Mukai and Apples (1996) were the first to apply in situ polymerase chain reaction (in situ PCR) FISH for mapping plant genes. This method combines the extreme sensitivity of PCR with the cytological location of DNA sequences provided by in situ hybridization.The in situ locations of the rye-specific spacer region were determined on metaphase chromosomes using two pairs of primers designed for rye Nor-R1and rye 5S-Rrna-R1.The sizes of amplificants are 386 bp and 107 bp, respectively.Rye NOR primers generated strong signal in the nucleolus organizing region of rye chromosome 1R and an additional faint signal in the long arm of chromosome 4R. With rye 5S primers, signals appeared on the satellite segment of chromosome 1R and the short arm of chromosome 3R. No signals were detected on the short arm of chromosome 5R which has a previously described locus, 5S-Rrna-R2. The absence of a 5S site is due to sequence differences between the different 5S rDNA lineages. In the same gene family we could detect chromosome specific sequences by selecting primers specific to the chromosome. Thus in situ PCR is expected to be a useful method for amplifying the small region of DNA sequences of specific plant chromosomes and for mapping low copy genes of interest (Mukai and Yamamoto 1998).

Physical mapping of agronomically important genes

Agronomically important genes of wheat are mostly unique or low copy sequences. The information about the exact physical location of agronomically important genes could be useful in breeding programs as well as in understanding the organization of cereal genomes. On human chromosomes, single copy sequences as small as 1 kb could be routinely detected by the standard FISH technique, but in wheat it is difficult to locate 10 kb sequences even when amplification of FISH signals is applied to enhance high resolution FISH efficiency. As a suitable alternative, large genomic clones such as lambda phages, cosmids, BACs, and PACs could be successfully used: the large amount of repeated sequences contained in them would facilitate homologous hybridization, signals detection of which would be suppressed by competitive hybridization with unlabeled C₀t -1 DNA or total genomic DNA enabling the unique sequences of interest as expressively detected.

Single or low copy sequences such as genes controlling wheat grain quality have been routinely mapped on wheat chromosomes using lambda phage clones containing 10 to 20 kb insert of genomic DNA sequences. Starch consists of amylose and amylopectin, which differ in the degree of branching; amylopectin is much more branched than amylose. There are two types of starch branching enzymes, I (SBEI) and II (SEMI), which control the pattern and degree of branching in starch in consonance with the various starch syntheses and debranching enzymes. SBEll is further subdivided into Ila and IIb. Many lambda clones involved in starch synthesis were isolated from genomic libraries of Ae. squarrosa var. strangulata and hexaploid wheat. FISH experiments with the lambda phage done, E7, containing SBE I gene showed a single major site in both Ae. squarrosa and hexaploid wheat (Rabman et al. 1997). The SBE I gene was located at the distal tip of the long arm of chromosome 7D (homoeologous to 7AL and 7BL) (Fig. 1). Notwithstanding that there are about 10 genes for SBEI based on Southern analysis in wheat, there were no signals from 7A or 7B. This suggests that there may be considerable sequence variation in homoeologous loci. FISH on Ae. squarrosa with the lambda done, F₂, containing SBElla gene shows that the SBEIIa locus was mapped to the proximal region of the long arm of chromosome 2D (34% of the physical length of 2DL from centromere) (Rahman et al. 2001). The clone clearly hybridized to the same region of the long arm of chromosome 2D of Chinese Spring wheat (Fig. 2). No signals were observed on any other chromosome including A- and B-genome chromosomes.

Wheat contains at least four classes of starch synthases in the endosperm, granule bound starch synthases I (GBSSI) and starch synthases I, II, and III (SSI, SSII, SSIII). FISH using the lambda phage done G1 was performed to physically locate the genes for SSII on Ae. squarrosa chromosomes (Li et al . 2003). The results show that the genes are located in the middle of the short arm of chromosome 7D (Fig. 3).

Wheat grains can be classified as having either a soft or hard endosperm texture. The hardness of the grain is controlled by a major locus, Ha, on the short arm of chromosome 5D. GSP-I and puroindolines a and b are regarded as good markers for grain hardness and have been proposed as candidate genes for that trait. Two (GSP-1 clones, RG1 and fl-1, were isolated from a soft bread wheat and Ae. squarrosa lambda phage libraries (Turner et al. 1999). As genes for GSP-1 are found on all group 5 chromosomes, it was important to identify the genomic origin of RG1. This was achieved through FISH. There was a clear signal on the end of the short arm of chromosome 5A (Fig. 4). A weak signal was also observed occasionally at the tip of the short arm of chromosome 5B; the sensitivity of the technique was not sufficient to detect the GSP-1 locus on chromosome 5D. On the other hand, attempts to localize Tt-1 to the short arm of chromosome 5D were not successful as the genomic clone included repetitive sequences that hybridized to all chromosomes of wheat even if competitive DNA was added. These results emphasize the differences between the GSP-1 genes in the A and D genomes, with regards to neighboring sequences.

Although the genes for puroindoline-a, puroindoline-b, and GSP-1 are known to be tightly linked to the Ha locus, but the physical location of the genes for puroindoline-a and puroindoline-b has not yet been demonstrated. An extensive study comparing corresponding sequences of diploid and polyploid wheats has unraveled the molecular basis of the evolutionary events observed at the Ha locus (Chantret et al. 2005). FISH was carried out with lambda clones for puroindoline-a and puroindoline-b, but only puroindoline-b localized to the short arm of chromosome 5D (Mukai et al. 1998). FISH was therefore carried out with PCR products covering the gene and sequences approximately 1 kb upstream or downstream of the gene. The total length of the probe was 2.1 kb for puroindoline-a, 2.8 kb for puroindoline-b, and 1.8 kb for GSP-1. With these probes the sequences for these three genes were shown to be physically located at the distal end of the short arm of chromosome 5 of Ae. squarrosa (Turnbull et al. 2003). This showed that there was correspondence between the genetic and physical maps obtained for this chromosome and the tight linkage between the three markers was not merely due to anomalously low recombination.

Two clones having a debranching enzyme gene (a sugary-type isoamylase gene), DBEI and 12, were isolated from Ae. squarrosa BAC and lambda phage libraries, respectively (Rahman et al. 2003), and their chromosomal localizations were confirmed by FISH using PCR amplificants as probes. Consecutive FISH of Ae. squarrosa chromosomes, using DBEI BAC clone probe first (Fig. 5a) and then lambda done 12 probe (Fig. 5b), did not yield any discrete signals. On the other hand, their PCR fragments having only the gene sequences amplified, with no amplification of repetitive sequences, unraveled clear homologous hybridization signals confirming their localization in the proximal region of the short arm of chromosome 7D (Fig. 5c).

Genome analysis at a glance

The classical methods of genome analysis, like the investigations on chromosome pairing at meiosis and pollen fertility of F₁ hybrids have various limitations, e.g. difficulty in obtaining hybrid plants in trees, effect of Ph genes, and so on. The genomic in situ hybridization (GISH) that uses total genomic DNA as the probe is one of the excellent technologies suitable for the visualization of whole genome in the species hybrids and polyploids. GISH has overcome the problems associated with classical methods of genome analysis and successfully discriminated the genomes of many polyploid crops including wheat, and even woody species where it takes long time to observe the chromosome association at meiosis (Raina et al. 1998), Multicolor FISH using total genomic DNA probes is called multicolor GISH and has been applied to genome analysis of polyploid Aegilops and Triticum species and allopolyploids of other taxa (Mukai 1996).

Mukai et al. (1993a) succeeded in simultaneous discrimination of the three genomes in hexaploid wheat using multicolor FISH. Biotin-labeled total genomic DNA of the A-genome progenitor Triticum urartu, digoxigenin-labeled genomic DNA of the D-genome progenitorAe. squarrosa, and unlabeled genomic DNA of one of the possible B-genome progenitors,Ae. speltoides, were used as probes and hybridized to chromosome DNA of T. aestivum cv. Chinese Spring. For detection, two fluorochromes, FITC-conjugated avidin and rhodamine-conjugated anti-digoxigenin, were used. The biotin-labeled probe hybridized to the A -genome chromosomes of wheat could be detected by yellow fluorescence, and the digoxigenin-labeled probe hybridized to the D-genome chromosomes of wheat detected by orange fluorescence. The B-genome chromosome showed faint fluorescence as a result of cross-hybridization of the A- and D-genome probes. As such, the A-, B-, and D-genome chromosomes could be simultaneously detected by their yellow, brown, and orange fluorescence.

Application of BAC-FISH

As bacterial artificial chromosome (BAC) vector can accommodate large-insert genomic DNA, BAC libraries are used most widely at present for analyzing complex plant genomes. Physical mapping by FISH using BAC clones as probes is called BAC-FISH and has been successful in plant species with relatively small genomes, such as sorghum, cotton, rice, Arabidopsis, tomato, potato, and wild sweet potato. The large-scale BAC-FISH analysis in the large genome plants, such as wheat and onion, has been studied at conventional molecular cytogenetical level (Suzuki et al. 2001; Suzuki and Mukai 2004; Zhang et al. 2004a, 2004b).

Randomly selected 202 BAC clones of Ae. squarrosa were applied to FISH analysis on chromosomes of common wheat in the absence of C₀t -1 genomic DNA (Suzuki and Mukai 2004). They did not use C₀t -1 genomic DNA as competitor in the BAC-FISH screening because cytogenetic markers detecting FISH signal without competitive genomic DNA are useful in simultaneous detection of the target loci and the markers by multicolor FISH. Most of the clones (163 clones) were hybridized with entire chromosomes as dispersed signals that maybe due to the repetitive sequences dispersed on the wheat genome. These clones were classified into three types based on the signal pattern. Signals of the 91 clones were detected uniformly on the chromosomes. On the other hand, mottled signals on the chromosomes were detected for 45 clones. Interestingly, signals of 27 clones are more specific to the D genome; these clones might contain D-genome specific repetitive sequences. Localized signals were obtained in 19 clones. The signals of 10 of the 19 clones showed centromeric localization, that of two clones showed sub-telomeric localization, and banded signals were obtained in the remaining seven clones. The clones showing the localized signals on centromeres or subtelomeres are useful to characterize the location of the centromeres and telomeres during the mitosis and meiosis. In the clone showing banded signals, one clone showed the signals on nine regions of five chromosomes; two separate loci on different arms of chromosome 1D, each set of two closely linked loci on chromosomes 4D, 5D and 6D, and one locus on chromosome 2D. However, there are no clones detecting a single locus by the BAC-FISH, suggesting that the FISH signals on the specific region are hard to be obtained in this method.

Zhang et al. (2004a, 2004b) selected 56 RFLP locus-specific BAC clones from libraries of Ae. squarrosa and A-genome diploid T. monococcum. Different types of repeated sequences were identified using BAC-FISH: two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to pAs1; one BAC clone hybridized to the centromeric regions of wheat; one BAC clone hybridized to five D-genome chromosomes pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These clones can be used for chromosome identification. In addition to the above tandem repeats, three dispersed repeats were identified: the BAC clone 676D4 from the T. monococcum library preferentially hybridized to the A-genome chromosomes of wheat and two BAC clones, 9110 and 9M13, from the Ae. squarrosa library hybridized to the D-genome chromosomes. These clones are useful in simultaneously discriminating the three different genomes in hexaploid wheat, and in identifying intergenomic translocations in wheat. Sequencing data show that all these repeats are transposable elements, indicating the important role of retrotransposons in the genome evolution of wheat.

FISH analysis on DNA fibers in Triticeae

DNA probes of the 18S-5.8S-26S rRNA and 5S rRNA genes in common wheat (Chinese Spring) were used in fiber FISH experiments to estimate the sizes of these multigene families (Yamamoto and Mukai 1998). The hybridization signals of the 18S-5.8S-26S rRNA genes in EDFs were traced with lengths of up to 625 μm. Taking the value of the B-form DNA equivalent to 3.27 kb for 1 μm (Fransz et al. 1996), the microscopical length of the 18S-5.8S-26S rRNA signals was estimated to correspond to a molecular size of 2 An. In Chinese Spring wheat, there are 9,100 copies of the gene per haploid genome (Flavell and O'Dell 1979). About 100 - 5,000 copies are present at nucleolar organizing region of each satellited chromosome. The length of the repeating unit of rDNA is 8.9 kb. The length of 2 Mb DNA corresponds to 225 copies of one rDNA unit.

The 5S RNA genes are independent of the rDNA and are organized in tandem repeats. In wheat, there are two lineages for the 5S RNA multigenes having repeats of different lengths of 500 bp and 400 bp. There are two types of signals: continuous and discontinuous fluorescent strings. Three to ten discontinuous strings within the 50-150 kb size range were lined up in tandem (a total of 300 to 950 kb). On the other hand, continuous signals were observed as fluorescent strings with lengths of 380 to 1,200 kb. The maximum size (1,200 kb) of 5S rRNA gene cluster is estimated to be 2,400 to 3,000 copies.

FISH analysis of the centromeric region on EDFs visualized that the Ty3/gypsy retrotransposon-like elements are tandemly repeated in wheat centromeres (Fukui et al.2001). The centromeric retrotransposon-like elements consist of highly conserved integrase and CCS1 sequences. Fluorescent signals of each sequence were clearly detected as a dot although the integrase and CCS1 fragments used as probes were about 800 bp and 1.7 kb, respectively. The average interval between the integrase sequences was 50-65 kb.

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by FISH to extended DNA fibers of rye, employing DNA probes of lambda phage clones containing the w-secalin gene (Yamamoto and Mukai 1998, 2005). The fluorescent signals on extended DNA fibers of rye revealed continuous strings of 45 μm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2 kb fragment including the entire coding region of the w-secalin gene and a 1.0 kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. Physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of Sec-1 locus having 15 copies of the gene. The findings based on fiber FISH analysis support the contention that the w-secalin genes are arranged in a head to tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb (Clarke et al. 1996). In transgenic plants, fiber FISH can physically map the transgenes directly on extended DNA fibers. This method is complementary to PCR, Southern blot and sequence analyses (Jackson et al. 2001; Nakano et al. 2005) and is discussed later in this article.

Recently molecular combing has made progress as a new technique to map directly cloned DNA sequences on individual stretched DNA molecules. Application of molecular combing FISH facilitates determining the structure of a clone, copy number of genes, and the order of genes and specific sequences. A small fragment (2.5 kb) of the starch branching enzyme I gene (Sbe-1) was mapped on DNA molecules of a BAC clone (SBEI) by FISH (Suzuki et al. 2003). Rahman et al. (1997) presumed existence of 4-5 copies of the Sbe-1 gene in the Ae. squarrosa genome, and mapped the genes on the short arm of chromosome 7D. Three clear hybridization signals within 30 kb of each other were observed on the majority of the insert fragments of BAC SBEI molecules (Fig. 6). The results indicate that DNA fragments as small as 2.5 kb can be visualized on BAC molecules using this technique and there exist at least three copies of the gene in BAC SBEI.

The orientation of the seed storage protein and grain-hardness genes was analyzed on wheat BAC DNA molecules (circular and linear types) using BAC clones forming a contig (Yamamoto and Mukai 2004). The accurate determination of the overlap between the adjacent clones has also been elucidated. This saves time and labor in analysis and provides useful information for construction of BAC contig. Thus, the visual information by high resolution mapping will be useful for detailed analysis of structure and organization of large insert clones.

Application of FISH to wheat breeding

Limitation of GISH was reported in routine screening for alien introgression in wheat (Lukaszewski et al. 2005). GISH technique failed to reveal the presence of some distally located breakpoints. The limit of resolution was at least 3.5 cM (7%) of the relative genetic lengths of chromosome arms in configurations with proximal rye and terminal wheat segments when rye DNA was used as a probe. When wheat DNA was used as a probe, a terminal wheat segment estimated to be 1.6 cM in length could not be visualized.

The most important task in current plant breeding research is to introduce a set of agronomically useful genes in a given crop across the barriers of taxonomy and reproductive isolation. It is therefore, necessary in practical plant genomics that suitable transformation systems are established that facilitate introduction / integration of large genomic fragments loaded with several desired gene functions from a distant gene source into the recipient crop beyond the barrier of reproductive isolation.

FISH could complement such efforts by supplementing information on size of genomic insert and its possible integration sites that will have value in understanding amenability of genomic insertion and its acceptability and expression. This is very important in practical plant breeding since very little is known about where transgenes land, and what effect do they impart onto the recipient organism in terms of stability and expression.

Synteny between wheat and rice genes

Genomics concerned with the systematic molecular characterization of whole genome is important for obtaining an overview of the genome organization and to provide the basic information for isolating specific genes or DNA sequences. At present various projects in many higher plants are at various stages of sequencing and genome analysis.

Genomic sequencing provides information about global genome structure and organization, regulatory regions, transposable elements, and non-coding sequences. Comparative genomics is the study of the similarities and differences in structure and function of genetic information across taxa at the DNA level using molecular tools (Paterson et al. 2000). In cereals, covering most of the world's food and feed crops, a consensus map of 12 grass genomes is now available and it represents the chromosome segment of each genome relative to rice on the basis of the mapping of anchor DNA markers (Gale and Devos 1998; Devos and Gale 2000). However, the comparison of gene location along chromosomes has been mainly conducted in terms of genetic map, and not physical map. Therefore we need to determine the accurate position of genes of interest by physical mapping by FISH.

Cereal genomes are highly variable in size, ranging from 430 Mb in rice to 16,700 Mb in hexaploid wheat. Comparative genomic analysis at the genetic-map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene-coding regions between cereals showed that the distance between the genes is correlated with the genome size. In comparison to rice, large amount of DNA is lying in between the genes on wheat chromosomes (Table 1, Fig. 7). On the other hand, rice and wheat genomes contain regions that are highly enriched in genes with very little or no repetitive DNA. Telomeric regions of both genomes revealed a density of one gene per 5~15 kb, very similar to the gene density in Arabidopsis thaliana. For example, at least eight genes were found in 94 kb DNA of the wheat Ha locus region (Chantret et al. 2005). The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species.

We have mapped several genes on wheat chromosomes by FISH involved in starch synthesis and compared the position of these genes on linkage map of rice (Li et al. 2003). The results show that gene synteny was well conserved in the wheat and rice genomes, including even in large blocks often comprising entire chromosome, despite huge differences in the genome size (Fig. 8).

Trans-introduction of large wheat genomic fragments into rice

In order to expand genetic variability in rice, we have attempted to introduce the large fragments of genomic DNA containing agronomically important genes of wheat into rice. We have successively introduced huge DNA fragments via BAC clones from wheat into rice by 'genome fusion' method. In order to achieve this, as a first step, we developed a system that facilitates efficient introduction of huge mass of wheat genomic DNA into rice. Our prime goal is to realize bread making and udon noodles features in rice powder, for which we have focused our efforts on the introduction of three types of gene clusters from wheat into rice, i.e. starch synthesis-related genes, grain hardness-related genes and prolamin-related genes. It is surmised that full realization of targeted objectives would result in expanded rice consumption, necessitating increased rice cultivation, facilitating environmental conservation, and also help import-substitution for wheat flour.

The 75 kb of Ae. squarrosa genome insert containing the wheat isoamylase 1 (TaISA1) gene was transformed to rice by Agrobacterium -mediated transformation method using BAC vector (Kubo et al. 2005). The presence of the transgenes in regenerated plants was confirmed by PCR analysis using primers for a part of the gene. Then, to detect wheat DNA fragments cytologically, we carried out FISH experiments using the original large DNA fragment as a probe in several lines of transgenic rice. Two hybridization signals were observed in metaphase chromosomes in the homozygous T₂ plants of each line (Fig. 9). Most signals appeared at the terminal or distal regions of rice chromosomes. The fragments of wheat genome DNA were stably transmitted to offspring and the transgenes were expressed in rice.

Little information is available about the organization and stability of large fragments of foreign DNA in transgenic plants. We also introduced a 92-kb DNA fragment of the wheat Ha-locus region into rice by Agrobacterium -mediated transformation (Nakano et al. 2005). The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers. A long and contiguous red signal with two short green signals was observed on the DNA fiber (Fig. 10). The length of the integrated region was estimated to be 140 kb. The possible integration events are summarized as follows: two copies of the BAC 8 construct have been integrated in inverted orientation, a deletion has occurred in one of the copies, and short segments of T-DNA and insert DNA have been duplicated and transferred to the terminal region. Rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. This result suggested that the large T-DNAs integrated by Agrobacterium -mediated transformation are subject to be reorganized in transgenic rice plants. The integration of intact T-DNA should, therefore, be routinely confirmed by molecular cytogenetic analysis in the. case of transgenic plants transformed with large transgenes using Agrobacterium.

Concluding remarks

Molecular approaches to wheat cytogenetics have generated useful knowledge base on alien relatives and genomic resources of valuable gene pool, enriching wheat genomics to complement breeding research. Isolation and molecular characterization of multiple repetitive sequences, each representing a substantial fraction of the genome followed by their physical localization has provided a novel top-down chromosomal approach to complement bottom-up DNA markers to facilitate clone based genome analysis to visualize genomic organization, chromosome structure and landmarks for looking at genes, their clustering and orientation. This has far reaching repercussions on transfer and stable integration of transgenes across the taxonomic barriers. Availability of genome fusion technology opens new vistas on transfer of fully functional genes to realize novel genomic complementation. Such developments have opened immense possibilities in plant genomics research in general and wheat genomics in particular. To make best use of molecular cytogenetics approaches to wheat research in the 21st century, it would be most desirable to focus our efforts on identification of agronomic / qualitative and developmentally useful genes in wheat, its alien relatives as well as wild resources, and assign them to their physical linkage groups with targeted objectives of their transgene integration for value addition. With the current state of technological advancement and future possibilities, this could be conveniently done following molecular cytogenetic analysis (1) from chromosome level to DNA fiber level; (2) from individual analysis to comparative analysis; (3) from structural analysis to functional analysis; (4) from segregated comprehension to integrated comprehension; and (5) from basic study to applied study, following an integrated multidisciplinary approach.

Acknowledgements

I consider it my pleasant duty to record the support of my associates in the laboratory, Drs. Maki Yamamoto, Sadiquer Rahman and Go Suzuki, and other young researchers over a period of time, who contributed and sustained our research efforts in understanding molecular cytogenetics of wheat.

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