Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.     

Reassignment of chicken W chromosome sequences to the Z chromosome by fluorescence in situ hybridization (FISH)

There is much interest in the gene content of the small heterochromatic W chromosome of the chicken, on the supposition that it may contain sex-determining genes. A considerable region in the chicken genome has been assigned to the W chromosome on the basis of its repetitive sequences. Using fluorescent in situ hybridization (FISH) we localized five Bacterial Artificial Chromosomes (BACs) onto female chicken metaphase spreads. We physically mapped these BACs to the Z chromosome. The chicken genome database, however, assigned all five BACs to the W chromosome. Our results demonstrate that the 17 genes on these BACs are Z-specific, and points to the inadequacy of assigning regions of the genome based exclusively on repetitive sequences.     

Decompactization of chromosome 1 in the artificially decondensed human sperm nuclei: overall topology and non-random location of chromosome bends

We have used FISH with microdissected chromosome arm-specific DNA probes that allow direct visualization of chromosome 1 folding in human sperm nuclei. We described the overall topology and the mode of packaging of sperm chromosome 1 and thus established new elements of ordered genome architecture in these specialized cells.     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

Molecular cytogenetic analysis of the chicken and red-legged partridge chromosome 4 repatterning

Conserved genome homologies between the chicken and partridge have been demonstrated for chromosomes 1 and Z in previous studies. Morphological differences between the chicken and partridge for chromosome 4 have also been identified. The chicken chromosome 4 is submetacentric while the partridge chromosome 4 is acrocentric. We now report that in spite of this morphological difference, both species share extensive homology for chromosome 4 as determined by fluorescent in situ hybridization (FISH). Since only two chromosomes of the partridge karyotype showed FISH signals, our observation suggests that a chromosome rearrangement (peri- or paracentric inversion) has occurred in the partridge chromosome 4.     

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Étude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5–10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics. YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution. We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs. Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.     

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.     

Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes.

Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.     

Chromosome reshuffling in birds of prey: the karyotype of the world's largest eagle (Harpy eagle, Harpia harpyja) compared to that of the chicken (Gallus gallus)

Like various other diurnal birds of prey, the world's largest eagle, the Harpy (Harpia harpyja), presents an atypical bird karyotype with 2n=58 chromosomes. There is little knowledge about the dramatic changes in the genomic reorganization of these species compared to other birds. Since recently, the chicken provides a “default map” for various birds including the first genomic DNA sequence of a bird species. Obviously, the gross division of the chicken genome into relatively gene-poor macrochromosomes and predominantly gene-rich microchromosomes has been conserved for more than 150 million years in most bird species. Here, we present classical features of the Harpy eagle karyotype but also chromosomal homologies between H. harpyja and the chicken by chromosome painting and comparison to the chicken genome map. We used two different sets of painting probes: (1) chicken chromosomes were divided into three size categories: (a) macrochromosomes 1–5 and Z, (b) medium-sized chromosomes 6–10, and (c) 19 microchromosomes; (2) combinatorially labeled chicken chromosome paints 1–6 and Z. Both probe sets were visualized on H. harpyja chromosomes by multicolor fluorescence in situ hybridization (FISH). Our data show how the organization into micro- and macrochromosomes has been lost in the Harpy eagle, seemingly without any preference or constraints.     

Localization of DNA probes for human ribosomal genes on barley chromosomes]

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig |m~ hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced. Received: 25 April 1995 / Accepted: 1 September 1995     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

FISH on avian lampbrush chromosomes produces higher resolution gene mapping

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1–6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.     

Fluorescence in situ hybridization mapping of Avena sativa L. cv. SunII and its monosomic lines using cloned repetitive DNA sequences.

Fluorescent in situ hybridization (FISH) employing multiple probes was used with mitotic or meiotic chromosome spreads of Avena sativa L. cv. SunII and its monosomic lines to produce physical chromosome maps. The probes used were Avena strigosa pAs120a (which hybridizes exclusively to A-genome chromosomes), Avena murphyi pAm1 (which hybridizes exclusively to C-genome chromosomes), A. strigosa pAs121 (which hybridizes exclusively to A- and D-genome chromosomes), and the wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH employing two-by-two combinations of these probes allowed the unequivocal identification and genome assignation of all chromosomes. Ten pairs were found carrying intergenomic translocations: (i) between the A and C genomes (chromosome pair 5A); (ii) between the C and D genomes (pairs 1C, 2C, 4C, 10C, and 16C); and (iii) between the D and C genomes (pairs 9D, 11D, 13D, and 14D). The existence of a reciprocal intergenomic translocation (10C-14D) is also proposed. Comparing these results with those of other hexaploids, three intergenomic translocations (10C, 9D, and 14D) were found to be unique to A. sativa cv. SunII, supporting the view that 'SunII' is genetically distinct from other hexaploid Avena species and from cultivars of the A. sativa species. FISH mapping using meiotic and mitotic metaphases facilitated the genomic and chromosomal identification of the aneuploid chromosome in each monosomic line. Of the 18 analyzed, only 11 distinct monosomic lines were actually found, corresponding to 5 lines of the A genome, 2 lines of the C genome, and 4 lines of the D genome. The presence or absence of the 10C-14D interchange was also monitored in these lines.     

Localization of Human Ribosomal Gene DNA Probes on Barley Chromosomes

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S–28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barleyHordeum vulgareL. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 70% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.     

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F₁ hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.     

BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment

Fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) with large genomic DNA inserts as probes (BAC 'landing') is a powerful means by which eukaryotic genomes can be physically mapped and compared. Here we report a BAC landing protocol that has been developed specifically for the weedy grass species Brachypodium distachyon, which has been adopted recently by the scientific community as an alternative model for the temperate cereals and grasses. The protocol describes the preparation of somatic and meiotic chromosome substrates for FISH, the labeling of BACs, a chromosome mapping strategy, empirical conditions for optimal in situ hybridization and stringency washing, the detection of probes and the capturing and processing of images. The expected outcome of the protocol is the specific assignment of BACs containing single-copy inserts to one of the five linkage groups of the genome of this species. Once somatic or meiotic material is available, the entire protocol can be completed in about 3 d. The protocol has been customized empirically for B. distachyon and its near relatives, but it can be adapted with minor modifications to diverse plant species.     

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.     

Comet-FISH for the evaluation of plant DNA damage after mutagenic treatments

The aim of this study was to perform a comparative investigation of the actions of three mutagens that are widely used in plant mutagenesis using the comet-FISH technique. The comet-FISH technique was used for the analysis of DNA damage and the kinetics of repair within specific DNA sequences. FISH with rDNA and telomeric/centromeric DNA probes was applied to comets that were obtained from an alkaline/neutral comet assay. Migration within specific DNA sequences was analysed after treatment with two chemical mutagens-maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU), and γ-rays. Barley was used as a model plant in this study. The possible utility of specific DNA sequences in a comparative assessment of the distribution of DNA damage within a plant genome was evaluated. This study proved that the comet-FISH technique is suitable for a detailed quantification of DNA damage and repair within specific DNA sequences in plant mutagenesis. The analysis of FISH signals demonstrated that the involvement of specific DNA sequences in DNA damage was different and was dependent on the mutagen used. We showed that 5S rDNA and telomeric DNA sequences are more sensitive to mutagenic treatment, which was expressed by a stronger fragmentation and migration in comparison to the other probes used in the study. We found that 5S rDNA and telomeric DNA probes are more suitable for testing the genotoxicity of environmental factors. A comparison of the involvement of specific chromosome domains in direct DNA breakage/repair and in chromosome aberration formation after mutagen treatment indicates the compatibility of the results.