Evolution of satellite DNA sequences in two tribes of Bovidae: A cautionary tale

Two clones, Bt1 from Bos taurus and Om1 from Ovis orientalis musimon, were used as probes for hybridization on genomic DNA and on metaphase chromosomes in members of Bovini and Caprini tribes. Bt1 and Om1 are sequences respectively belonging to the 1.715 and 1.714 DNA satellite I families. Southern blots and fluorescence in situ hybridization experiments showed completely coherent results: the Bovini probe Bt1 hybridized only to members of the Bovini tribe and not to members of Caprini. Likewise, the Caprini probe Om1 hybridized only to members of the Caprini tribe and not to members of Bovini. Hybridization signals were detected in the heterochromatic regions of every acrocentric autosome, except for two pairs of autosomes from Capra hircus that did not show hybridization to probe Om1. No signal was detected on X and Y chromosomes or on bi-armed autosomes. Remarkably, probe Om1 showed almost 100% homology with a bacterial sequence reported in Helicobacter pylori.     

Evidence Against Amplification of Four Genes in Giant Cells Induced by Meloidogyne incognita

Giant-cell DNA was isolated from pea (Pisum sativum) inoculated with Meloidogyne incognita and used in slot blots to test for selective sequence amplification. Four sequences representing low (ribulose 1,5-bisphosphate carboxylase and actin), mid-level (histone 3), and highly repetitive (large ribosomal repeat) sequence DNA were used as probes. Known amounts of root-tip DNA and giant-cell DNA were blotted onto hybridization membranes and probed. The signal strength on autoradiographs containing equal amounts of root-tip DNA and giant-cell DNA were compared with a scanning densitometer. No difference in signal strength between equal amounts of root-tip DNA and giant-cell DNA was found. Thus, for the probes tested, there is no difference in copy number and, hence, no selective DNA sequence amplification has occurred.     

A remarkable autosomal heteromorphism in Pseudoryzomys simplex 2n = 56; FN = 54–55 (Rodentia,Sigmodontinae)

Pseudoryzomys simplex, the false rice rat, is a monotypic genus of the Oryzomyini tribe (Sigmodontinae) distributed in part of Bolivia, Paraguay, Argentina and Brazil. Its diploid number has been described as 56 acrocentric chromosomes decreasing in size and no karyotype figure has been depicted. Herein, we present karyotypic data on P. simplex, including chromosome banding and molecular fluorescent in situ hybridization using telomeric sequences and the whole X-chromosome of its sister clade Holochilus brasiliensis (HBR) as probes. A case of remarkable autosomal heteromorphism due to the presence of a whole heterochromatic arm leading to the variability of FN is reported, as well as the occurrence of regions of homology between the X and Y chromosomes (pseudoautosomal regions) after chromosome painting with the HBR X probe on P. simplex metaphases.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Genomic RFLP Analysis of Meloidogyne arenaria Race 2 Populations

Traditional morphological methods of Meloidogyne identification have been unsuccessful in distinguishing three South Carolina, USA Meloidogyne arenaria race 2 populations—Govan, Pelion, and Florence. These populations differ greatly in reproductive rate and aggressiveness on soybean hosts. Total genomic DNA from eggs of each population was digested with the restriction endonuclease Eco RI and Southern hybridization analyses were performed with single-copy and interspersed multi-copy cloned probes. Probes were isolated from a genomic library of Eco RI, M. arenaria DNA fragments cloned into pUC8. One probe, designated pE1.6A, when hybridized to Southern blots of M. arenaria genomic DNAs, displayed an interspersed repetitive pattern, and the RFLPs distinguished the Govan population from the Pelion and Florence populations. Another clone, pE6.0A, carrying moderately repeated sequences, distinguished the Pelion and Florence isolates. This communication demonstrates the utility of genomic RFLP analysis for distinguishing populations of the same race within the same species. To test the possible utility of these moderately repeated sequence probes for detecting the presence of nematode DNA in DNA samples from roots inoculated with varying numbers of nematodes, dot blot hybridization analyses were performed. It is possible to detect as few as 30 nematodes per root sample with these cloned probes.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.     

Chromosomal evolution in naked catfishes (Bagridae,Siluriformes): A comparative chromosome mapping study

We analyzed the distribution of repetitive DNA sequences on the chromosomes of nine species of the Bagridae from Thailand, i.e., Hemibagrus filamentus; H. nemurus; H. wyckii; H. wyckioides; Mystus atrifasciatus; M. multiradiatus; M. mysticetus; M. bocourti and Pseudomystus siamensis. Two classes of microsatellites and one transposable element (TE) were mapped by fluorescence in situ hybridization. The distribution of the repetitive sequences was comparatively analyzed in view to investigate their contribution in the chromosomal evolution of this fish group. In all species the microsatellites (CA)₁₅ and (GA)₁₅ are abundantly distributed in all chromosomes, usually in the telomeric regions. The retrotransposable element Rex 1 is widely distributed over the whole genome including heterochromatin and euchromatin, but with an unexpected accumulation in one chromosome pair in some species. In fact, some species–specific patterns could be observed considering both microsatellites and TE distribution. The results demonstrated that the compartmentalization of repeated elements is not simply restricted to heterochromatic regions, as it has been postulated in the first concepts of the genomic organization of repetitive elements in genomes. Moreover, the organization of these repeats seems to reflect their intense and specific evolutionary pathway, providing new insights about the chromosomal evolution in the Bagridae.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

Background and Aims

Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.

Methods

In order to isolate highly repetitive sequences, a c₀t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).

Key Results

Characterization of the c₀t-1 library resulted in the detection of repetitive sequences including the (GA)_n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.

Conclusions

The repeated sequences isolated from D. moschatum c₀t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.     

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

Intra- and Interpopulation Genome Variation In Meloidogyne arenaria

The genetic heterogeneity of two M. arenaria race 2 populations (designated Pelion and Govan) was examined using RFLP analysis of 12 clonal lines established from single egg masses (six distinct clonal lines from each population). These populations are essentially identical by traditional biochemical and race identification schemes; however, the Govan population is more aggressive than the Pelion population, producing larger galls and exhibiting greater reproductive capabilities on many soybean cultivars and experimental accessions. Variation at the genomic DNA level was examined using probes representative of expressed DNA sequences present in the eukaryotic genome. Ribosomal DNA, interspersed repeated sequences, and cDNA probes were tested for detection of polymorphism within and between single egg mass lines of each population. Cloned cDNAs and ribosomal intergenic spacer sequences detect polymorphism both within and between populations, demonstrating the usefulness of these sequence classes for molecular genetic analysis of population structure and genome evolution.     

Differential detection of transposable elements between Saccharum species

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 – 16x = 40 – 128). These accessions have 100 to 130 chromosomes, 80–85% of which are derived from S. officinarum, 10–15% from S. spontaneum, and 5–10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.     

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.     

Genomic Differences among Pathotypes of Bursaphelenchus xylophilus

Total genomic DNA from Bursaphelenchus xylophilus pathotypes MPSy-1 and VPSt-1 and from B. mucronatus was digested with restriction endonucleases. DNA fragments were electrophoretically separated, Southern blotted to nitrocellulose, and hybridized to genomic DNA from one of the isolates. The resulting hybridization patterns indicate genomic differences in repetitive DNA sequences among these populations. Greatest differences were seen between B. xylophilus and B. mucronatus, but genomic differences were also apparent between B. xylophilus pathotypes MPSy-1 and VPSt-1 and between a population from P. nigra in New Jersey and a population of a mucronate form from Abies balsamea in Quebec, Canada.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect

Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C_ot-1 DNA). Contrary to expectation, we demonstrated that C_ot-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C_ot-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C_ot-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using C_ot-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of C_ot-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for C_ot-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.     

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years.

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.     

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes ¹. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions ^2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes ^{4, 5, 6}. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti ⁷ and Cx. quinquefasciatus ⁸, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates ⁹. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti ¹⁰, the accumulation of multiple chromosomal rearrangements in cell line chromosomes ¹¹ makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4^th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol ¹² is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.