Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo.

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F1 hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat x maize hybrid embryos.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides

Clusters of four simple sequence repeats (SSRs), AAC, AAG, AG and CAT, have been mapped physically to hexaploid wheat chromosomes; 15—24-bp synthetic oligonucleotides were labelled by random-primer labelling and used as probes for fluorescent in situ hybridization with standard formamide and low-salt conditions. AAC hybridized strongly to the pericentromeric regions and several intercalary sites of all seven chromosomes of the B-genome corresponding to N bands and enabling their identification. Most of the AAC sites also co-localize with AAG, although the strength of the AAC and AAG signal was often different at the same location. Not all heterochromatic bands showed AAC signals and a few AAC sites were detected that are neither AAG nor N band positive, revealing the complex and heterogeneous genome organization of wheat and identifying the four most frequent classes of banded chromatin. Clusters characterised by a high concentration of AG repeats were detected on chromosome arms 3BS, 4BL, 5BS and 5BL, adjacent to AAG sites. The only detectable CAT cluster was found on chromosome arm 3BL, making this oligonucleotide valuable in identifying this particular chromosome. SSR in situ hybridization is useful as a diagnostic tool in cytogenetics and for understanding genome organization in wheat. Received: 21 September 1999 / Accepted: 19 March 2000     

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization

Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E^b of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E^b short arm and four are located on the 5E^b long arm. These RAPD markers have been used to confirm the identity of putative 5E^b (5A) and 5E^b (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.     

An improved method of genomic in situ hybridization (GISH) for distinguishing closely related genomes of tetraploid and hexaploid wheat species

An improved modification of genomic in situ hybridization (GISH) was proposed. It allows clear and reproducible discrimination between closely related genomes of both tetraploid and hexaploid wheat species due to preannealing of labeled DNA probes and prehybridization of chromosomal samples with blocking DNA. The method was applied to analyze intergenomic translocations 6A:6B and 1A:6B identified in the IG46147 and IG116188 samples of tetraploid wheat Triticum dicoccoides by C-banding. The structure of the rearranged chromosomes was defined for two translocation variants, and the breakpoints were identified on the chromosome arms. Possible application of the developed GISH variant to study genome reorganizations during speciation of allopolyploid plants in evolution is discussed.     

Ultrastructural detection of ribosomal RNA by in situ hybridization using a biotinylated probe on ultrathin sections of cultured animal cells

A genomic probe containing ribosomal sequences and labelled with biotin was used to hybridize rRNA molecules in ultrathin sections of animal cells embedded in Lowicryl K4M. After detection with streptavidin conjugated with 10 nm gold particles, ribosomal target sequences were localized preferentially in the dense fibrillar component of the nucleolus and in the polyribosome structures of the cytoplasm. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression at the ultrastructural level, particularly under different physiological and pathological conditions.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.     

QTL analysis of bread-making quality in wheat using a doubled haploid population

A set of 187 doubled haploid lines derived from the cross between cvs. Courtot and Chinese Spring was explored for QTLs for three bread-making quality tests: hardness, protein content and strength of the dough (W of alveograph). The scores of the parental lines were quite different except for protein content, and the population showed a wide range of variation. About 350 molecular and biochemical markers were used to establish the genetic map, and technological criteria were evaluated in 1 to 3 years. QTL detection was performed by the ”marker regression” method. The most significant unlinked markers were used in the model as covariates, and the results were tested by bootstrap resampling. For hardness, we confirmed a previously tagged major QTL on chromosome 5DS, and two additional minor QTLs were found on chromosome 1A and 6D, respectively. For protein content two main QTLs were identified on chromosomes 1B and 6A, respectively. For W, three consistent QTLs were detected: two at the same location as those for hardness, on chromosomes 1A and 5D; the third one on chromosome 3B. Therefore, it appeared that except for the Glu-1A locus, storage protein loci were not clearly involved in the genetic control of the criteria studied in the present work. Despite the reasonable size of the population no QTL with interactive effects could be substantially established as measured. All computations were carried out using home-made programmes in Splus language, and these are available upon request. Received: 16 May 1999 / Accepted: 15 October 1999     

Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n=42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。     

Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene

The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar ’Bobwhite’ by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T₁ progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T₁ plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T₁, T₂ and T₃ generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP. Received: 15 December 1998 / Accepted: 30 January 1999     

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.     

Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.

A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.     

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.