In situ hybridization of biotinylated DNA probes to cotton meiotic chromosomes

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference filters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes of cotton (Gossypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization. In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resoltion of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.     

New ribosomal RNA gene locations in Gossypium hirsutum mapped by meiotic FISH

In this study we have mapped newly identified rDNA loci in Gossypium hirsutum. Four new minor 18S-26S rDNA loci, in addition to the sites previously identified, were mapped using fluorescence in situ hybridization (FISH) to heterozygous translocation (NT) quadrivalents (IVs). The newly detected 18S-26S rDNA loci were mapped to the right arms of chromosomes 8, 9, 15, 17, 19, 20, and 23 and the left arms of chromosomes 5, 11, 12, and 14. Using the rDNA loci as common reference points, we detected several erroneous arm assignments in the previously published map of NT breakpoints. The data are summarized in the form of an integrated map for all 17 known rDNA loci, relative to centromeres, telomeres, and NT breakpoints. This information will facilitate future locus-specific research on rRNA gene evolution and function. Received: 26 January 1998; in revised form: 28 May 1998 / Accepted: 12 February 1999     

In situ hybridization of iodinated ribosomal RNA to human chromosomes

Iodinated ribosomal RNA was hybridized to human metaphase chromosomes in a test of the effectiveness of iodinated products for in situ hybridization studies in a diploid system. The results indicate that ¹²⁵I is a feasible alternative as a source of radioactivity for autoradiographic mapping studies.     

Integrative mapping of Gossypium hirsutum L. by meiotic fluorescent in situ hybridization of a tandemly repetitive sequence (B77)

We determined the relative positions of the tandem-repeat molecular cytogenetic marker B77, translocation breakpoints, and telosome arms in Gossypium hirsutum cytogenetic stocks by fluorescence in situ hybridization (FISH) analysis of meiotic quadrivalents in 16 single and 2 double translocation heterozygotes and five monotelodisomics. Results delimited the B77 FISH locus to the right arm of the D-subgenome chromosome 14 (14R) and the short arm (14sh), respectively. By equating 14R with 14sh and 14L (left) with 14Lo (long), the findings established a unified nomenclature for the arms of chromosome 14. Previously reported chromosome 14 arm locations were confirmed for four of the five translocations involving chromosome 14, namely NT1L-14L (2780), NT2R-14R (2B-1), NT14L-23R (2777), and NT14R-24R (2781), whereas the location of breakpoint T6L-14L was not confirmed and was reassigned to arm 14R. When used as a probe on Southern blots, the B77 signal was associated with a terminus of the D-subgenome RFLP linkage group (LG) D04 by linkage analysis of an interspecific F(2) population, now known to be chromosome 20. However, additional codominant DNA marker information in the affected region excluded the B77 polymorphism detected by Southern blot hybridization from chromosome 20 and, indeed, from the remainder of the genome.     

Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.     

Complete assignment of the chromosomes of Gossypium hirsutum L. by translocation and fluorescence in situ hybridization mapping

Significant progress has been made in the construction of genetic maps in the tetraploid cotton Gossypium hirsutum. However, six linkage groups (LGs) have still not been assigned to specific chromosomes, which is a hindrance for integrated genetic map construction. In the present research, specific bacterial artificial chromosome (BAC) clones constructed in G. hirsutum acc. TM-1 for these six LGs were identified by screening the BAC library using linkage group-specific simple-sequence repeats markers. These BAC clones were hybridized to ten translocation heterozygotes of G. hirsutum. L as BAC-fluorescence in situ hybridization probes, which allowed us to assign these six LGs A01, A02, A03, D02, D03, and D08 to chromosomes 13, 8, 11, 21, 24, and 19, respectively. Therefore, the 13 homeologous chromosome pairs have been established, and we have proposed a new chromosome nomenclature for tetraploid cotton.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.     

DNA methylation alterations of upland cotton (Gossypium hirsutum) in response to cold stress

DNA methylation plays an important role in regulating gene expression in plants. In the experiment, we studied effects of cold on DNA methylation variation in upland cotton. Using the methylation-sensitive amplified polymorphism procedure, we chose 66 pairs of selective amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, cold triggered the demethylation of hemimethylated or internally full methylated cytosine. With the prolongation of cold treatment, the demethylation loci increased and the methylation loci decreased. Nevertheless, this change could be reverted when cotton was subsequently recovered under normal temperature. In addition, 29 polymorphic bands that appeared in the electrophoretogram were sequenced. By homologous alignment analysis, most of these 29 fragments were identified as genes or DNA clones involved in abiotic stress response. The variation in methylation loci existed at both coding and non-coding regions. Furthermore, the expression of the abiotic stress-related genes, GhCLSD (Seq21), GhARK (Seq22), GhARM (Seq15, Seq18, Seq19 and Seq21) and GhTPS (Seq8), were tested. The results revealed that cold treatment induced down-regulation of GhCLSD, GhARK and GhARM, but up-regulated the expression of GhTPS. These changes were in accordance with the alteration of DNA methylation. Thus, cold may affect the gene expression via changing the methylation status in the cytosine nucleotide.     

Identification of a third fuzzless seed locus in upland cotton (Gossypium hirsutum L.)

Segregating populations were developed to evaluate the inheritance of the fuzzless seed phenotypes in upland cotton (Gossypium hirsutum L.). Accession 143 of the Mississippi Obsolete Variety Collection (MOVC) has a fuzzless seed phenotype. This line carries the n(2) locus which is recessive to the seed fuzz phenotype. Data from the F(2), BC(1)F(1), F(2:3), and BC(1)F(2) populations of DP 5690 x 143 fit a two-loci model for expression of the recessive fuzzless seed phenotype. Fuzzless seeds were obtained in n(2)n(2) plants when a second recessive locus (n(3)) was present. The dominant N(3) allele found in DP 5690 confers the fuzzy seed phenotype in homozygous n(2) plants. Accession 243 of the MOVC carries the N(1) locus, which is dominant to the presence of seed coat fuzz. No variation from expected ratios was observed in the F(2), BC(1)F(1), F(2:3), and BC(1)F(2) populations of the DP 5690 x 243 cross. The N(3) allele had no apparent effect on the expression of the N(1) locus. In a cross between accessions 243 x 143, a few plants were observed which were completely devoid of lint and fuzz fiber (fiberless). A fiberless line was developed from one of these fiberless plants. This line was designated MD 17 fiberless. In a cross between DP 5690 x MD 17 fiberless, we demonstrated that at least three loci were involved in the expression of the fiberless phenotype. The involvement of n(2) and n(3) in the expression of this fiberless phenotype was demonstrated in the F(2) progeny of the cross between 143 x MD 17 fiberless. This is the first demonstration that N(1), n(2), and n(3) interacted to produce fiberless seed.     

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.     

DNA in situ hybridization on polytene chromosomes of Simulium sanctipauli at loci relevant to insecticide resistance

A DNA technique for in situ hybridization developed by Kumar & Collins (1994) for use on polytene chromosomes of adult Anopheles mosquitoes (Diptera: Culicidae) was modified for use with Simulium larval salivary gland chromosomes (Diptera: Simuliidae). Cloned fragments of several Simulium genes (coding for aspartate amino transferase, cytochrome P450 and DNA polymerase) were successfully mapped physically by assigning specific band locations in Simulim sanctipauli V. & D. This represents the first attempt at locating genes beyond the resolution of linkage to inversions in any blackfly species.     

Identification of flow-sorted chromosomes by G-banding and in situ hybridization

The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.     

Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization

 Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25 S rDNA probes obtained by nested PCR. Several hybridization sites were detected per nucleus and an internuclear variability was observed in the number of loci. This is the first report of successful application of FISH to analyse the genomes of glomalean fungi. Accepted: 16 September 1998     

Characterization of supernumerary ring marker chromosomes by fluorescence in situ hybridization (FISH).

Five cases with small supernumerary ring chromosomes are characterized at the molecular level. Routine chromosome banding analysis was insufficient for identification of the ring chromosomes, and none of them was DA/DAPI positive. Fluorescence in situ hybridization utilizing repetitive centromeric probes for all chromosomes has determined that one of these five ring chromosomes originates in each of chromosomes 4, 7, 8, 9, and 20. Chromosome painting with chromosome-specific libraries has confirmed this and excluded the involvement of additional chromosomes in the rearrangements.