BACKGROUND
chromosome maps of higher plants were originally constructed by analyzing markers obtained from differences in qualitative traits, such as seed shape or cotyledon color.
although these maps were rather sparse because the number of traits that could serve as markers was limited, they were effective in determining the distance between and order of loci related to these traits.
in the past two decades, it has become possible to construct high-density maps for almost all areas of chromosomes using dna markers based on sequence polymorphisms.
such chromosome maps have become essential tools for linkage analysis of important traits, as well as for genome evolution analysis.
compared to amplified fragment length polymorphism  and random amplified polymorphic dna  markers, gene-derived markers are more useful for comparative genomics, and can also serve as phenotype-linked functional markers  <cit> .
grass species are very closely related to one another in comparison to plants in other families  <cit> , and as a result, a high degree of similarity is expected in the structure and sequence of grass orthologous genes.
consequently, a great deal of information has been obtained regarding intergenomic synteny and collinearity by using orthologous genes as anchor markers  <cit> .
rice has the smallest genome size among all cereal crops, and much genetic information related to agriculturally important traits has been obtained for this crop.
the complete genome of the rice cultivar "nipponbare" has been sequenced and annotated  <cit> , and this data has been used for comparative genomic studies with other grass species.
common wheat  evolved by polyploidization about  <dig>  years ago, after which it quickly spread and was domesticated throughout the world  <cit> .
globally, it is now the most widely cultivated grain, and a large volume of data has been collected regarding genetic factors involved in important traits such as yield, quality and biotic/abiotic stress resistance  <cit> .
due to both its high agricultural importance and rapid evolution, the level of interest in genomic research on wheat is high.
the international triticeae mapping initiative  <cit>  has led a large-scale chromosome mapping project, and data have been organized in the form of the graingenes public database  <cit> .
using various grass plant-derived cdnas as probes, approximately  <dig>  rflp markers have been mapped on linkage maps to date.
furthermore,  <dig>  wheat est sequences have been located on physical maps of wheat by using these ests as probes in southern hybridizations  <cit> .
in comparison to rflp markers, pcr-based markers require less dna and facilitate high throughput analysis.
thus, the pcr-based marker has become the main tool for genetic analysis.
in recent years, numerous pcr-based markers, referred to as "est-pcr markers", have been developed by designing primers based on est sequences.
already more than  <dig>  wheat est sequences have been registered with public databases.
most wheat est-pcr markers were designed from ests that contained a simple sequence repeat   <cit> , and ests with ssrs  are estimated to represent  <dig> % of total wheat unigenes  <cit> .
in order to develop new est-pcr markers of wheat, it is necessary to make the best use of the abundant wheat est resources, specifically the remaining  <dig> % of unigenes.
in allohexaploid wheat, pcr products derived from homoeologous genes are often amplified simultaneously.
additionally, if an est sequence used for marker design is derived from a gene that has paralogues, the number of amplified products is likely to be magnified in a polyploid plant species such as wheat.
such problems hinder the mapping of est-pcr markers on chromosomes.
recently, sophisticated methodologies for the development of est-pcr markers for plants have been reported  <cit> .
however, in polyploid species, the production of multiple pcr products is unavoidable even when using these methods.
for example, feltus et al.
<cit>  reported that in the development of est-pcr markers of orphan crops, up to four products derived from alleles at a particular locus were thought to be produced in an autotetraploid line of cynodon dactylon, and problems were encountered in separating these products.
consequently, when developing est-pcr markers for wheat, it is essential to eliminate est sequences derived from paralogous genes and to establish a method to efficiently sort products derived from homoeologous genes.
although high degrees of similarity are found among wheat homoeologous genes, intron regions have a greater degree of polymorphism in terms of insertions/deletions and base substitutions than do exon regions  <cit> .
correspondingly, in the seven sets of wheat genes for which the genomic sequences of the three homoeologous genes are known , insertions/deletions and base substitutions are more common within intron than within exon sequences .
therefore, if pcr products incorporate intron regions, it may be possible to separate products derived from homoeologous genes using a simple technique such as agarose gel electrophoresis.
when designing primers for est-pcr markers, primers that span exon-exon junctions in the ests prevent amplification of the product from the target gene.
to increase the success rate of pcr, a technique for predicting exon-exon junctions in ests is required.
based on the assumption that exon regions and exon-intron structures of orthologous genes are highly conserved amongst grass species, we developed an interactive system to design primers in exon regions flanking an intron, using a wheat est dataset selected by a proprietary program.
we confirmed that the primer sets thus designed amplified specific pcr products from target genes, and that the products derived from homoeologous genes could be effectively separated on agarose gels.
RESULTS
establishment of pcr-based landmark unique gene  system
as shown in the flowchart in figure  <dig>  we developed an interactive system for designing wheat pcr primers.
with this system, single-copy genes were extracted by a blastn search from among all rice cdna sequences of the gene models on tigr pseudomolecules version  <dig> .
we defined the loci corresponding to these genes as landmark unique gene loci .
the system extracted a total of  <dig>  lugs, which account for  <dig> %  of the all predicted gene loci  <cit> , and there were  <dig>  lugs with high homology to wheat ests in the unigene databases.
from these  <dig>  lugs,  <dig>  taest-lugs were selected as template loci for potential plug markers, as they reached our designated minimum length threshold and spanned an area that included an intron in the corresponding rice gene .
the number of taest-lugs accounted for  <dig> % of the lugs or  <dig> % of the total rice gene loci.
figure  <dig> shows the distribution of lugs and taest-lugs for each rice chromosome .
higher densities of lugs and taest-lugs were generally observed in the distal as opposed to the proximal regions of the chromosome arms.
the numbers of taest-lugs also varied greatly among chromosomes: high on chromosomes  <dig> and  <dig>  but low on chromosomes  <dig> and  <dig>  the number of taest-lugs on chromosome  <dig> was more than five times that for chromosome  <dig> .
to estimate the number of potential plug markers on each wheat chromosome group, taest-lugs were assigned to wheat chromosomes based on previously reported data for rice and wheat synteny  <cit> .
as an example, rice chromosomes  <dig> and  <dig>  which have fewer taest-lugs, showed synteny with regions of the wheat chromosome groups  <dig> and  <dig>  respectively .
however, wheat chromosome group  <dig> also corresponds to a large part of rice chromosome  <dig>  while group  <dig> corresponds to the remaining part of chromosome  <dig> and all of chromosome  <dig>  therefore, the distribution bias of taest-lugs on wheat chromosome groups is lower than that for rice chromosomes, and as a result, the number of potential plug markers per wheat chromosome group was estimated to be 450– <dig> .
pcr amplification with plug primers
to demonstrate the performance of the plug system, one marker was randomly selected from the short and long arms of each of the  <dig> rice chromosomes, giving a total of  <dig> markers.
to design pcr primers for these markers, the following settings were used for the plug system: melting temperature was 55–65°c , primer length was 18– <dig> bases , and the desired size of amplified fragments estimated based on the rice genome was approximately  <dig> kb .
one taest-lug was randomly selected from each arm of the  <dig> rice chromosomes, and from these loci,  <dig> plug primer sets were produced.
the table shows the tigr rice locus id, the accession numbers of wheat ests exhibiting homology , the sequences of forward and reverse primers, the size of the region flanked by the primers along the rice genomic sequence, and the size of the region flanked by the primer along the wheat est sequence.
pcr was performed with the  <dig> primer sets using genomic dna from the wheat variety chinese spring as a template.
electrophoresis on a 1% agarose gel clearly separated the pcr products into one to three bands: three bands were obtained with two primer sets  , two bands with five primer sets  , and a single band with the remaining  <dig> primer sets  .
the size of these products ranged from  <dig> to  <dig>  bp, and each product was larger than the size predicted from the wheat est , suggesting that all pcr products contained introns.
with type i primer sets, it is likely that the three products corresponded to products derived from three homoeologous genes.
with type ii and iii primers, products from more than one gene were likely present in some of the bands detected in the 1% agarose gel.
assignment of plug markers to wheat chromosomes
to assign the  <dig> plug markers to wheat chromosomes, pcr products obtained from nullisomic-tetrasomic lines were either separated on 1% agarose gels, or were digested with either haeiii or taqi restriction enzyme then separated on 4% agarose gels.
with type i marker no.
<dig>  we determined that the three bands obtained were amplified from chromosomes 1a, 1b and 1d, while with type i marker no.
<dig>  products were from chromosomes 7a, 7b and 7d .
types i, ii and iii indicate that 1% agarose gel electrophoresis of pcr products resulted in the separation of three, two, or single bands, respectively.
plug markers were assigned to chromosomes by electrophoresis on 1% agarose gels, or by electrophoresis of haeiii- or taqi-digested fragments on 4% agarose gels.
the table also shows the annotations of taest-lugs that were used for marker development.
for marker no.
<dig> , the longer band on the 1% agarose gel was the product derived from chromosome 5a .
after separating taqi-digested products on a 4% agarose gel, we confirmed that the shorter band contained two products derived from chromosomes 5b and 5d .
for the other type ii markers, we identified three chromosomes for marker no.
<dig>   <dig> and  <dig>  and a single chromosome for marker no.
<dig> .
according to pcr-rflp analyses, the single band detected on a 1% agarose gel with marker no.
<dig> consisted of the products derived from chromosomes 2a, 2b and 2d .
similarly, for the other type iii markers, we determined that four markers  could be assigned to three chromosomes, five markers  to two chromosomes, and two markers  to a single chromosome .
in total,  <dig> of the  <dig> markers were assigned to at least one chromosome.
sequence comparison of the plug markers
to confirm that the  <dig> markers were derived from the target genes, the pcr products were cloned and sequenced.
sequences of the clones were compared to the wheat est sequences used for primer design, confirming that all markers were derived from the target genes and contained at least one intron.
the sequences of the exon regions were compared to the est sequences, and very high degrees of similarity  were observed.
five of the markers contained two related but not identical sequences, while  <dig> of the markers contained three related sequences.
marker no.
<dig> was unusual in that five related sequences were found among the clones derived from this product .
for all markers, sequencing of products confirmed that haeiii or taqi restriction sites occurred at the appropriate positions to result in the restriction fragment size polymorphisms that were used to assign markers to chromosomes .
for marker no.
<dig>  which could not be assigned to a chromosome, the three sequences obtained were compared in a pair-wise manner.
the degrees of similarity between pairs ranged from  <dig> to 97% in the exon regions and from  <dig> to 92% in the intron region.
these sequence alignments also indicated that insertion/deletion polymorphisms existed in the intron regions, but not in the exon regions .
using hinfi recognition site polymorphisms, the marker could be assigned to chromosomes 5a, 5b and 5d.
for each of the  <dig> primer sets, similarity between sequences derived from homoeologous genes was lower in the intron regions  than in the exon regions .
it is notable that all insertion/deletion polymorphisms among homoeologous gene sequences were found in the intron regions.
discussion
gene expression data indicates that in rice, about 48% of all predicted genes are expressed, based on the ratio of expressed transcriptional units  to total tu .
according to this proportion, the number of lugs corresponding to expressed genes is approximately  <dig> , whereas in this study, a total of  <dig>  lugs showed high homology to available wheat ests.
since the wheat unigene datasets consist of numerous est sequences derived from multiple libraries, and are therefore considered to cover most expressed genes, the difference in these numbers is thought to be primarily due to sequence diversity between wheat and rice, rather than the number of available wheat unigenes.
therefore, using the same blastn search threshold levels as were used in this study, a significant increase in the number of lugs with homology to wheat ests is not likely to be observed in the future.
in contrast, improvements in cdna sequence databases would result in an increase in the proportion of these lugs that could be used for marker development.
the availability of sequences from a wheat full-length cdna library, for example, should result in a large increase in taest-lugs.
the plug markers were produced from  <dig> randomly selected taest-lugs, yet all of them amplified specific products.
therefore, when designing primers from taest-lugs, quite a high success rate can be expected.
our success rate was higher than rates achieved with pearl millet and sorghum est-pcr markers based on the rice genome database  <cit> .
for example, feltus et al.
<cit>  compared the rice genome and sorghum unigenes and prepared  <dig> conserved intron-spanning primer  sets.
these cisp sets achieved a success rate of 81%.
a high success rate was observed in our study because, unlike the automatic pipeline systems for the cisps, the plug system allows pcr product length and primer picking condition data to be entered independently .
this manual procedure enables us to avoid areas close to exon-exon junctions where sequences can be ambiguously aligned, and also allows us to identify highly conserved regions.
in addition, an optimal primer set can be selected from the three candidate primer sets recommended by the system.
furthermore, by adjusting the position of the primer, the size of the expected product can be set from  <dig> to  <dig>  bp.
this flexibility is useful in redesigning primers to amplify different regions of the taest-lugs.
in wheat, est-pcr markers often yield multiple products originating from paralogous genes, as well as from homoeologous genes.
thus, we developed a system based on rice single-copy gene loci under the hypothesis that the copy number of genes was highly conserved between rice and wheat.
this hypothesis appears to be correct, as in the majority of cases the number of products amplified with the plug primers was only two or three.
in addition, sequence analysis confirmed that plug amplification products were derived from target genes and contained introns in the expected positions.
the genes represented by taest-lugs were highly conserved between wheat and rice, not only in terms of copy number but also in sequence and exon-intron structure, and the plug markers appeared to represent the wheat orthologs of the genes on the taest-lugs.
nullisomic-tetrasomic analysis indicated that some amplification products could not be clearly separated and assigned to chromosomes using the basic methods of this study.
however, comparisons between homoeologous sequences allowed us to detect insertions/deletions and base substitutions related to recognition site polymorphisms other than haeiii and taqi.
therefore, by using additional restriction endonucleases in the pcr-rflp analysis, it should be possible to assign all sequences to chromosomes.
indeed, using hinfi, marker no.
<dig> was assigned to the group  <dig> chromosomes .
furthermore, sequence analysis of all twenty-four markers revealed that while exon regions were generally equal in length, intron regions had insertion/deletion polymorphisms every  <dig> bp on average .
this suggests that products derived from homoeologous genes can be sorted based on differences in length, without resorting to sequencing or rflp analysis.
generally, to detect slight size differences in pcr products, high-resolution electrophoresis, which is both time-consuming and expensive, is required.
however, in recent years, a high-resolution low-cost capillary electrophoresis device has become available  <cit> .
such a device may enable high throughput analysis, which would be required for the development of genome-wide plug markers.
once markers are assigned to chromosomes, a series of deletion lines  <cit>  is available for determining marker locations within the respective chromosomes.
nineteen of the  <dig> markers could be assigned to at least one chromosome by the simple detection methods of either electrophoresis on a 1% agarose gel or restriction digestion with haeiii or taqi followed by electrophoresis on a 4% agarose gel.
sixteen markers were assigned to the same chromosomes as was predicted by the previously reported synteny data for rice and wheat  <cit> .
for example, synteny has been reported between rice chromosome  <dig> and wheat chromosome group  <dig>  and in the present study, two markers  that were produced based on taest-lugs from rice chromosome  <dig> were located on wheat chromosomes 3a, 3b, and 3d .
marker no.
<dig>  which was located on chromosomes 5a and 4d, was developed based on the taest-lug in the short arm of rice chromosome  <dig>  which corresponds to wheat chromosome group  <dig>  therefore, it can be assumed that marker no.
<dig> is located on a known reciprocal translocation region between 4a and 5a  <cit> .
these results indicate that the synteny between rice and wheat facilitates the effective development of est-pcr markers on targeted wheat chromosomes.
on the other hand, markers no.
<dig> and  <dig> were predicted to be located on chromosome groups  <dig> and  <dig>  respectively; however, they were actually located on chromosome group  <dig> and chromosome 7a, indicating some synteny perturbation between rice and wheat in these areas.
furthermore, marker no.
<dig> was predicted to be located on chromosome group  <dig>  but it was actually located on 3a, 5a, and 5b chromosomes.
further investigation revealed that the marker was also located on 3b and 5d chromosomes .
this suggests that marker no.
<dig> is derived from paralogous genes resulting from a gene duplication event in the wheat genome; furthermore, these genes are located in regions where the synteny between rice and wheat is perturbed.
the est sequences of the plug markers were used as queries in blastn searches of the graingenes  <cit>  databases.
results indicated that  <dig> of the  <dig> est sequences showed high similarity with bin-mapped ests, three ests showed high similarity with est-ssr sequences, and three ests showed high similarity with sequences in the sequenced probe database , with some marker sequences being present in more than one of these databases.
totally  <dig> out of  <dig> markers were based on ests that have not previously been used for marker development.
of the remaining  <dig> markers,  <dig> markers are derived from genes that were assigned to wheat chromosomes in previous studies  <cit> .
no contradictions in the assigned chromosomes were observed between this study and previous studies; however, the numbers of chromosomes the corresponding genes were assigned to varied .
for example, in a previous bin mapping study, the gene corresponding to marker no.
<dig> was located on chromosome 3b; however, in this study, it was located on chromosomes 3a, 3b, and 3d.
hence, mapping information was further enriched with mutually complementary data.
in summary, by designing primers from the approximately  <dig>  taest-lugs extracted using the system, it would be expected that  <dig>  markers could be assigned to at least one chromosome by the same methods used here, and that  <dig>  of these markers would be novel.
furthermore, even if the markers developed are not new, they may be useful for enriching mapping information.
in this study, we showed that the plug system could be used to develop new markers on specifically targeted wheat chromosomes by taking advantage of synteny with rice chromosomes.
this implies that the plug markers can accurately show the chromosomal locations of wheat genes that are orthologous to rice genes, and that the markers can act as scaffolds for comparative genomics.
furthermore, locus specific sequences can be readily obtained from the plug markers, and by utilizing plug markers as probes, it will be possible to quickly identify target clones from the huge bac library for wheat.
the locus-specific sequences obtained from plug markers are also potentially very useful for surveying sequence polymorphisms among wheat cultivars, allowing the development of new markers near quantitative trait loci .
we are presently using the taest-lugs to develop plug markers for the entire wheat genome and to carry out bin mapping.
a web site is under construction to allow public access to the plug system.
CONCLUSIONS
with the plug system,  <dig>  taest-lugs were shown to be useful as reliable standards for wheat est-pcr marker development.
in a study using  <dig> randomly selected taest-lugs, half showed high homology to wheat est sequences that have not previously been used for marker development.
therefore, by carrying out large-scale wheat marker development using the plug system, we can potentially double the number of gene-derived markers.
in comparisons of intron sequences from wheat homoeologous genes, insertion/deletion polymorphisms were found in almost all cases.
this suggests that the sorting of multiple pcr products derived from homoeologous genes, which is a major block for est-pcr marker development, can be resolved by including an intron sequence in pcr products.
therefore, the plug system, which semi-automatically extracts primer sets flanking an intron sequence, is a very effective tool in wheat marker development.
with this system, it is now possible to sort homoeologous genes using a low-cost and convenient separation method, thus allowing large-scale pcr-based marker development for wheat.
since the plug system is based on orthologous gene conservation, markers produced using the system can also be used as accurate anchor markers for genomic research comparing rice and wheat.
furthermore, plug markers can show the positional relationships of wheat homoeologous genes, and as a result, these markers are expected to contribute greatly to research on synteny dissociation among wheat homoeologous chromosomes.

 chromosome maps of higher plants were originally constructed by analyzing markers obtained from differences in qualitative traits, such as seed shape or cotyledon color. although these maps were rather sparse because the number of traits that could serve as markers was limited, they were effective in determining the distance between and order of loci related to these traits. in the past two decades, it has become possible to construct high-density maps for almost all areas of chromosomes using dna markers based on sequence polymorphisms. such chromosome maps have become essential tools for linkage analysis of important traits, as well as for genome evolution analysis. compared to amplified fragment length polymorphism and random amplified polymorphic dna markers, gene-derived markers are more useful for comparative genomics, and can also serve as phenotype-linked functional markers. grass species are very closely related to one another in comparison to plants in other families, and as a result, a high degree of similarity is expected in the structure and sequence of grass orthologous genes. consequently, a great deal of information has been obtained regarding intergenomic synteny and collinearity by using orthologous genes as anchor markers. rice has the smallest genome size among all cereal crops, and much genetic information related to agriculturally important traits has been obtained for this crop. the complete genome of the rice cultivar "nipponbare" has been sequenced and annotated, and this data has been used for comparative genomic studies with other grass species. common wheat evolved by polyploidization about years ago, after which it quickly spread and was domesticated throughout the world. globally, it is now the most widely cultivated grain, and a large volume of data has been collected regarding genetic factors involved in important traits such as yield, quality and biotic/abiotic stress resistance. due to both its high agricultural importance and rapid evolution, the level of interest in genomic research on wheat is high. the international triticeae mapping initiative has led a large-scale chromosome mapping project, and data have been organized in the form of the graingenes public database. using various grass plant-derived cdnas as probes, approximately rflp markers have been mapped on linkage maps to date. furthermore, wheat est sequences have been located on physical maps of wheat by using these ests as probes in southern hybridizations. in comparison to rflp markers, pcr-based markers require less dna and facilitate high throughput analysis. thus, the pcr-based marker has become the main tool for genetic analysis. in recent years, numerous pcr-based markers, referred to as "est-pcr markers", have been developed by designing primers based on est sequences. already more than wheat est sequences have been registered with public databases. most wheat est-pcr markers were designed from ests that contained a simple sequence repeat, and ests with ssrs are estimated to represent % of total wheat unigenes. in order to develop new est-pcr markers of wheat, it is necessary to make the best use of the abundant wheat est resources, specifically the remaining % of unigenes. in allohexaploid wheat, pcr products derived from homoeologous genes are often amplified simultaneously. additionally, if an est sequence used for marker design is derived from a gene that has paralogues, the number of amplified products is likely to be magnified in a polyploid plant species such as wheat. such problems hinder the mapping of est-pcr markers on chromosomes. recently, sophisticated methodologies for the development of est-pcr markers for plants have been reported. however, in polyploid species, the production of multiple pcr products is unavoidable even when using these methods. for example, feltus et al. reported that in the development of est-pcr markers of orphan crops, up to four products derived from alleles at a particular locus were thought to be produced in an autotetraploid line of cynodon dactylon, and problems were encountered in separating these products. consequently, when developing est-pcr markers for wheat, it is essential to eliminate est sequences derived from paralogous genes and to establish a method to efficiently sort products derived from homoeologous genes. although high degrees of similarity are found among wheat homoeologous genes, intron regions have a greater degree of polymorphism in terms of insertions/deletions and base substitutions than do exon regions. correspondingly, in the seven sets of wheat genes for which the genomic sequences of the three homoeologous genes are known, insertions/deletions and base substitutions are more common within intron than within exon sequences. therefore, if pcr products incorporate intron regions, it may be possible to separate products derived from homoeologous genes using a simple technique such as agarose gel electrophoresis. when designing primers for est-pcr markers, primers that span exon-exon junctions in the ests prevent amplification of the product from the target gene. to increase the success rate of pcr, a technique for predicting exon-exon junctions in ests is required. based on the assumption that exon regions and exon-intron structures of orthologous genes are highly conserved amongst grass species, we developed an interactive system to design primers in exon regions flanking an intron, using a wheat est dataset selected by a proprietary program. we confirmed that the primer sets thus designed amplified specific pcr products from target genes, and that the products derived from homoeologous genes could be effectively separated on agarose gels.  establishment of pcr-based landmark unique gene system as shown in the flowchart in figure we developed an interactive system for designing wheat pcr primers. with this system, single-copy genes were extracted by a blastn search from among all rice cdna sequences of the gene models on tigr pseudomolecules version. we defined the loci corresponding to these genes as landmark unique gene loci. the system extracted a total of lugs, which account for % of the all predicted gene loci, and there were lugs with high homology to wheat ests in the unigene databases. from these lugs, taest-lugs were selected as template loci for potential plug markers, as they reached our designated minimum length threshold and spanned an area that included an intron in the corresponding rice gene. the number of taest-lugs accounted for % of the lugs or % of the total rice gene loci. figure shows the distribution of lugs and taest-lugs for each rice chromosome. higher densities of lugs and taest-lugs were generally observed in the distal as opposed to the proximal regions of the chromosome arms. the numbers of taest-lugs also varied greatly among chromosomes: high on chromosomes and but low on chromosomes and the number of taest-lugs on chromosome was more than five times that for chromosome. to estimate the number of potential plug markers on each wheat chromosome group, taest-lugs were assigned to wheat chromosomes based on previously reported data for rice and wheat synteny. as an example, rice chromosomes and which have fewer taest-lugs, showed synteny with regions of the wheat chromosome groups and respectively. however, wheat chromosome group also corresponds to a large part of rice chromosome while group corresponds to the remaining part of chromosome and all of chromosome therefore, the distribution bias of taest-lugs on wheat chromosome groups is lower than that for rice chromosomes, and as a result, the number of potential plug markers per wheat chromosome group was estimated to be 450–. pcr amplification with plug primers to demonstrate the performance of the plug system, one marker was randomly selected from the short and long arms of each of the rice chromosomes, giving a total of markers. to design pcr primers for these markers, the following settings were used for the plug system: melting temperature was 55–65°c, primer length was 18– bases, and the desired size of amplified fragments estimated based on the rice genome was approximately kb. one taest-lug was randomly selected from each arm of the rice chromosomes, and from these loci, plug primer sets were produced. the table shows the tigr rice locus id, the accession numbers of wheat ests exhibiting homology, the sequences of forward and reverse primers, the size of the region flanked by the primers along the rice genomic sequence, and the size of the region flanked by the primer along the wheat est sequence. pcr was performed with the primer sets using genomic dna from the wheat variety chinese spring as a template. electrophoresis on a 1% agarose gel clearly separated the pcr products into one to three bands: three bands were obtained with two primer sets, two bands with five primer sets, and a single band with the remaining primer sets. the size of these products ranged from to bp, and each product was larger than the size predicted from the wheat est, suggesting that all pcr products contained introns. with type i primer sets, it is likely that the three products corresponded to products derived from three homoeologous genes. with type ii and iii primers, products from more than one gene were likely present in some of the bands detected in the 1% agarose gel. assignment of plug markers to wheat chromosomes to assign the plug markers to wheat chromosomes, pcr products obtained from nullisomic-tetrasomic lines were either separated on 1% agarose gels, or were digested with either haeiii or taqi restriction enzyme then separated on 4% agarose gels. with type i marker no. we determined that the three bands obtained were amplified from chromosomes 1a, 1b and 1d, while with type i marker no. products were from chromosomes 7a, 7b and 7d. types i, ii and iii indicate that 1% agarose gel electrophoresis of pcr products resulted in the separation of three, two, or single bands, respectively. plug markers were assigned to chromosomes by electrophoresis on 1% agarose gels, or by electrophoresis of haeiii- or taqi-digested fragments on 4% agarose gels. the table also shows the annotations of taest-lugs that were used for marker development. for marker no., the longer band on the 1% agarose gel was the product derived from chromosome 5a. after separating taqi-digested products on a 4% agarose gel, we confirmed that the shorter band contained two products derived from chromosomes 5b and 5d. for the other type ii markers, we identified three chromosomes for marker no. and and a single chromosome for marker no.. according to pcr-rflp analyses, the single band detected on a 1% agarose gel with marker no. consisted of the products derived from chromosomes 2a, 2b and 2d. similarly, for the other type iii markers, we determined that four markers could be assigned to three chromosomes, five markers to two chromosomes, and two markers to a single chromosome. in total, of the markers were assigned to at least one chromosome. sequence comparison of the plug markers to confirm that the markers were derived from the target genes, the pcr products were cloned and sequenced. sequences of the clones were compared to the wheat est sequences used for primer design, confirming that all markers were derived from the target genes and contained at least one intron. the sequences of the exon regions were compared to the est sequences, and very high degrees of similarity were observed. five of the markers contained two related but not identical sequences, while of the markers contained three related sequences. marker no. was unusual in that five related sequences were found among the clones derived from this product. for all markers, sequencing of products confirmed that haeiii or taqi restriction sites occurred at the appropriate positions to result in the restriction fragment size polymorphisms that were used to assign markers to chromosomes. for marker no. which could not be assigned to a chromosome, the three sequences obtained were compared in a pair-wise manner. the degrees of similarity between pairs ranged from to 97% in the exon regions and from to 92% in the intron region. these sequence alignments also indicated that insertion/deletion polymorphisms existed in the intron regions, but not in the exon regions. using hinfi recognition site polymorphisms, the marker could be assigned to chromosomes 5a, 5b and 5d. for each of the primer sets, similarity between sequences derived from homoeologous genes was lower in the intron regions than in the exon regions. it is notable that all insertion/deletion polymorphisms among homoeologous gene sequences were found in the intron regions. discussion gene expression data indicates that in rice, about 48% of all predicted genes are expressed, based on the ratio of expressed transcriptional units to total tu. according to this proportion, the number of lugs corresponding to expressed genes is approximately, whereas in this study, a total of lugs showed high homology to available wheat ests. since the wheat unigene datasets consist of numerous est sequences derived from multiple libraries, and are therefore considered to cover most expressed genes, the difference in these numbers is thought to be primarily due to sequence diversity between wheat and rice, rather than the number of available wheat unigenes. therefore, using the same blastn search threshold levels as were used in this study, a significant increase in the number of lugs with homology to wheat ests is not likely to be observed in the future. in contrast, improvements in cdna sequence databases would result in an increase in the proportion of these lugs that could be used for marker development. the availability of sequences from a wheat full-length cdna library, for example, should result in a large increase in taest-lugs. the plug markers were produced from randomly selected taest-lugs, yet all of them amplified specific products. therefore, when designing primers from taest-lugs, quite a high success rate can be expected. our success rate was higher than rates achieved with pearl millet and sorghum est-pcr markers based on the rice genome database. for example, feltus et al. compared the rice genome and sorghum unigenes and prepared conserved intron-spanning primer sets. these cisp sets achieved a success rate of 81%. a high success rate was observed in our study because, unlike the automatic pipeline systems for the cisps, the plug system allows pcr product length and primer picking condition data to be entered independently. this manual procedure enables us to avoid areas close to exon-exon junctions where sequences can be ambiguously aligned, and also allows us to identify highly conserved regions. in addition, an optimal primer set can be selected from the three candidate primer sets recommended by the system. furthermore, by adjusting the position of the primer, the size of the expected product can be set from to bp. this flexibility is useful in redesigning primers to amplify different regions of the taest-lugs. in wheat, est-pcr markers often yield multiple products originating from paralogous genes, as well as from homoeologous genes. thus, we developed a system based on rice single-copy gene loci under the hypothesis that the copy number of genes was highly conserved between rice and wheat. this hypothesis appears to be correct, as in the majority of cases the number of products amplified with the plug primers was only two or three. in addition, sequence analysis confirmed that plug amplification products were derived from target genes and contained introns in the expected positions. the genes represented by taest-lugs were highly conserved between wheat and rice, not only in terms of copy number but also in sequence and exon-intron structure, and the plug markers appeared to represent the wheat orthologs of the genes on the taest-lugs. nullisomic-tetrasomic analysis indicated that some amplification products could not be clearly separated and assigned to chromosomes using the basic methods of this study. however, comparisons between homoeologous sequences allowed us to detect insertions/deletions and base substitutions related to recognition site polymorphisms other than haeiii and taqi. therefore, by using additional restriction endonucleases in the pcr-rflp analysis, it should be possible to assign all sequences to chromosomes. indeed, using hinfi, marker no. was assigned to the group chromosomes. furthermore, sequence analysis of all twenty-four markers revealed that while exon regions were generally equal in length, intron regions had insertion/deletion polymorphisms every bp on average. this suggests that products derived from homoeologous genes can be sorted based on differences in length, without resorting to sequencing or rflp analysis. generally, to detect slight size differences in pcr products, high-resolution electrophoresis, which is both time-consuming and expensive, is required. however, in recent years, a high-resolution low-cost capillary electrophoresis device has become available. such a device may enable high throughput analysis, which would be required for the development of genome-wide plug markers. once markers are assigned to chromosomes, a series of deletion lines is available for determining marker locations within the respective chromosomes. nineteen of the markers could be assigned to at least one chromosome by the simple detection methods of either electrophoresis on a 1% agarose gel or restriction digestion with haeiii or taqi followed by electrophoresis on a 4% agarose gel. sixteen markers were assigned to the same chromosomes as was predicted by the previously reported synteny data for rice and wheat. for example, synteny has been reported between rice chromosome and wheat chromosome group and in the present study, two markers that were produced based on taest-lugs from rice chromosome were located on wheat chromosomes 3a, 3b, and 3d. marker no. which was located on chromosomes 5a and 4d, was developed based on the taest-lug in the short arm of rice chromosome which corresponds to wheat chromosome group therefore, it can be assumed that marker no. is located on a known reciprocal translocation region between 4a and 5a. these results indicate that the synteny between rice and wheat facilitates the effective development of est-pcr markers on targeted wheat chromosomes. on the other hand, markers no. and were predicted to be located on chromosome groups and respectively; however, they were actually located on chromosome group and chromosome 7a, indicating some synteny perturbation between rice and wheat in these areas. furthermore, marker no. was predicted to be located on chromosome group but it was actually located on 3a, 5a, and 5b chromosomes. further investigation revealed that the marker was also located on 3b and 5d chromosomes. this suggests that marker no. is derived from paralogous genes resulting from a gene duplication event in the wheat genome; furthermore, these genes are located in regions where the synteny between rice and wheat is perturbed. the est sequences of the plug markers were used as queries in blastn searches of the graingenes databases. results indicated that of the est sequences showed high similarity with bin-mapped ests, three ests showed high similarity with est-ssr sequences, and three ests showed high similarity with sequences in the sequenced probe database, with some marker sequences being present in more than one of these databases. totally out of markers were based on ests that have not previously been used for marker development. of the remaining markers, markers are derived from genes that were assigned to wheat chromosomes in previous studies. no contradictions in the assigned chromosomes were observed between this study and previous studies; however, the numbers of chromosomes the corresponding genes were assigned to varied. for example, in a previous bin mapping study, the gene corresponding to marker no. was located on chromosome 3b; however, in this study, it was located on chromosomes 3a, 3b, and 3d. hence, mapping information was further enriched with mutually complementary data. in summary, by designing primers from the approximately taest-lugs extracted using the system, it would be expected that markers could be assigned to at least one chromosome by the same methods used here, and that of these markers would be novel. furthermore, even if the markers developed are not new, they may be useful for enriching mapping information. in this study, we showed that the plug system could be used to develop new markers on specifically targeted wheat chromosomes by taking advantage of synteny with rice chromosomes. this implies that the plug markers can accurately show the chromosomal locations of wheat genes that are orthologous to rice genes, and that the markers can act as scaffolds for comparative genomics. furthermore, locus specific sequences can be readily obtained from the plug markers, and by utilizing plug markers as probes, it will be possible to quickly identify target clones from the huge bac library for wheat. the locus-specific sequences obtained from plug markers are also potentially very useful for surveying sequence polymorphisms among wheat cultivars, allowing the development of new markers near quantitative trait loci. we are presently using the taest-lugs to develop plug markers for the entire wheat genome and to carry out bin mapping. a web site is under construction to allow public access to the plug system.  with the plug system, taest-lugs were shown to be useful as reliable standards for wheat est-pcr marker development. in a study using randomly selected taest-lugs, half showed high homology to wheat est sequences that have not previously been used for marker development. therefore, by carrying out large-scale wheat marker development using the plug system, we can potentially double the number of gene-derived markers. in comparisons of intron sequences from wheat homoeologous genes, insertion/deletion polymorphisms were found in almost all cases. this suggests that the sorting of multiple pcr products derived from homoeologous genes, which is a major block for est-pcr marker development, can be resolved by including an intron sequence in pcr products. therefore, the plug system, which semi-automatically extracts primer sets flanking an intron sequence, is a very effective tool in wheat marker development. with this system, it is now possible to sort homoeologous genes using a low-cost and convenient separation method, thus allowing large-scale pcr-based marker development for wheat. since the plug system is based on orthologous gene conservation, markers produced using the system can also be used as accurate anchor markers for genomic research comparing rice and wheat. furthermore, plug markers can show the positional relationships of wheat homoeologous genes, and as a result, these markers are expected to contribute greatly to research on synteny dissociation among wheat homoeologous chromosomes. 
such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. we then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes. by using the similarities in gene structures between rice and wheat, we developed a system called plug to design primers so that pcr products include intron sequences. in the development of wheat est-pcr markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. using the plug system, wheat est sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. est-pcr markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. the plug system consists of the following steps: single-copy rice genes exhibiting high degrees of homology to wheat unigene sequences are extracted; alignment analysis is carried out using the lugs and wheat unigene sequences to predict exon-exon junctions, and lugs which can be used to design wheat primers flanking introns are extracted; and primers are designed in an interactive manner. however, because wheat is an allohexaploid plant, pcr products derived from homoeologous genes are often simultaneously amplified. with all of these primer sets, we obtained specific, intron-containing products from the target genes. hence, the plug system is an effective tool for large-scale marker development. by pcr-rflp analysis using agarose gel electrophoresis, of the markers were located on at least one chromosome. from a total of taest-lugs, loci were randomly selected and used to design primers.
 est-pcr markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. however, because wheat is an allohexaploid plant, pcr products derived from homoeologous genes are often simultaneously amplified. such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. however, genomic sequence data for wheat are limited; therefore it is difficult to predict the location of introns. by using the similarities in gene structures between rice and wheat, we developed a system called plug to design primers so that pcr products include intron sequences. we then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes.  the plug system consists of the following steps: single-copy rice genes exhibiting high degrees of homology to wheat unigene sequences are extracted; alignment analysis is carried out using the lugs and wheat unigene sequences to predict exon-exon junctions, and lugs which can be used to design wheat primers flanking introns are extracted; and primers are designed in an interactive manner. from a total of taest-lugs, loci were randomly selected and used to design primers. with all of these primer sets, we obtained specific, intron-containing products from the target genes. these markers were assigned to chromosomes using wheat nullisomic-tetrasomic lines. by pcr-rflp analysis using agarose gel electrophoresis, of the markers were located on at least one chromosome.  in the development of wheat est-pcr markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. using the plug system, wheat est sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. hence, the plug system is an effective tool for large-scale marker development. 