was identified, and other minor differences were found between the lines. Received: 11 August 1998 / Accepted: 28 November 1998     

Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci. Received: 19 November 1996; in revised form: 10 January 1997 / Accepted: 10 January 1997     

Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human x Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.     

Sexing of porcine embryo by in situ hybridization using chromosome Y- and 1-specific DNA probes

This study was carried out to determine if a rapid, simultaneous detection system using chromosome Y- and 1-bearing boar spermatozoa was applicable for sexing embryos. Porcine embryos were recovered from gilts and sows 4 to 6 d after mating, and whole embryos or biopsy cells were mounted on a glass slide with a small amount of fixative (methanol: acetic acid: distilled water = 9:1:4). The samples were then stained by means of a fluorescence in situ hybridization (FISH) procedure developed specifically for the detection of Y-bearing spermatozoa. Hybridization was performed using digoxigenin (dig)-labeled chromosome Y- specific DNA, and biotin-labeled chromosome 1-specific DNA sequences were detected as a signal of FITC and Texas Red on nucleus visualized DAPI-stain. Proportions of whole embryos labeled with chromosome 1-probe were 17 and 97% at the 3 to 16 and > or = 32 cell stage, respectively. Of the 93 biopsied embryos analyzed by FISH, 85 embryos (91%) could be accurately classified as male or female. Of the 65 biopsied embryos, 60 embryos (92%) had a clear blastocoele and a inner cell mass after 48 h of culture in vitro, and these embryos were evaluated as available embryos. One out of 4 recipient gilts which received sexed embryos at transfer farrowed 12 piglets of the expected sex. The results of this study demonstrated that porcine embryos at the > or = 32 cell stage can be sexed within 2 h using the FISH method. Moreover further development of the FISH technique could make it an effective tool for the study of early porcine embryos and for the control of porcine sex.