1 INTRODUCTION
Human visual cues are estimated to account for 70%‒80% of all external stimuli received by humans from their surroundings. As a result, information that can be perceived visually is important in the field of quantitative biology. Although visual information is generally extensive and difficult to understand, scientists have continually sought different approaches to present such information by converting it to numerical values and measurements such as length, angle and area, and the type of visual information used. Some visual information, such as chromosome structure, is particularly difficult to evaluate without a subjective measure of the available information. Consequently, analyses of visual information have posed a challenge for scientists. Because only biological expert researchers can evaluate results with their naked eye, objective evaluation methods are highly demanded. The computer technology has been rapidly developing since the late 20th century. In fact, computers have become very helpful, sometimes even indispensable, tools for research in the field of image analysis; this is because they can be used to process visual information objectively. In the past, due to the high cost and need for dedicated instruments, only few researchers had access to systems for digital image analysis. As image analysis is now popular and necessary in several biology fields, it is important to understand the implications of this approach.
The development of chromosome karyotyping using image analysis is important for genetic evolution and diversification. In fact, a detailed chromosome map is thought to contribute to the plant breeding, and the study of fundamental biology and genetics of organisms. Chromosome mapping is carried out with a locus that allows the band to appear on a chromosome using differential staining techniques, such as G-band or C-band [
1]. Forty-six human chromosome identifications representing clear G-bands treated with trypsin have been established [
2,
3]. Rely on the varying genome sizes among plants species, although individual banding patterns show on the chromosomes of important crops,
e.g., wheat (average genome size, 807 Mb/each chromosome [
4]), barley (1026 Mb), and field bean (3324 Mb) which are pretty large and easily observed chromosomes, in the comparison of such as rice (41 Mb),
Lotus japonicas (78 Mb), red clover (63 Mb) and soybean (55 Mb) do not show the clear band patterns. Those species with a small chromosome (less 100 Mb/chromosome) have been able to identify chromosomes by performing image analysis and FISH detection on the typical condensation pattern of chromosomes.
Fukui
et al. [
5] developed first chromosome image analyzing system (CHIAS) and a chromosome map by analyzing the chromatin condensation pattern. A quantitative barley N-banded chromosome map was also developed using CHIAS to integrate both the genetics and chromosome of barley [
6]. CHIAS II is an improved version of the original CHIAS. It includes a mainframe upgraded from professional and special equipment to personal software in a PC [
7–
9]. CHIAS II was also used to develop quantitative chromosome maps [
10]. CHIAS II developed to analyze
Brassica chromosomes and was subsequently used to identify three diploid
Brassica chromosomes (
B. rapa, B. nigra, and
B. oleracea, with AA, BB, and CC genomes, respectively). All chromosomes of
B. napus (2
n = 38, AACC genome) were identified by CHIAS II, and a quantitative chromosome map or idiogram was developed. Nevertheless, the image analysis technology was only available to few researchers owing to the absence of affordable image-processing equipment.
Applications of a wide range of visualization procedures in plant genome studies are now essential for obtaining quantitative data on genomes and their evolution. Chromosomal maps are useful for efficient plant genome analysis. To develop a chromosomal map, an image analysis method that performs objective analysis while avoiding human subjective judgments and ambiguous expression, is extremely effective. Previously, a detailed chromosome map was developed for each small plant chromosome [
11,
12]. Extracting features from an image is common in an image analysis method for gene and genome mapping. Fluorescence
in situ hybridization (FISH), which involves the labeling of a fluorescent dye to a gene, was previously developed [
13]. By transducing the cooled CCD camera that could detect a very small amount of light, the digitization of the chromosome image progressed rapidly, and the affinity with the image analysis became greater. To carry out an effective study of the plant genome, total genomics combined with chromosome image analysis and the FISH method should be performed. Monocotyledon and dicotyledon model plants, such as rice and
Arabidopsis, were previously subjected to genome projects [
11,
12]. Integrating the genetic information of DNA sequence and deriving a linkage map and cytological quantitative chromosome map could further improve the genetic value of these plants.
In this review, we evaluated the effectiveness of digitization using the image analysis technology. Chromosome studies are required to establish and certify the importance of imaging methods in the development of new genetic models. Quantitative analysis of chromosomes and chromatins includes information about the correct positional information of a connected chromatin conformation, such as heterochromatin and euchromatin for genomic elucidation; such analysis could be carried out using the image analysis program developed in previous studies. We also sought to describe the development of an image analysis system for small chromosome FISH mapping and karyotyping, nucleus structure, fiber DNA and nano-level chromatin structure using an advanced helium ion microscope. Finally, we discussed the development of an effective image analysis application for the chromosome and chromatin structure of plants.
2 IMAGING THE IDIOGRAM OF SMALL PLANT CHROMOSOMES
Small chromosomes in plants such as Gramineae and Leguminosae do not display clear bands in conventional banding methods, such as N and C banding methods. This is because the tiny and similar-sized chromosomes in the mitotic metaphase impedes the identification of individual chromosomes. The morphological characteristics of the chromosomes are difficult to analyze. Therefore, a characteristic condensation pattern (CP) has been reported in prometaphase chromosomes during mitosis in rice [
5]. CP is the differentiation between chromatin condensed and non-condensed regions especially in small plant chromosomes at the prometaphase stage. The CP shows the individual characteristic chromosome feathers on each of the 12 chromosomes in rice instead of C-, N-, or G-banding patterns. Using characteristic CP as a parameter for image analysis enables the identification of chromosomes and the construction of a quantitative chromosome map. Chromosomal maps of
Lotus japonicus and red clover (
Trifolium pratense L.) have also been prepared using CHIAS III [
12]. These maps were continuously upgraded with the latest version of the software (currently CHIAS IV) [
14].
Fabaceae syn. Leguminosae, the third-largest family of flowering plants, is comprised of 700 genera and 20,000 species of flowering plants, including the most common type of flowering plant found globally. Fabaceae encompasses various species, such as peas (
Pisum sativum, 2n = 2x = 14; genome size per haploid, 5000 Mb), alfalfa (
Medicago sativa, 2n = 2x = 16; genome size per haploid, 1600 Mb), and soybeans (
Glycine max, 2n = 2x = 40; genome size per haploid, 1100 Mb), whose genomes have been studied for years [
15]. Fabaceae plant types exhibit remarkable variety (ranging from small annual herbs to massive tropical trees) [
16]. The subfamily, Papilionidae, in Fabaceae has the most pulse crops, including major grain legumes, oilseed crops, forage crops, ornamental crops, medicinal crops, and agroforestry species.
The quantification of chromosome density by CHIAS, in situ localization of repetitive sequences, and high-resolution mapping of genes and/or markers by FISH are expected to facilitate the analysis of gene density, segment duplication, and other chromosome rearrangements, and to yield integrated maps for legumes. In particular, chromosome analyses that are applicable for Lotus japonicas L. and red clover would help to fabricate a framework for common legume genomics.
L. japonicus is characterized by a small genome (2n = 2x = 12; genome size per haploid, 472 Mb), relatively short life cycle (2‒3 months), and ease of genetic manipulation (
e.g., transformation as an autogamous diploid plant) [
17,
18]. Sato
et al. [
19] constructed a high-density genetic linkage map of
L. japonicus and mapped numerous transformation-competent artificial chromosome (TAC) genomic markers. These markers are indispensable for Leguminosae studies in various fields, including comparative genomics, gene identification, gene isolation, and marker-assisted breeding. Consequently, several microsatellite and simple sequence repeat (SSR) markers, as well as derived cleaved amplified polymorphic sequences (dCAPS), have been genetically and physically mapped onto the
L. japonicus genome. Ohmido
et al. [
20] integrated quantitative pachytene chromosome maps using six
L. japonicus chromosomes based on length, centromeric position, heterochromatin, and euchromatin distribution pattern, as well as the position of major repetitive sequences by using FISH and an imaging method in CHIASIII. In addition, they used high-resolution pachytene chromosomes to determine the precise integration between the genetic and physical distances in the
L. japonicus genome.
Red clover (
Trifolium pratense L.) is a forage legume and an allogamous diploid plant (2n = 14; 440 Mb). Kataoka
et al. [
21] examined the seven prometaphase chromosomes of red clover using FISH with several repetitive sequences and bacterial artificial chromosomes (BAC). The investigation using specific BAC clones to determine the chromosome specific regions identified by Sato
et al. [
22]. The position of the hybridization signals and the chromosome condensation patterns were quantified using CHIAS IV. Fourteen BAC clones belonging to the seven linkage groups hybridized to individual chromosomes were identified, thereby establishing the relationship between the linkage groups and the karyotype. Quantitative analysis using FISH mapping and chromosome analysis using CHIAS IV enabled the construction of a quantitative idiogram. This idiogram was comprised of a comprehensive chromosome map of red clover chromosome sequences derived from the same chromosome in each individual and the corresponding chromosome sequence in each individual. Based on the findings, chromosomal collinearity was identified among allogamous red clover varieties. This integration of genetic linkage and quantitative chromosome maps should provide valuable insights into allogamous legume genetics.
3 IMAGING THE GENOME SIZES AND CHROMATIN CONFORMATION IN INTERPHASE NUCLEI
Genome size plays an important role in the evolution of plants and animals as changes in genome size might accompany, if not facilitate, evolutionary adaptation to environmental conditions. Genome sizes have been analyzed via flow cytometry, a method extensively used to determine plant genome size using fluorescence intensity [
23]. Plant DNA flow cytometry is a very popular method with applications ranging from basic and applied research to industry. However, image analysis of interphase nuclei is important to understand characteristics chromatin condensation and the structure of nuclei.
How does the nuclei organization and genome sizes relate in plant cells? Both fluorescent and conventional nuclear staining methods can reveal uneven orientation in a nucleus. Previously, the Rabl orientation was believed to be a basic interphase structure of different plant nuclei [
24,
25]. However, some plant species with genome sizes less than 1 × 10
4 Mb (2C value), such as
Arabidopsis thaliana and
Oryza sativa, are known to have a non-Rabl orientation [
26–
29]. Small genome species have chromosomes in their interphase nuclei disposed in diffused chromosome territories, without the Rabl arrangement. However, in 2C value larger than 4 × 10
4 Mb genomes, the chromosomes run in a string-like manner through the nucleus with a Rabl orientation displayed throughout the cell cycle. Dong and Jiang [
27] revealed that Gramineae species with larger 2C values, such as wheat, rye, barley and oats, have a clear Rabl nucleus orientation. They also suggested that a relationship exists between Rabl orientation and genome size in maize with middle 2C value and incomplete formation of the Rabl orientation. Plant species with a 2C value larger than 4 × 10
4 Mb were found to display Rabl orientation. Furthermore, Fujimoto
et al. [
30] demonstrated that
Lilium and
Fritillaria plants with over 7 × 10
4 Mb (2C values) unexpectedly displayed telomere in both hemispheres; however,
H. vulgare was found to display a deviation in one hemisphere. The images were deconvoluted to obtain clear 3D images using a deconvolution program. Preservation of the nuclear 3D structure was confirmed with the reconstructed 3D images. As a result, the Rabl orientation was found to not represent a common 3D structure of genomic DNA, even in plants with large 2C values. Several studies have indicated that whether the chromosomes adopt a non-Rabl configuration is not dependent on the genome size in plants (Tab.1) [
30,
31]. Nonetheless, an upper limitation of the ratio of DNA volume to nuclear volume exists in plants. In fact, the constant ratio between the DNA volume and nuclear volume should not exceed 3%.
4 IMAGING THE CHROMOCENTERS AND 3D STRUCTURE OF INTERPHASE NUCLEI
Computational image analysis provides precise, objective, and reproducible quantitative data from images. Images of interphase nuclei have been analyzed using various methods. In fact, both the fluorescent and conventional nuclear staining methods can be used to reveal uneven DNA density in a nucleus. The nucleus at the interphase can be characterized by distinct morphological features and the distribution pattern of certain chromosomal regions, such as chromocenters (CCs). The nuclei at the interphase are well known to exhibit different distribution patterns in their heterochromatic CCs, which can be used as an indicator of the cytological characteristics of each strain, species, or genus in plants [
32]. In some plants, heterochromatic CCs are displayed as heavily-stained granular regions. Tanaka [
33] categorized the distribution patterns of CCs within an interphase nucleus into seven types of “resting chromosomes” in Orchidaceae. As a result, species with a similar form of resting chromosomes would occupy close phylogenic relatives. The numerical parameters of CCs within a nucleus are used to determine the type of resting chromosome, which is a useful cytological characteristic that can serve as an indicator of cross compatibility among plant species. However, it is difficult to objectively determine the size of the types of resting chromosomes through visual inspection of the nuclei under a microscope. Further, individual human biases in the visual determination of the nucleus types cannot be prevented. Quantitative estimation and comparison of the types are almost impossible under normal visual inspection as the comparison of data from different studies cannot be ignored. Acquiring reproducible and quantitative data on the chromocenters within the nucleus is however the most difficult part of objective typing. The image data for the types of resting chromosomes can be separated into four parameters: the number of CCs; area of CCs; percentage of total area of CCs in a nucleus; and distribution of CCs, which are represented by X and Y coordinates for the center of gravity of each CC. The interphase nuclei of Orchidaeceae show many CCs with different sizes using conventional aceto-orcein staining methods [
25]. An adaptive thresholding method, which was modified with unsharp mask filtering, was applied previously in the reports [
34,
35]. Further, this method could be used to analyze the distribution pattern of CCs in the interphase nuclei. The numerical parameters of CCs can provide useful genetic information for phylogenetic studies of plant diversity.
Kikuchi
et al. [
36] observed parental centromere behavior during mitosis and the meiotic prophase. To understand the positioning of parental centromeres in a fertilized egg, genome separation in the nuclei of epidermal cells and apical meristem cells from F
1 hybrids was statistically analyzed between
Torenia fournieri and
T. baillonii. To determine the separation between two species centromere signal sets in the nuclei, a 3D distinction computer program [
25] was used. FISH with centromere repeats would reveal the relationship between the specific sequence and nuclear structure. Therefore, the separation and association between two parental centromeres in the nuclei of the hybrids were revealed. The 3D program measures and calculates the average nearest distance from a centromere of one species to that of another species in the observed nuclei and digitally-simulated nuclei. The association of centromeres from a single genome was observed at a higher frequency than that with another genome. Such finding suggests that centromeres within one genome are spatially separated from those within the other.
To link nuclear structure and function, imaging tools that quantify nuclear morphology as well as the positioning and organization of chromatin domains in 3D are warranted. Poulet
et al. [
37] performed 3D interphase image analysis using NucleusJ plugin. NucleusJ is an ImageJ plugin in Java language released as a jar file for the ImageJ platform. They examined the shape and size of the nuclei as well as intra-nuclear objects and their position within the nucleus using 3D quantitative measurements from single images or large data sets. Expertise in image analysis with
Arabidopsis was not required. NucleusJ is useful to users interested in quantifying the size and shape of nuclei, nuclear objects or chromatin domains as well as the positioning of the latter in the nuclear space. The developers expect to improve NucleusJ to quantify FISH and immunochemistry signals and to define automatic segmentation of CCs. NucleusJ can also be adapted to segment FISH signals [
37]. Dubos
et al. [
38] introduce the optimized NucleusJ 2.0 to improve the nuclei analyzing speed automated steps. NucleusJ 2.0 was then used to characterize nuclei stained with DNA dyes or labeled with 3D-DNA FISH in whole-mount tissue of a plant mutant with strong alteration of nuclear morphology and chromatin organization.
5 IMAGING ENVIRONMENTAL EPIGENETICS OF THE INTERPHASE NUCLEI
The CCs in the model plant,
A. thaliana, are discrete nuclear domains of mainly pericentric heterochromatic regions. Heterochromatin is confined to small pericentromeric regions of all five chromosomes and the nucleolus organizing regions. This clear differentiation enables the study of spatial arrangement and functional properties of individual chromatin domains in the interphase nuclei [
39]. Heterochromatin segments are organized as condensed CCs containing heavily cytosine methylated, mainly repetitive DNA sequences. Further,
Arabidopsis CCs harbor major tandem and dispersed repetitive sequences [
39]. Epigenetic markers, such as DNA methylation and histone methylation and acetylation, play vital roles in plant cell growth and development. Differentiated cells exhibit relatively complex histone modification patterns while stem cells display fewer and simpler modifications [
40]. Fluorescent protein is also used to visualize specific nuclear protein [
41,
42].
Arabidopsis CCs carry epigenetic markers of silent chromatin, such as histone H3 dimethylated at lysine K9 (H3K9me2) and 5 methyl cytosine (5mC) [
39,
43]. Immunostaining can also be used to visualize specific molecules modification, such as methylated cytosine and specific nuclear protein development process [
44–
46], photo response [
47], and abiotic stress response [
48]. Chromatin plasticity has been proposed to be associated with plant acclimation to environmental changes.
The plant root apical meristem can be divided into three main parts: the meristem, elongation, and maturation zones. There is new evidence that specific epigenetic changes are related to the growth and development of plants. Histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and 5mC have unique and specific patterns in barley meristem tissues [
45]. The heterochromatin marker, H3K9me2, has a high concentration in the differentiated cell, boundary meristem-elongation zone, and epidermis cell; however, 5mC is dominant in the root cap and distal meristem. Neither heterochromatin marker is abundant in the proximal meristem region. However, most euchromatin markers (H3K5ac and H3K4me2) are highly concentrated in peri-meristematic regions undergoing highly active cell division. Polosoro
et al. [
48] measured the signal intensities of each histone epigenetic marker on the nuclei of abiotic stress response iron-treated rice plants using CHIAS. Their intensities in proximal meristem nuclei were also compared to those in the elongation zone. Under iron treatment, the concentration of H3K9me2 increased by approximately 7-fold until its level in the elongation-zone nuclei was identical between control and treated plants. However, the intensity of H3K4me2 in these nuclei declined by approximately 7-fold to levels similar between the two groups of plants. According to a nuclear size analysis by image analysis, the nuclei in the proximal meristem region became smaller and were identical to those in the elongation zone. Regardless of their iron sensitivity, the rice cultivars had generally similar response patterns, which implied that their primary epigenetic responses were similar, although not identical, to those of the other cultivars.
6 IMAGING THE STRETCHING OF DNA FIBERS
Chromosomes and nuclei consist of DNA fiber, histone proteins, and non-histone proteins [
49–
51]. The basic unit of chromatin fibers is the nucleosome, which consists of approximately 147 bp of DNA units coiled around a core of octamer histone molecules (two subunits of H2A, H2B, H3, and H4) [
52] associated with the linker histone, H1. Nucleosome units connect with other units to form 10-nm fibers, known as “beads on a string” [
53]. FISH resolution on the highly condensed somatic metaphase and pachytene chromosomes in plants is estimated to be 4‒5 Mb and 1.2 Mb, respectively [
54]. Therefore, two DNA sequences located less than 1 Mb apart cannot be reliably resolved. FISH resolution is estimated to be 20‒30 kb in human interphase nuclei. Thus, two target sequences that are closer in distance will show overlapped FISH signals on chromosomes and nuclei. To increase the optical restriction of the resolution of light microscopy (about less than 100 nm),
e.g., structured illumination microscopy (SIM), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED) offering new insights into molecular structures, interactions and functions of DNA and protein were developed [
55]. Recently, Kubalová
et al. [
56] had achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying SIM doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm.
In contrast to somatic metaphase chromosomes that provide an average resolution of 1 Mb, extended chromatin fibers can be extracted from somatic cells. Fiber-FISH can be used to achieve the highest resolution of mapping of adjacent DNA sequences at distances of only 1 kb [
57]. Further, it can be used to achieve a resolution that bridges the megabase molecular techniques, such as pulsed-field gel electrophoresis and sequence analysis of genomes [
54]. Fiber-FISH was thus successfully used to perform a detailed investigation of chromosomal domains in
Arabidopsis [
57], rice [
58], tomato [
59], and sugar beet [
60]. By considering the stretching degree of chromatin fibers of 3.27 kb µm
−1, multicolor fiber-FISH could be used to measure the physical distance between the DNA sequences in repetitive regions of plant chromosomes, which cannot be assembled in genome sequencing projects. Multi-color FISH on extended DNA fibers (EDFs) allows the placement and visualization of markers, genes, and repetitive sequences on each of the chromosomes that cannot be mapped by recombination-based methods or direct contig assembly.
Stretched chromatin fibers have been used as an effective alternative procedure for mapping adjacent DNA probes with a higher spatial resolution [
57,
61]. Fiber-FISH was mainly used to analyze the structure and organization of repetitive DNA sequences and determine the length of long DNA molecules [
62]. EDFs combined with FISH has the highest spatial resolution for obtaining genome structure; this is because all proteins that interact with DNA compaction are removed and the genome DNAs are extended. This method was used to detect the molecular organization of genome structures. FISH was thus applied to detect in japonica and indica rice [
26]. As a result, a rice A genome-specific tandem repeat sequence (TrsA) FISH signals were found to be localized in the terminal regions of the long arms of rice chromosomes. In dual-color FISH experiments on chromosomes, these signals were found to overlap with the telomeric-specific signals in the terminal regions (Fig.1). Fiber-FISH enables accurate analysis of repetitive sequences in the eukaryotic genomes and is valuable in large genome sequencing projects. Information for DNA or protein in EDFs and nuclei that can be observed visually, can also be analyzed quantitatively using image analysis methods.
As mentioned above, information for DNA or protein in EDFs and nuclei that can be observed visually, can also be quantitatively analyzed using image analysis methods. CHIAS-Straight can be used to carry out quantitative analysis of fiber signals on EDFs (Fig.1). CHIAS-Straight is a free application that is used to measure the imaging performance of fiber-FISH and immunostained chromatin fibers (Fig.1, Tab.1) [
58,
60,
63]. Routinely, 25‒100 signal tracks are screened to measure the length of the fluorescent signal tracks on EDFs (Fig.1, D). The CHIAS-Straight plugin file can be installed after a user is registered in the website of Ohmido lab and the CHIAS-Straight manual can be founded in Ohmido lab. CHIAS IV runs as a plug-in in the public-domain image-processing software, ImageJ (National Institute of Health (NIH) Bethesda, MD, USA) using the Java programming language [
64]. ImageJ is the host application for CHIAS IV [
14]; for both operating systems, the software requires ImageJ version 1.51 or later.
Fiber-FISH can be employed to carry out an analysis of tandem duplications associated with large genomic duplicates. In fact, fiber-FISH characterization of a 620-kb mitochondrial DNA (mtDNA) fragment inserted into chromosome 2 of
A. thaliana [
65] was achieved early after its introduction. This large mtDNA insert was derived from complex duplication and deletion events, which could not be characterized using traditional methods, such as PCR or southern blot hybridization. Koo
et al. [
66] developed fiber-FISH imaging to detect mtDNA fragments in the nuclear genome. Somatic human cells contain thousands of copies of mtDNA. In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mtDNA copies. As a result, Koo and colleagues developed fiber-FISH imaging to carry out mtDNA repeat calculations to analyze nuclear mtDNA insertions in the entire human genome. This method is called mtDNA FIBER-FISH and is employed for cancer cell lines and primary tumors and in advance basic and translational cancer research.
7 IMAGING THE NANOSTRUCTURE OF PLANT CHROMOSOMES
Chromosome compaction of chromatin fibers results in the formation of a nucleosome consisting of a DNA unit coiled around a core of histone molecules associated with a linker histone. Since the discovery of chromosomes in the 19th century, the compaction of chromatin fibers has been controversial. Moreover, higher levels of chromatin fibers appear as shallow supercoiled 30 nm structures, known as solenoids [
67]. Helium ion microscopy (HIM) or scanning helium ion microscope is a state-of-the-art nanofabrication imaging technique that harnesses the positive charge of helium ions interacting on the surface of samples [
68]. Owing to the use of high beam energy, this technique allows helium ions to be deposited deep into biological samples as the mass of the ion is greater than that of the electron. As a result, a small beam spot and high secondary electrons (SEs) are produced, thereby demonstrating the brightness of SE emissions [
68]. Recently, HIM was used to study the effects of magnesium ions on the structure of the human chromosome, which is composed of 11-nm chromatin fibers. This technology did not require a metal coating owing to sufficient SEs and low beam damage [
69]. HIM is thus a powerful tool for nano-level imaging as well as the analysis of the surfaces of biological soft samples. However, HIM has yet to be used to study the structure of plant chromosomes.
Sartsanga
et al. [
70] developed a simple technique to isolate intact barley (
H. vulgare) chromosomes in high yield. As a result, chromosomes could be observed with a high-resolution scanning ion microscope and the HIM imaging technology, which is based on a scanning helium ion beam. HIM images captured from the surface chromatin fibers of the barley chromosome were analyzed to determine the size and alignment of the chromatin fibers via image analysis. Image analysis was also used to analyze the chromatin fiber images and based on Wako
et al. [
71], chromosome images obtained by HIM were subjected to several imaging processes. A series of steps were carried out to analyze the images obtained by HIM (Fig.2). Briefly, the area of the chromatin fiber was defined using the HIM image (steps 1 to 3) (Fig.2‒C). Mid lib lines were then drawn on the chromatin area (steps 4 and 5) (Fig.2). In steps 6 to 8, the width lines of the chromatin fiber were drawn perpendicular to the mid lib lines (Fig.2). Finally, the size of the width lines from the chromatin area was measured (step 9). The width lines were denoted by various colors based on their lengths (Fig.2). The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Such findings suggest that the surface structure of the chromosome consists of a chromatic fiber network [
74]. Compacting the surface structure of barley via a chromatin network and observation via HIM are thus powerful tools for investigating chromatin structure. The procedures discussed herein can be applied to other biomaterials, such as the inner structure of the chromatin axis with DNA and protein interactions. Accordingly, we will investigate the outer and inner structures of chromatin in a future study to fully elucidate the process of chromatin compaction in plant chromosome.
The integrity of condensed chromatin is reported to depend on the binding of cations [
75]. Calcium and magnesium are the most abundant cations within a cell. In addition, these cations are known to critically contribute to the chromosome condensation process in eukaryotic cells [
76]. Previous studies have revealed the decondensed structure of the chromosome following cation depletion [
69,
77,
78]. However, a qualitative data regarding the effects of both ions on chromosome structure are currently available. Previously, Dwiranti
et al. [
77] investigated the effects of Ca
2+ and Mg
2+ on the morphology of chromosomes. In addition, the structural changes that occur in chromosome and chromatin after divalent cation depletion were further analyzed quantitatively using the chelating agent, ethylenediaminetetraacetic acid (EDTA). For this purpose, CHIAS was employed to quantitatively determine the effect of Ca
2+ and Mg
2+ depletion after bent chromosomes were straightened as a result of cation depletion. Successful straightening of the bent chromosomes was achieved using CHIAS IV. Such straightening could also provide quantitative data regarding the chromosome. The average length of the chromosomes was decondensed and double that of the chromosomes treated with EDTA. Collectively, by employing CHIAS IV to straighten the bent chromosomes, the study has demonstrated the effects of cation depletion. Such finding indicates the advantages of CHIAS IV in carrying out chromosome analysis and highlights the fundamental effects of cations on chromosome condensation.
8 APPLICATION OF OPTICAL IMAGING ANALYSIS FOR NEW PLANT GENOME RESEARCHES
Recent achievements and future applications of genome mapping and oligo-FISH using imaging technology are discussed below. Next-generation genome mapping through fiber-FISH imaging analysis using optical mapping of plant genomes brings genome assemblies to the ‘nearly-finished’ level for reliable and detailed gene annotations and assessment of structural variations (Gaiero
et al. 2018). An improved high-throughput optical mapping technology is being applied to image analysis in whole genome mapping [
79]. Optical mapping can also be applied to dynamic chromosome structure variations (SVs), such as inversions, deletions, translocations, and duplications. The optical mapping also applied to the genome assembly investigated by the next generation sequence of the large scale scaffold included accurate length information for the direction of the contig and the gap region by combining genome map data, and then misassembled data was corrected. Optical mapping for plant genome analyses has been performed to identify BAC contigs and correct genome assembly errors over the past decade. Optical mapping techniques had been applied in rice [
80], medicago [
81], maize [
82] and tomato [
83]. Concerning the identification of the large SVs at chromosomal scale, modern systems based on optical mapping technology such as the Bionano Genomics Saphyr system have remarkable sensitivity towards detection of genome-wide SVs. Shearer
et al. [
83] demonstrated that tomato genome scaffold arrangement based on BAC-FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. The results of BAC-FISH and optical mapping strongly indicate that many scaffolds were arranged incorrectly, the FISH based arrangement of scaffolds is being used in the new release of the tomato genome. In a role reversal, the new scaffold arrangement based on physical methods should be used to rearrange hundreds of markers in the linkage map. As a result, it is possible to generate and handle the massive quantities of optical mapping data that are required for the assembly of a large and complex plant genome.
Idziak
et al. (2014) studied the karyotype evolution of small genome grasses using BAC-FISH based comparative chromosome barcoding in four
Brachypodium species. The well-developed cytogenetic platform for
Brachypodium species permits two complementary approaches to comparative mapping—chromosome barcoding and chromosome painting. BAC-FISH-based chromosome barcoding in seven
Solanum species of the Solanaceae revealed inversions that affect specific lineages of the genus and thus enabled the construction of the ancestral chromosome [
84].
An important recent development is the application of FISH probes based on synthetic oligos, which can be used to create synthetic oligos. Oligo based probes can be designed either from repetitive DNA elements or from single-copy DNA sequences, a paradigm shift from the traditional FISH probes using cloned DNA sequences. Oligo probes designed from simple-sequence repeats or satellite repeats synthetic oligo-FISH probes were first developed to physically map simple-sequence repeats (SSRs) in plants [
85]. Synthesized oligos specific to specific chromosomal regions, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping. The advances in synthetic oligo probes will significantly expand the applications of FISH in many plant and crop species.
9 CONCLUSIONS
In order to understand the functions of genes and the genome, it is essential to understand the structure of chromosomes and nuclei. To help in the clarification of the taxonomy status of plants, imaging analysis for chromosome ideogram is important. Chromatin conformation and 3D structure analysis could be helpful the genome cosmic nuclei.
Recently, RNA-guided endonuclease—
in situ labelling (RGEN-ISL) was reported as visualizing instead of FISH [
86]. RGEN-ISL is a simple method to visualize repetitive DNA sequences using the CRISPR/Cas9 system. It is versatile and simple to use, and has been used to visualize DNA sequences in nuclei or chromosomes without the denature process which destroys the structures of cytological samples. RGEN-ISL is capable of simultaneously detecting DNA loci and immunohistochemical signals, including histone modification, in various types of plant tissues and species [
87].
The studies described in this review sought to measure and evaluate chromosome and chromatin using image analysis methods, which may reduce measurement time and improve resolution. Further, we discussed the development of an effective image analysis to evaluate the structure of chromosome and chromatin in chromosome and nucleus. The study of cell biology and the genetics of plants using image analysis methods is expected to be a major propeller in the development of new applications.
The Author(s). Published by Higher Education Press.
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