Recent development of image analysis methods in plant chromosome research

Image analysis methods have provided effective tools in chromosome research along with the development both in computer software and hardware. A chromosome image analyzing system, CHIAS, for plant chromosomes was developed in 1985 and was subsequently revised so that with CHIAS3 one can take advantage of Internet use for downloading the program. In this review, the recent developments of imaging methods in plant chromosome research for automating chromosome identification, constructing a map of a pachytene chromosome, and patterning of interphase nuclei are described.     

Integration of Linkage and Chromosome Maps of Red Clover ( Trifolium pratense L.)

Red clover (Trifolium pratense L.) is a forage legume and an allogamous diploid plant (2n = 14; 440 Mb). Here, we examine the 7 prometaphase chromosomes of red clover using fluorescence in situ hybridization (FISH) with ribosomal RNA sequences, pericentromeric and telomeric repeats, as well as bacterial artificial chromosome (BAC) clones. Position of hybridization signals and chromosome condensation patterns were quantified by the help of the chromosome image analysis system ver. 4.0 (CHIAS IV). Fourteen BAC clones belonging to linkage groups (LG) 1-7 hybridized to individual chromosomes 4, 2, 6, 5, 1, 7, and 3, respectively. Quantitative analysis of FISH mapping and chromosome analysis using CHIAS IV allowed us to construct a quantitative idiogram that constitutes the comprehensive chromosome map of red clover. Chromosomal positions of the 26S rDNA locus were detected at a heterozygous locus on chromosome 6 in the variety HR, and polymorphisms of rDNA loci were observed in other varieties, although chromosomal positions of some BAC clones did not vary among HR and other varieties. These results demonstrate chromosomal collinearity among allogamous red clover varieties. This integration of genetic linkage and quantitative chromosome maps should provide valuable insight into allogamous legume genetics.     

Marker chromosomes commonly observed in the genus Glycine

Summary Soybean [Glycine max (L.) Merr.] chromosomes were analyzed using the chromosome image analyzing system, CHIAS, and seven groups, including subgroups, were identified based on morphological characteristics. Two pairs of chromosomes were conspicuous in their morphological traits. One pair of chromosomes, which had the largest arm ratio among all the chromosomes, was commonly observed in the species in all three subgenera of the genus Glycine. These chromosomes also displayed a unique pattern after N-banding and were detected as marker chromosomes. G. soja, which is considered to be the ancestor of G. max, has two types of marker chromosomes. The lines that carry the same type as G. max may be the ancestors of G. max among the lines of G. soja. The morphological differences of the marker chromosomes within the species in the subgenus Soja are discussed in relation to the domestication process of soybean.     

High-resolution FISH on super-stretched flow-sorted plant chromosomes

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

西藏巨柏核型的图象自动分析与识别的研究

我们应用图象自动分析和识别的原理和方法,建立了植物染色体自动分析CHROMHUK软件系统,并首次对西藏巨柏进行了核型自动分析,抽取了染色体多个特征多数:相对长度、臂比、着丝点指数、相对体密度和次缢痕相对长度.对247个巨柏体细胞进行多参数数据统计和分析,并设置95°.置信判别区域和树分类判别.实现了染色体自动配对和分类,并由计算机直接输出染色体的组型图和核型模式图.分析结果表明:巨柏体细胞的染色体数目为2n=22,按Levan的分类标准,其核型公式为2n=4m(SC)+16m+2sm.据Stabbins分类为1A型.     

Software development of the EMI head scanner.

A method has been developed to run the general purpose operating system RDOS on the same disc of the head scanner computer as is used for scanner software and data. This made it possible to develop additional software in high level programming language for image processing, based on original image data on the disc. All new images produced by the program are stored on the disc in the same format as the original images. This makes it possible to handle processed images exactly as the original ones and to do multiple operations. The following processing has been included in the program so far: subtraction, smoothing, density profiles, vertical reconstructions, magnification and labelling. A set of operator commands has been developed which are very similar to the ordinary commands for the scanner, which makes the program to appear being a direct extension of the standard scanner software.     

High resolution bands in maize chromosomes by G-banding methods

Summary It was demonstrated that G-bands are unequivocally present in plant chromosomes, in contrast to what had been formerly believed by plant cytologists. Maize chromosomes prepared by an enzymatic maceration method and treated with trypsin or SDS showed clear G-bands spreading along the chromosomes. The most critical point during the G-banding procedures was the post-fixation with glutaraldehyde solution. Banding patterns were processed by using the chromosome image analyzing system and a clearer image was obtained. Gbanding technique and the image manipulation method described here can be applied to many plant species, and would contribute new information in the field of plant cytology and genetics.     

Analysis off cereal chromosomes by atomic force microscopy.

Atomic force microscopy has been applied to the study of plant chromosomes from cereal grasses Triticum aestivum (bread wheat), Triticum tauschii, and Hordeum vulgare (barley). Using standard mitotic metaphase squashes, high resolution images have been obtained of untreated chromosomes and also of chromosomes after C-banding, N-banding, and in situ hybridization. The true 3-dimensional nature of the images permits detailed analysis of the surface structure and, on untreated uncoated chromosomes, surface features on a length scale consistent with nucleosome structures have been observed. C+ and N+ regions are manifest as areas of high relief on a slightly collapsed chromosome structure. In situ hybridization leads to a more severe degradation of the native structure, although it is still possible to correlate the optical signal with the topography of the hybridized chromosome. Key words : atomic force microscope, AFM, chromosomes, C-banding, in situ hybridization.     

Automatic karyotyping of plant chromosomes by imaging techniques

Chromosomes of Crepis capillaris (L.) Wallr. were analyzed using a newly developed chromosome image analyzing system, CHIAS-mini. The CHIAS-mini is a desktop system which consists of an ordinary 16-bit personal computer and an image processor as the main frames. Data acquisition, karyotyping and idiogramming of the plant chromosomes were automatically carried out with some manual interaction. The data obtained were comparable to those obtained manually and by a standard image analyzer already developed.     

Computer-assisted karyotyping with human interaction.

A system for machine assisted karyotyping and chromosome analysis has been developed. The system uses a drum- or TV-scanner as input device, runs provisionally in 32 K memory, and also allows human interaction on several stages. The accuracy with which banded chromosomes are karyotyped depends strongly on the type of classifier and varies from 40 up to 80%. The accuracy of the human assisted classifier (98%) comes close to that of a skilled technician (99.5%) using manual chromosomal analysis. Due to technical and memory limitations, the time necessary for the karyotyping of one cell is too long and depends on the interaction time; however karyotyping within 5 min, including human interaction, will be possible in the near future.     

From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.     

Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification

The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region.     

DNA image-fluorimetry of individual human chromosomes

A microfluorimetric method has been developed for determination of DNA content in individual human chromosomes. The method is based on a preliminary identification of chromosomes with Hoechst 33258 followed by staining of the chromosomes with Feulgen reaction by using Schiff’s reagent type ethidium bromide-SO₂ and then by measuring the fluorescence intensity of the chromosomes by using an image analyzer. The method allows determining the DNA content of individual chromosomes with an accuracy up to 4.5 fg. The DNA content of individual human chromosomes and their p-and q-arms, as well as homologous chromosomes, were measured by using the developed method. It has been shown that the DNA content in chromosomes of the normal human karyotype is unstable and can fluctuate in some chromosomes within 35–40 fg.     

Direct preparation and G-banding of chromosomes of mouse liver cells

A method has been developed for the direct preparation and G-banding of chromosomes of mouse liver cells by combining the techniques of liver perfusion and preparation of G-banded chromosomes with partial hepatectomy and colcemid treatment of the animal. The results indicate that cytogenetic investigations of isolated preneoplastic liver cells are possible. The method offers an increased possibility for the use of the liver as an in vivo test system for mutagens and carcinogens.     

Molecular cytogenetic characterization of the Antirrhinum majus genome

As a model system in classical plant genetics, the genus Antirrhinum has been well studied, especially in gametophytic self-incompatibility, flower development biology, and transposon-induced mutation. In contrast to the advances in genetic and molecular studies, little is known about Antirrhinum cytogenetics. In this study, we isolated two tandem repetitive sequences, CentA1 and CentA2, from the centromeric regions of Antirrhinum chromosomes. A standard karyotype has been established by anchoring these centromeric repeats on meiotic pachytene chromosome using FISH. An ideogram based on the DAPI-staining pattern of pachytene chromosomes was developed to depict the distribution of heterochromatin in the Antirrhinum majus genome. To integrate the genetic and chromosomal maps, we selected one or two molecular markers from each linkage group to screen an Antirrhinum transformation-competent artificial chromosome (TAC) library. These genetically anchored TAC clones were labeled as FISH probes to hybridize to pachytene chromosomes of A. majus. As a result, the relationship between chromosomes and the linkage groups (LGs) in Antirrhinum has been established.     

A quick enzyme squash technique for detailed studies on female meiosis in Solanum

A simple enzyme squash technique that enables detailed studies of meiosis in potato ovules has been developed. Fixation of ovules in iron-propionic-ethanol followed by enzymatic maceration and squashing in acetocarmine yielded numerous well preserved megasporocytes with nicely spread chromosomes. Resolution was sufficient, allowing detailed analysis of chromosome pairing and chiasma formation and readily permitting distinction between normal and desynaptic mutant plants. Whereas the use of previously developed ovule squash techniques has been restricted to cytogenetic analyses of plant species with relatively large megasporocytes and large chromosomes, the present technique is potentially more useful for analyses of species with small megasporocytes and small chromosomes.     

Quantitative karyotyping of three diploid Brassica species by imaging methods and localization of 45s rDNA loci on the identified chromosomes

 Chromosomes of the three diploid Brassica species, B. rapa (AA), B. nigra (BB) and B. oleracea (CC), were identified based on their morphological characteristics, especially on the condensation pattern appearing at the somatic pro-metaphase stage. The morphological features of the pro-metaphase chromosomes of the three Brassica spp. were quantified by imaging methods using chromosome image analyzing system II (CHIAS 2). As a result, quantitative chromosome maps or idiograms of the three diploid Brassica spp. were developed. The fluorescence in situ hybridization (FISH) method revealed the location of 45s rDNA (the 26s-5.8s-18s ribosomal RNA gene cluster) on the chromosomes involved. The number of 45s rDNA loci in the B. rapa, B. nigra and B. oleracea are five, three and two, respectively. The loci detected were systematically mapped on the idiograms of the three Brassica spp. Received: 5 September 1997 / Accepted: 6 October 1997     

A Quick Enzyme Squash Technique for Detailed Studies on Female Meiosis in Solanum: Stain Technology

A simple enzyme squash technique that enables detailed studies of meiosis in potato ovules has been developed. Fixation of ovules in iron-propionic-ethanol followed by enzymatic maceration and squashing in acetocarmine yielded numerous well preserved meg-asporocytes with nicely spread chromosomes. Resolution was sufficient, allowing detailed analysis of chromosome pairing and chiasms formation and readily permitting distinction between normal and desynaptic mutant plants. Whereas the use of previously developed ovule squash techniques has been restricted to cytogenetic analyses of plant species with relatively large megasporocytes and large chromosomes, the present technique is potentially more useful for analyses of species with small megasporocytes and small chromosomes.     

Molecular evidence for transcription of genes on a B chromosome in Crepis capillaris

Dispensable, supernumerary (B) chromosomes are found in diverse eukaryotic species. The origin and genetic consequences of B chromosomes have been the subjects of speculation for more than a century. Until now, there has been no molecular evidence that B chromosome DNA is transcribed and there is no unequivocal evidence as to their origin. B chromosomes are considered to be genetically inert although they appear to cause a variety of phenotypic effects. We report that members of one of two ribosomal RNA gene families that are confined to the B chromosomes of a plant, Crepis capillaris, are transcribed--thus providing the first molecular evidence of gene activity on B chromosomes. Sequence analysis of part of the A and B chromosome rRNA genes, together with comparisons with related species, indicates that the B chromosome rRNA genes originate from the A chromosome.