Keywords

6.1 Introduction

6.1.1 Classical Cytogenetics

The term cytogenetics is referred to the study of genetic consequences in terms of chromosome number, structure, and behavior vis-à-vis speciation and evolution. Cytogenetics has been proved to be an integral part of genome mapping projects owing to magnificent chromosomal dynamics during mitosis and meiosis. The field of plant cytogenetics was heavily induced by Barbara McClintock’s pioneering work on maize (Zea mays) (McClintock 1929, 1932, 1938, 1941a, b, 1984). McClintock used carmine for staining and uniquely identified all of the individual chromosomes from a single meiotic nucleus with a combination of two metrics, i.e., the relative lengths and arm ratios of the chromosomes. Her studies on unequivocal identification of individual chromosomes established a milestone in the scientific community, which allowed neo-discoveries regarding the dynamic structure and behavior of the maize genome (McClintock 1929, 1932, 1938, 1941a, b, 1984). Further, development of chromosome-banding techniques greatly improved the usefulness of chromosome biology to understand the basic genome architecture. In this context, Caspersson et al. (1968) proposed Q-banding pattern using the fluorescent dye quinacrine on plant chromosomes. Vosa and Marchi (1972) compared Giemsa C-banding to Q-banding on the chromosomes of bean (Vicia faba), keeled garlic (Allium carinatum), and maize. Further, Giemsa staining technique also showed its utility to identify individual rice prometaphase chromosomes (Kurata and Omura 1978), karyotype development for diploid rye (Secale cereale) (Gill and Kimber 1974), and barley (Hordeum vulgare) (Linde-Laursen 1975). With the advent of information on DNA and its characteristics, modifications of DNA staining dyes and banding techniques were adapted and optimized for cytogenetic characterization of different plant species. These classical approaches have proven invaluable for chromosome characterization, but the development of in situ hybridization, which allows for direct visualization of specific DNA sequences on chromosomes, forms a quantum leap forward for cytogenetics by combining cytology with molecular biology (Gill and Friebe 1998; Harper and Cande 2000).

6.1.2 Molecular Cytogenetics

A combination of ‘classical cytogenetics’ and ‘recombinant DNA technology’ gave birth to a versatile multicolored fluorescence engineering-based chromosome biology called ‘molecular cytogenetics.’ During initial development, such technique had been performed by using radioactive nucleic acid probes for the detection of specific DNA or RNA sequences in metaphase chromosomes or interphase. Subsequently, in the late nineties, methods for labeling nucleic acids with non-radioactive haptens such as biotin became available and adopted widely (Jiang and Gill 1994). The advantages of non-radioactive probes over radioactive probes include increased stability, safe handling, rapid, precise spatial localization, less back ground, and most importantly the ability to use multiple colors on a single chromosome preparation.

The development of in situ hybridization (ISH) techniques opened up opportunities for cytogenetic analysis of any species, regardless of its inherent chromosome morphology (Gall and Pardue 1969; Pardue and Gall 1975; John et al. 1969). In plants, the use of radioactive tagged or modified nucleotides (labeled with biotin, digoxigenin, or fluorescent haptens) and FISH probes also permits microscopic visualization and localization of complementary sequences in cells/nuclei and on individual chromosomes (Mukai et al. 1991; Fransz et al. 1996a; Mukai and Yamamoto 1998). Basic FISH makes use of green and red fluorochromes for probe detection and DAPI (4,6-diamidino-2-phenylindole) for counterstaining the chromosomal DNA. Although FISH is commonly used to map unique or low-copy-number sequences, however it also showed its potential to localize repetitive sequence in order to produce chromosome-specific landmarks or explore genome relations in polyploidy/closely related plant species (Lysak et al. 2001, 2003; Kato et al. 2004; Lamb and Birchler 2006). FISH has been found most successful in mapping the repetitive and single-copy DNA sequences on prometaphase chromosomes, interphase nuclei, pachytene complements, chromatin fibers, and naked DNA molecules. Accurate localization of repetitive and tandem arrays plays a major role in chromosome identification and karyotype analysis in plants (Mukai and Yamamoto 1998). The broad applications of FISH in structural, comparative, and functional genomics place plant cytogenetics in an important place to complement, accelerate, or guide plant genome research (Lamb et al. 2007; Danilova and Birchler 2008; Nagaki et al. 2012b). On the other hand, genomic in situ hybridization (GISH) (Le et al. 1989; Mukai and Gill 1991), a special type of FISH that uses genomic DNA of a donor species as a probe in combination with an excess amount of unlabeled blocking DNA, provides a powerful technique to monitor chromatin introgression during interspecific hybridization. In addition, the GISH technique allows the study of genome affinity between polyploid species and their progenitors (Mukai et al. 1993b; Raina et al. 1998; Raina and Mukai 1999). GISH is thus a valuable supplemental technique to traditional genome analysis such as conventional meiotic pairing analysis.

Molecular cytogenetics has now become an indispensible tool and a conceptual foundation for modern genome projects by providing significant information on individual chromosome portfolio of the organism under investigation.

6.2 Advances in Molecular Cytogenetic Techniques

Rapid developments in genetics, molecular genetics, molecular biology, and genomics, together with molecular cytogenetics , have driven major conceptual advances in mitotic, meiotic analysis, chromosome structure, and chromosome manipulation. Along such development although the principal steps of the FISH technique have remained same, various technical developments have been adapted in plant molecular cytogenetics . The basic development was the use of several colors for labeling the probes which provide holistic view of genome structure at a single glance, i.e., McFISH and McGISH (Mukai et al. 1993b; Mukai 1996). Some of the recent developments in the field of plant molecular cytogenetics in order to understand genome architecture and organization at ultra-resolution are described below.

6.2.1 Tyr-FISH

Tyr-FISH was developed to improve the detection sensitivity of FISH experiments. This method allows signal amplification by using a peroxidase-conjugated antibody as the first layer of signal detection. Fluorochrome-labeled tyramides as peroxidase substrate are used to generate and deposit many fluorochromes close to the in situ bound peroxidase (Raap et al. 1995). The sensitivity of the basic FISH technique can be increased by 10–100 times using such modification. DNA probes smaller than 1 kb were successfully visualized on plant chromosomes using Tyr-FISH (Khrustaleva and Kik 2001; Stephens et al. 2004).

6.2.2 DNA Fiber-FISH

The DNA fiber-FISH technology is applied to characterize complex genomic arrangements in plant nuclei by using decondensed chromatin and highly extended intact DNA fibers on microscopic slides (Fransz et al. 1996a). The method involves the release of DNA molecules from lysed nuclei followed by spreading them on the surface of a microscope slide and the hybridization of probes using a standard FISH method. Applying FISH probes to the stretched DNA molecules provides the higher spatial resolution with increased detection sensitivity. DNA prepared from BAC clones or plant tissues extends approximately 2.5–3.5 kb/μm on slides and provides fine-mapping resolution of up to a few kilobases. The drawback of the technique is that chromosome identification requires control DNA sequences, since there is no chromosome structure. In plants, Fransz et al. (1996b) demonstrated the utility of the extended DNA fiber-FISH (EDF-FISH) technology to characterize Arabidopsis thaliana and tomato genome. Later, this method was applied on other plants (e.g., rye, rice, and maize) in order to characterize complex genomic arrangements (Nagaki et al. 2004; Jin et al. 2004; Nakano et al. 2005; Yamamoto and Mukai 2007). Fiber-FISH is particularly informative when the exact position and ordering of DNA clones are needed. It can also evaluate the distances and overlaps between neighboring sequences (Ersfeld 2004; Suzuki et al. 2004; Yamamoto and Mukai 2007). The minimum target DNA size that can be distinguished unambiguously in plants is 10 kb (representing a ~3 μm fluorescent signal, de Jong et al. 1999); however, good flanking markers are crucial in order to differentiate and identify shorter DNA stretches.

6.2.3 Three-Dimensional (3D) FISH

The 3D-FISH technique had developed by Bass et al. (1997). Meiotic cells of maize were fixed in a buffer to preserve chromosome structure. Pollen mother cells were also gently extruded out of the fixed anthers and embedded in optically clear polyacrylamide for staining and imaging. Stacks of FISH images were taken and composed into a single 3D image. Individual chromosomes bearing the FISH signals were traced out and computationally straightened (Harper and Cande 2000). Since the chromosome structure can be preserved using this technique, it is advantageous for the identification of precise location of DNA probes on the chromosomes as well as within the nucleus.

6.2.4 FISH on Super-Stretched Chromosomes

Interphase nuclei, super-stretched mitotic metaphase chromosomes, and meiotic pachytene chromosome provide intermediate resolving power for FISH mapping. The relative positions of clone separated by <100 kb can be resolved on these cytological targets (Jiang et al. 1996; Wang et al. 2006). Pachytene chromosomes are particularly versatile targets for FISH mapping. Late pachytene chromosomes can be used to orient the telomere–centromere positions of the adjacent clones, whereas early pachytene chromosomes can be used to resolve even partially overlapped BAC clones. Nevertheless, pachytene chromosomes are not amenable for cytological analysis in many plant species.

On the other hand, flow-sorted plant chromosome at meiotic metaphase can be stretched to more than 100 times of their original size (Valarik et al. 2004). FISH on stretched chromosomes showed brighter signals than on the untreated control presumably as a result of better probe accessibility to the stretched chromatin. FISH on super-stretched metaphase chromosomes provides a mapping resolution of up to 70 kb (Valarik et al. 2004), similar to the resolution on meiotic pachytene chromosomes (Cheng et al. 2002). Thus, this modification of FISH provides an alternative mapping target for those plant species where meiotic pachytene chromosomes are not suitable for cytological analysis.

6.2.5 BAC-FISH

For the genome-wide sequencing project, a genomic library-holding large DNA fragments is an important tool for physical mapping or positional cloning of important chromosome landmarks. BAC-FISH, a unique tool of molecular cytogenetics , uses genomic DNA cloned in large-insert vectors such as bacterial artificial chromosomes (BACs) (Shizuya et al. 1992) in combination with FISH. This technique has shown its tremendous potential for physically mapping of specific DNA sequences and identifying individual chromosomes in plants (Suzuki and Mukai 2004). The BAC clones provide efficient resources for chromosome-specific FISH markers especially for plant species having small genomes such as rice, cotton, and sorghum. BAC-FISH favors the large clone as a probe for better resolution. The conventional FISH analysis on plant chromosomes employing probes containing over 10-kb insert DNA provides stable and distinct signals (Mukai and Yamamoto 1998; Suzuki et al. 2010). It is quite difficult to detect a single locus by using a plasmid clone of several kbs as the FISH probe. In this regard, the BAC clones containing around 50–100 kb fragments are suitable for probe of the FISH analysis.

6.3 Molecular Cytogenetics in Plant Genome Research

6.3.1 Physical Mapping and In Situ PCR

Plant genome are known for abundance of repeat sequences and cytogenetic or physical mapping of such repetitive DNA sequences decipher their genomic distribution and precisely identify the typical chromosome or set of chromosomes. These repeated rDNA gene clusters are being widely used and a common starting point for FISH-based mapping (Mukai et al. 1991; Yamamoto and Mukai 1991; Fransz et al. 1996a; Mukai and Yamamoto 1998; Sharma et al. 2012). The two types of ribosomal RNA genes (rDNA), 18S-5.8S-26S rDNA and 5S rDNA, have been extensively used as probes for physical mapping in higher plants due to their arrangement in tandem arrays (Mukai 1999). FISH mapping of rDNA clusters has provided a number of chromosomal markers that proved their efficacy in exploration of chromosome evolution and species interrelationships.

In hexaploid wheat, the six loci of 5S rRNA genes were identified on the short arm of the chromosomes of homoeologous group 1 and 5 (1A, 1B, 1D, 5A, 5B, and 5D) (Mukai et al. 1990), whereas 18S-5.8S-26S rDNA loci were mapped on the short arm of 1A, 1B, 6B, and 5D chromosomes and the long arm of 7D chromosome (Mukai et al. 1991) (Fig. 6.1). The rRNA genes are associated with the nucleolar organizing region (NOR), and the visualization of such repeat clusters at interphase represents the number of active rDNA loci. Multicolor FISH (McFISH) approach targeting repetitive DNA and rDNA probes also serves as chromosome identification markers in many plant species, for example, common wheat (Fig. 6.2). Similarly, Xu and Earle (1996) mapped the 45S rRNA DNA loci on to the tomato pachytene chromosomes, and Pedrosa et al. (2002) demonstrated the rDNA FISH for creating a karyotype of the model legume lotus. In addition, rDNA FISH in combination with other tandem repeats aids the generation of core cytogenetic maps, as demonstrated for maize, wheat (Jaing and Gill 1994), cotton (Hanson et al. 1996), tomato (Xu and Earle 1996), Pinus (Hizume et al. 2002), and Arabidopsis (Koornneef et al. 2003). The rDNA sequences are conserved across most plant species, but other tandem repeats exhibit variable degree of conservation.

Fig. 6.1
figure 1

Multicolor FISH mapping of 5S rRNA and 18S-5.8S-26S rRNA genes on the chromosomes of bread wheat (Triticum aestivum, 2n = 6x = 42, AABBDD genome), and fluorescence signals can be seen for 5S (red) and 18S-5.8S-26S (green) rRNA genes, respectively (Mukai 2004)

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Fig. 6.2
figure 2

Seven-color FISH on a metaphase cell of common wheat. Seven DNA sequences-pSc119.2, pSc74, pAs1, telomere, 18S-26S rDNA, 5S rDNA, and gliadin were detected by red, bluish green, green, orange, pink, blue, and white fluorescence, respectively, and the photographs were taken by triple exposures (Mukai 1996)

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Further, the chromosomal localization of rDNA has been widely used for comparative characterization of polyploid plant species. A comparison of FISH patterns of polyploid species with those of diploid progenitors of Aegilops revealed natural amphiplasty, in which the active rDNA sites either transformed to inactive or silent (deleted) during polyplodization event (Yamamoto 1994). Similarly, the U genome mostly suppresses the NOR activity of other genomes in tetraploids. On the other hand, the NOR activity of the d-genome chromosomes is completely suppressed by other genomes. In hexaploid species, all rDNA sites on the third genome remain active, reflecting time lapse after polyploid formation.

Simultaneously, Mukai and Apples (1996) invented the in situ polymerase chain reaction (in situ PCR)-FISH for mapping plant genes. This method uses the extreme temperature gradient sensitivity of PCR along with the cytological location of DNA sequences by means of in situ hybridization. The in situ locations of the rye-specific spacer region were determined on metaphase chromosomes. In such experiment, two pairs of primers for rye, i.e., Nor-R1 and rye 5S-Rrna-R1, were amplified in situ, which resulted in 386- and 107-bp amplified products, respectively. Rye NOR primers (45S) were localized on chromosome 1R and 4R, while 5S primers showed signals on the chromosome 1R and 3R. Interestingly, a previously described locus chromosome 5R did not show any signal in this experiment. It was concluded that the absence of a 5S site could be due to the sequence differences between the different 5S rDNA lineages. Several chromosome-specific sequences were also identified through primers specific to the chromosome. Thus, in situ PCR proved its utility in amplification of DNA sequences of specific plant chromosomes and for mapping low-copy genes of interest (Mukai and Yamamoto 1998).

Centromeric and telomeric sequences are also widely used in FISH mapping studies. Telomere repeats are highly conserved in plant species and occur in at least two major variants, i.e., (TTAGGG)n and (TTTAGGG)n (Lapitan et al. 1989; Adams et al. 1998; Fajkus et al. 2005). Similarly, the centromere associated 156-bp tandem repeat of maize, Cent C, was first discovered by Ananiev et al. (1998) and has become an invaluable cytogenetic milestone for maize and many related grass species. Cent O, a 155-bp centromere-specific satellite repeat sequence, the 180-bp satellite repeat, and CEN38, a 140-bp repeat sequence, have proven useful for labeling the primary constriction in rice, Arabidopsis, and sorghum, respectively (Heslop-Harrison et al. 1999; Cheng et al. 2002; Nagaki et al. 2003; Kim et al. 2005).

Further, employing BAC clones as a probes in FISH experiments become revolutionizing inventory in the field of molecular cytogenetic and extensively used in many plant species including cotton (Hanson et al. 1995), rice (Jiang et al. 1995), tomato (Zhong et al. 1996), Arabidopsis (Fransz et al. 1996b), onion (Suzuki et al. 2001), and sorghum (Kim et al. 2005). This approach can also be used to acquire insight for ongoing genome-sequencing projects worldwide.

King et al. (2002) demonstrated a GISH -based approach for physical mapping to distinguish recombination events between chromosomes of Festuca pratensis and Lolium perenne. A similar approach has also been used for the integration of genetic and physical maps of two Allium chromosomes (Khrustaleva et al. 2005). This GISH-based mapping strategy is similar to physical mapping using deletion and translocation stocks. This approach overcomes the major drawback of the tedious and time-consuming process of developing a large number of deletion and translocation stocks.

On the other hand, DNA clones were also used as probes for comparative FISH mapping in relative species. Several cytogenetics researchers reported FISH mapping of A. thaliana BACs on chromosomes of Brassica species. Comparative FISH mapping between Arabidopsis and Brassica provided a direct visualization of the genome duplication within Brassica species (Howell et al. 2005; Lysak et al. 2005). In addition, comparative chromosome painting with pooled BAC probes was used to investigate ancestral relationships among species that diverged within the Brassicaceae (Lysak et al. 2001, 2003, 2005). Recently, Koo and Jiang (2009) developed a technique by stretching maize pachytene chromosomes mechanically more than 20 times longer than their original size. Such super-stretched pachytene chromosomes can be directly used in conventional as well as molecular cytogenetic experiments. Super-stretching of the chromosomes coupled with immunofluorescence in situ detection of DNA methylation can lead to a new dimension and higher resolving power to modern molecular cytogenetics research. Collectively, these studies revealed that such FISH-based plant cytogenetical tools are uniquely informative and beneficial for genome analysis.

6.3.2 Chromosome Identification Subject to Parentage, Hybridity, and Ploidy

Fluorescence signal allows the identification of chromosomes, specific sequences, segments, or whole set of chromosome to gain a genome-wide view at a single glance in order to understand the plant genome organization and behavior. Fluorescence signals of either a single repetitive DNA probe or a mixture of several probes can be utilized for hybridization independently to identify individual chromosomes within a species. Chromosome identification through FISH method has advantage over the traditional chromosome-banding techniques due to availability of several probes for a particular species. Many repetitive DNA elements can also generate specific FISH signal pattern on individual chromosome within a single species (Mukai et al. 1991; Mukai and Yamamoto 1998; Koo et al. 2005). In this context, Pedersen and Langridge (1997) demonstrated the identification of all 21 chromosomes of hexaploid wheat through fluorescence signals derived from two different repetitive DNA probes. Later, similar approach has been adopted in several plant species for chromosome identification (Franz et al. 1998; Hizume et al. 2002; Kato et al. 2004; Koo et al. 2004).

On the other hand, GISH provides a direct visual method for distinguishing parental genomes and analyzing genome organization in intra-/interspecific hybrids, allopolyploid species, and introgression lines. This technique has an incredible prospective to identify application in identifying alien chromatin introgression and to study chromosomal pairing and recombination between divergent genomes. GISH has validated its utility in recognizing synthetic Hordeum chilense × Secale africanum (Schwarzacher et al. 1989) and Triticum aestivum (wheat) × S. cereale (rye) (Le et al. 1989). Mukai and Gill (1991) showed that GISH optimally detects barley chromosomes in a wheat background and further identified A-, B-, and D-genomes of the common wheat (Mukai et al. 1993b) using the same approach (Fig. 6.3). Similarly, Raina et al. (1998) and Raina and Mukai (1999) conclusively revealed that Coffea congensis and C. eugenioides, and Arachis villosa and A. ipaensis are the diploid wild progenitors of allotetraploid C. arabica (2n = 4x = 44) and A. hypogaea (2n = 4x = 40), respectively, using GISH as a tool. GISH has also been widely used to characterize the genome constitution of natural hybrids and to identify the parental origin of specific loci. By following the same approach, Takahashi et al. (1999) categorized the ancestral genome donors in maize and examined inter-genomic translocations and homeologous chromosome pairing (Zwierzykowski et al. 2008), as well as chromosomal areas with large species-specific sequences (alien chromatin introgression) or translocation break points (Qi et al. 2008). Such versatile approach of molecular cytogenetics also provides insight into somaclonal variation, the origin of B chromosomes, control of chromosome pairing, and other aspects of chromosome evolution (Kato et al. 2005).

Fig. 6.3
figure 3

Chromosome identification of Triticum aestivum (2n = 6x = 42). The AABBDD genome was simultaneously discriminated using GISH technique in which the diploid A genome progenitor Triticum urartu (yellow), diploid B genome progenitor Aegilops speltoides (brown), and diploid D genome progenitor Aegilops squarrosa (orange) have been identified precisely

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6.3.3 Karyotype and Phylogenetic Analysis

FISH-based chromosome identification systems could lead to precise karyotyping and to understand the evolution of particular plant taxa by means of speciation from wild to cultivated ones. For example, several repetitive DNA probes generate specific hybridization pattern on chromosomes of wheat and related species (Mukai et al. 1993a; Pederson and Langridge 1997). The FISH karyotypes from some repetitive DNA probes are similar to karyotypes based on C- or N-banding analysis (Cuadrado et al. 1995; Pederson and Langridge 1997). FISH-based karyotyping also specifies the phylogenetic view of related plant species (Lim et al. 2000). A number of repetitive DNA probes had utilized to develop FISH karyotypes of several diploid and polyploid Triticum and Aegilops species by Badaeva et al. (1996a, b). Similarly, comparative FISH mapping using several repetitive DNA probes in Nicotiana species found N. tomentosiformis to be the T-genome donor (Lim et al. 2000). Comparison of such karyotypes evidently revealed chromosomal landmarks to understand the evolutionary relationship between these species. Karyotyping using repetitive DNA probes can also visualize inter-genomic chromosome translocations in polyploid species. Since molecular cytogenetic techniques are often used to compare the ability of different genomes to hybridize (homology of genomes), together with the use of interspecific hybrids and allopolyploids, there by can serve as a powerful tool to understand phylogenetic relationships between species that is independent of nucleotide sequence-based approaches.

6.3.4 Chromosome Painting

The basic principle of FISH was further exploited to ‘paint’ individual plant chromosomes. The ‘chromosome painting’ is one of the most powerful molecular cytogenetic techniques to analyze nuclear organization and genome structure through visualization of specific cytogenetic target regions or entire chromosomes using this technique (Pinkel et al. 1986). Such technique involves the hybridization of fluorescence-tagged chromosome-specific composite probe pools (generally BAC clones) to various cytological preparations. Lysak et al. (2001) painted the chromosome of dicotyledonous model organism A. thaliana for the first time by employing selected BACs as differential labeled probes. However, in plants, mainly due to the presence of large amounts of repetitive DNA sequences such technique is remained limited (Jiang and Gill 2006). Such technique was found to be useful to identify individual chromosome in the interphase nuclei and could reveal the spatial arrangement and functional properties of individual chromatin domains. Further, Han et al. (2003, 2004) modified the McGISH to identify closely related wheat-Thinopyrum intermediates. Such chromosome painting provides insight into genome duplication/multiplication and karyotype evolution in closely related taxa. Arabidopsis chromosome and/or segment-specific probes were hybridized to ‘paint’ the chromosomes from species related to A. thaliana (Lysak et al. 2005). In later studies, the chromosome painting technique was applied successfully in related Brassica species (Lysak et al. 2010). These experiments proved that the technique is feasible for the detailed investigation of the pairing behavior of homologous chromosomes during early prophase I. Painting by this method is found to be feasible on small B chromosomes as well as alien chromosomes that possess chromosome-specific repeats (Houben et al. 1996). Comparative chromosome painting is an efficient and powerful approach to study the partial genome duplications and karyotype evolution. This advantage of the technique has been used to investigate the mechanisms of chromosome number reduction in A. thaliana and related Brassicaceae species.

Successful interspecific chromosome painting experiments were carried out between sorghum and maize (Koumbaris and Bass 2003). Ma et al. (2010) used Brachypodium distachyon BAC-clone to map the barley genome. Recently, the evolution and taxonomic split of the model grass B. distachyon were analyzed, and substantial phenotypic, cytogenetic, and molecular differences were detected between three cytotypes with the help of chromosome painting (Catalán et al. 2012). The development of comparative chromosome painting paves the way toward comparative chromosome mapping in several crop taxa including Triticeae hexaploid wheat, thereby facilitating the formulation of meaningful breeding program in light of the gene pool diversity.

6.3.5 Alien Chromatin and Transgene Detection

Schwarzacher et al. (1992) ascertained the alien chromatin incorporated from Leymus, Thinopyrum, Hordeum, or Secale in five bread wheat lines by GISH analysis. Friebe et al. (1991) also used GISH to locate the translocation chromosomes in different leaf rust-resistant wheat using GISH technology. Mukai et al. (1993a) also noticed the rye chromatin transfer in wheat. This technique has been effectively applied to detect genome donors in Brassica allopolyploids (Snowdon et al. 1997).

FISH has also analyzed the structure of the transgene loci on interphase nuclei, metaphase chromosomes, and on extended DNA fibers (Forsbach et al. 2003; Chen et al. 2003). Particle bombardment often generates very large, high-copy-number transgenic arrays that can extend for megabases. Interestingly, earlier studies showed that dispersed metaphase FISH signals come together at interphase. By contrast, Agrobacterium transformation gives rise to lower transgene copy numbers and is usually characterized by single discrete FISH signals. Employing molecular cytogenetic approaches, transgenes have been identified in Arabidopsis, barley, and rice, respectively (Forsbach et al. 2003; Chen et al. 2003).

6.4 Modern Molecular Cytogenetics

A biological question may not be solved by a simple localization of DNA sequences in interphase nuclei or on chromosomes. However, physical localization of a DNA sequence together with its associated protein may dramatically enhance the power of FISH. The global patterns of chromatin modification (e.g., DNA methylation and histone tail modifications) along with nuclear size and shape, relative content and distribution of heterochromatin/euchromatin , and organization and structure of chromosomes (e.g., position and orientation) provides new insight into evolution of the particular plant species at chromosomal level. Therefore, it has acquired am important share in this newly developing research field of studying chromatin dynamics through localization of epigenetic signatures of histone/DNA modifications and methylation. It has also been emphasized that amino-terminal tails of histone proteins are targets for a series of posttranslational modifications (PTMs), including acetylation, phosphorylation, and methylation. These modifications regulate chromatin structure and gene expression (Jenuwein and Allis 2001).

6.4.1 Immuno-FISH

Several plant laboratories have developed techniques that combine FISH with immunoassay methods (Jasencakova et al. 2001; Zhong et al. 2002; Nagaki et al. 2005, 2012a, b; Lavania et al. 2012). Such modernization of cytogenetic technique involves an immunoassay of specific antibodies and cytological preparations followed by standard FISH procedure. Immuno-FISH has been used to reveal DNA methylation and histone modifications with specific genomic region. A number of antibodies are available for studying 5mC and histone modifications vis-à-vis chromatin status. Recently, several studies have been conducted on plants using immunohistochemical staining to elucidate chromosomal distribution pattern of the epigenetic marks including Arabidopsis (Zhang et al. 2008), Allium (Suzuki et al. 2010; Nagaki et al. 2012b), maize (Jin et al. 2008; Koo and Jiang 2009; Koo et al. 2011), rice (Yan et al. 2010), brassica (Wang et al. 2011), Barley (Sanei et al. 2011), tobacco (Nagaki et al. 2009), sugarcane (Nagaki et al. 2005), and other taxa (Lavania et al. 2012). Most of the studies suggest that H3K4me1,2,3 mostly mark euchromatin, while H3K9me1 and H3K27me1 mostly target heterochromatin (Fuchs et al. 2006). While H3K9me2 and H3K27me2,3 showed diverse distribution pattern among angiosperms (Fuchs et al. 2006). On the other hand, centromere-specific histone H3 (CENH3) is one of the most fundamental centromeric proteins known to be involved in recruiting other centromeric proteins. CENH3 was first identified as CENP-A in humans (Earnshaw and Rothfield 1985) and subsequently found in a large number of plant species including Brassicaceae, Solanaceae, Leguminosae, Poaceae, and Juncaceae species (Zhong et al. 2002; Telbert et al. 2002; Nagaki et al. 2004, 2005, 2009, 2012a; Sanei et al. 2011; Tek et al. 2011; Wang et al. 2011; Neumann et al. 2012). Since CENH3 comprises part of the core histone that binds directly to DNA at centromeres, centromeric DNA has been isolated from several plant species using antibodies against CENH3 (Nagaki et al. 2003, 2004, 2009, 2011, 2012b; Nagaki and Murata 2005; Tek et al. 2011; Zhong et al. 2002; Neumann et al. 2012; Houben et al. 2007). Immunostaining of chromosomes of Allium species using anti-AfiCENH3 antibody has been shown in Fig. 6.4. Such studies suggest that these histone variants have immense potential to generate extensive information about chromosomal distribution pattern of the epigenetic marks in a wide range of plant species (Sharma et al. 2015).

Fig. 6.4
figure 4

Immunostaining of chromosomes of Allium species using anti-AfiCENH3 antibody: DAPIstained chromosomes (blue) and visualization of immunosignals of anti-AfiCENH3 antibody (red)

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6.5 Future Prospects

Exciting advances in plant molecular cytogenetic tools and array-based techniques are changing the nature of chromosome biology, in both basic research and at molecular diagnostic levels. Cytogenetic analysis now extends beyond the simple description of the chromosomal status of a genome and allows the study of fundamental biological questions of chromosomal evolution underlying speciation and adaptation. One of the major challenges in plant cytogenetics includes the increment of the resolution power of in situ hybridization and immunostaining techniques to detect shorter nucleotide stretches or single antigen molecules reliably on metaphase chromosomes, extended chromatin fibers and/or in interphase nuclei. Further, improvement of efficient and effective fluorescent chromatin tags for in vivo studies is also needed. FISH may play a powerful role to delineate the structure and DNA composition of long track of highly repeated regions, for example, centromere as well as telomeric ends that are difficult to clone.

As discussed earlier in this article, DNA methylation, nucleosome remodeling (including histone modification and histone variants), and noncoding RNAs can organize chromatin into accessible (euchromatic) and inaccessible (heterochromatic) sub-domains. This extends the information potential of the genetic code, and one genome can generate many ‘epigenomes’ in time and space, during the life span of an organism. The implications of epigenetic research seek attention and efforts that should be targeted to epigenome in a variety of plant systems especially at chromosome inheritance level. In a recent study, it was shown that these epigenetic modifications are not as conserved as was once thought. Further, very little is known about histone/DNA methylation/modification in large genome plants (Houben et al. 2003), which make up the bulk of the angiosperms (Arumuganthan and Earle 1991). Immuno-FISH should be practiced worldwide that has potential to significantly increase the resolving power to reveal fine interaction between DNA and proteins.

6.6 Next-Generation FISH

Next-generation sequencing (NGS) technologies continue to develop at a fast pace, and whole genome sequence of several plants have either been released or to be released soon. NGS technologies of third-generation platforms could produce reads reaching up to a few kilobases, whereas read lengths presently range from 30 to 400 bp depending on the platform. NGS may also facilitate probe development for studies of chromosome using FISH . These genomic regions can be mapped on the chromosome for precise location information with reference to chromosome rearrangements and translocation events and to identify chromosome with/without physical gaps, if any. Further, transcriptome sequencing has also been engaged in construction of large datasets of nuclear genes. NGS is also making the rapid sequencing of complete nuclear genomes routine, thus transforming the genomics research field and opening up new avenues of systematic endeavor in comparative genomics. Further, research should be aimed at understanding the distribution, location, and copy number of the epigenetically inherited gene/genic regions identified through NGS data in several crop/plant species/families in order to shed light on the role of chromatin dynamics in speciation and evolution.