BACKGROUND
microsatellites or simple sequence repeats are tandemly repeated 1– <dig> base-pair  nucleotide motifs distributed across the genome in many prokaryotes and eukaryotes  <cit> .
an increasing number of microsatellites have been characterized in protein coding sequences  and non-coding untranslated regions  of genes for several plant species.
alterations in these microsatellite sequences are thought to have significant consequences with regard to gene function  <cit> .
variation in the length of microsatellite motifs in non-coding sequences of genes  may affect the process of transcription and translation through slippage, gene silencing and pre-mrna splicing as has been observed for many diseases in humans, including cancers and neuronal disorders  <cit> .
microsatellite markers based on such sequence motifs would be useful as "functional genetic markers" for various applications in genomics and crop breeding.
however, the identification and characterization of such microsatellites has been limited in plants.
completely sequenced genomes provide scope for designing a large number of gene based microsatellite markers.
rice  is the first cereal with a completely sequenced genome that has enabled the development of a large number of microsatellite markers  <cit> .
recently, zhang et al.
<cit>  developed  <dig>  microsatellite markers polymorphic between indica and japonica.
it is difficult to choose useful and informative microsatellite markers from large marker data-sets for genotyping applications in rice.
this can be overcome by constructing a smaller informative microsatellite marker database comprising markers located in potentially functional genic sequences with relatively high polymorphic potential.
however, genic microsatellite markers when derived from protein-coding sequences are constrained by purifying selection  <cit>  and thus have less potential for revealing polymorphism particularly at the intra-specific level  <cit> .
in contrast, markers derived from non-coding sequence components  are under moderate selection pressure and thus expected to be more polymorphic as genetic markers.
previous studies have shown non-random and distinct patterns of microsatellite distribution in non-coding sequence components of rice genes predicted in the completely sequenced rice genome  <cit> .
in view of the excellent genetic attributes and higher informativeness expected for genic non-coding microsatellite  markers, development of such markers from the protein coding genes predicted in the rice genome would be of practical significance.
a comparative analysis of non-coding sequences, known as phylogenetic footprinting  <cit> , has provided useful inferences about conserved non-coding microsatellite  repeat-containing regulatory sequence elements and their significance in gene regulation in plant-specific pathways  <cit> .
these studies have suggested the use of completely finished and recently annotated rice genomic sequences for intra- and inter-genomic phylogenetic footprinting to detect a large number of paralogous and orthologous cnms motifs, respectively, in the 5' non-coding promoter regions of genes among cereals and arabidopsis thaliana.
identification of such cnms motifs would help in understanding the pattern of regulatory or non-coding promoter sequence evolution in plant genomes.
we undertook this study to characterize the frequency and relative distribution of gnms repeat-motifs in different sequence components of protein coding rice genes; design primers flanking the gnms repeat-motifs; physically locate the markers on rice chromosomes, and evaluate their efficiency in the assessment of molecular diversity; detect and characterize cnms motifs in the putative promoter regions of rice genes using intra- and inter-genomic phylogenetic footprinting; and evaluate markers for their utility in comparative physical mapping and establishing molecular phylogenetic relationships among different rice cultivar groups.
RESULTS
frequency and relative abundance of gnms
we identified  <dig>  perfect gnms  in  <dig>  protein coding genes including  <dig>  putative promoter sequences predicted in the rice genome.
the density of perfect gnms varied from  <dig> /mb in chromosome  <dig> to 450/mb in chromosome  <dig> with an average of  <dig>  gnms/mb .
this included  <dig>   gnms repeats in putative promoters,  <dig>   in 5'utrs,  <dig>   in introns and  <dig>   in 3'utrs .
of the promoter-derived gnms repeats,  <dig>  were found in  <dig>  tata box containing promoter sequences.
the average density of compound gnms in the rice genes was  <dig> /mb with maximum density  in chromosome  <dig> and minimum density  in chromosome  <dig>  the perfect gnms density in the promoters was maximum  in chromosome  <dig> and minimum  in chromosome  <dig>  whereas in the 5'utrs it varied from  <dig> /mb in chromosome  <dig> to 1360/mb in chromosome  <dig> .
the intronic gnms density was maximum  in chromosome  <dig> and minimum  in chromosome  <dig>  while in the 3'utrs the gnms density ranged from 96/mb in chromosome  <dig> to  <dig> /mb in chromosome  <dig>  the average microsatellite density was maximum  in the 5'utrs followed by introns , promoters  and 3'utrs  compared to  <dig> /mb in the cds .
thus, the overall gnms density increased gradually from the upstream of the transcription start sites  and reached a peak  in the downstream of the tss .
subsequently, the density decreased and dropped down in the coding region and showed asymmetrical distribution along the direction of transcription.
however, this asymmetry arose because of high gnms density in the introns; about  <dig>  and  <dig>  times higher than that in the cds and 3'utrs, respectively.
our results are in contrast to an earlier report from fujimori et al.
<cit> , which suggested gradual lowering of microsatellite density along the direction of transcription based on the analyses of  <dig>  full-length cdna sequences.
this may be because we used annotated rice gene sets having utrs, introns and promoter sequences predicted from the completely sequenced rice genome that provided a better evaluation of gnms density in the different sequence components studied.
the most frequent occurrence of gnms motifs in the 5'utrs is consistent with earlier observations in rice and arabidopsis genomes  <cit> .
interestingly, the majority  of the gnms motifs in the 5'utrs were present in various transcription- and translation-related genes encoding transcription/translation initiation and elongation factors and ribosomal proteins.
this suggests a functional significance of the repeat motifs present in the 5' utrs, which needs to be further investigated.
nature and distribution of gnms
the gc-rich trinucleotide gnms repeat-motifs were the most prevalent class of microsatellites in the regulatory regions , whereas the at-rich trinucleotide repeats were distributed evenly in all the coding and non-coding sequence components.
however, the proportion of gc-rich trinucleotide motifs was maximum in the 5'utrs  followed by putative promoters , 3'utrs  and introns  compared to  <dig> % in the cds .
this trend corresponds to gc-rich microsatellites being frequently detected in the regions downstream of tss possibly due to higher gc content at the 5'end of the rice genes  <cit> .
the gc-rich trinucleotide gnms repeat-motifs in the 5'utrs and promoters perhaps serve as binding sites for nuclear proteins that are essential for regulating translation and gene expression, and thus are expected to occur more frequently in these sequences  <cit> .
the high frequency of trinucleotide gnms repeat-motifs in the coding regions could be due to selection against frameshift mutations that limits expansion of non-triplet microsatellites  <cit> .
these results agreed well with earlier observations on the relative abundance of gc-rich trinucleotide repeat-motifs in the expressed sequence tags and unigene sequences of cereal genomes  <cit> .
the dinucleotide and tetranucleotide repeat-motifs were predominant particularly in the intronic and 3'utr sequences .
the at-rich dinucleotide repeat-motifs were most in intronic sequences  followed by 3'utrs , whereas the proportion of at-rich tetranucleotide repeats was maximum in 3'utrs .
the purine-rich dinucleotide microsatellites, such as n, were abundant in 5'utrs  followed by promoters  compared to 28% in cds .
our observations are comparable to those from earlier studies on abundance of ga-rich dinucleotide repeat-motifs in the coding  <cit>  and 5'-end flanking regions  <cit>  and at-rich dinucleotide motifs in the intronic sequences in rice genes  <cit> .
the promoter sequences of rice genes frequently  contained pyrimidine-rich microsatellites, especially n dinucleotides , possibly due to their potential role in activation of promoters for transcription initiation  <cit> .
the microsatellite with longer repeat-motifs is expected to be more polymorphic due to high length dependent replication slippage  <cit> .
we identified  <dig>  class i gnms repeat-motifs in the protein coding genes predicted in the rice genome with an overall density of  <dig>  gnms/mb .
the density of the class i repeat-motif containing gnms varied from  <dig> /mb in chromosome  <dig> to  <dig> /mb in chromosome  <dig> whereas its proportion ranged from  <dig> %  in promoters to  <dig> %  in intronic sequences .
thus, the potential of microsatellite expansion in the genic non-coding sequences of rice genes is not correlated with the frequency of gc-rich trinucleotide repeat-motifs in these regions  <cit> .
our results revealed non-random and strongly biased distribution of gnms repeat-motifs across the regulatory and non-coding regions of the rice genes.
design and physical location of gnms markers
forward and reverse primers were designed from the flanking sequences of the identified microsatellite repeat-motifs in each of the four genic non-coding sequence components of rice genes .
the structural organization of the various sequence components and their implications for designing different types of gnms markers are shown in additional file  <dig>  primers were designed for  <dig>   genic non-coding sequences –  <dig>   in promoters,  <dig>   in 5'utrs,  <dig>   in introns and  <dig>  in 3'utrs.
the primer sequences for  <dig>   hypervariable class i gnms markers distributed across  <dig> rice chromosomes are provided in additional file  <dig> .
class i markers included  <dig> in promoters,  <dig> in 5'utrs,  <dig>  in introns and  <dig> in 3'utrs.
the gnms markers were present in the rice genes that regulate biological and cellular functions.
for example, we identified  <dig>  gnms markers including  <dig> class i types in various disease resistance genes predicted in rice chromosome  <dig>  and  <dig>  gnms markers including  <dig> class i types in rice chromosome  <dig> .
the gnms markers when genetically associated with the target traits would facilitate gene cloning and marker-assisted breeding, thereby accelerating rice genetic improvement.
we determined the distribution of class i gnms markers designed from the  <dig> genic non-coding sequence components based on their physical location  on rice chromosomes.
we divided each rice chromosome into  <dig> mb interval sized physical bins and integrated  <dig>  class i gnms markers present in  <dig>  rice genes based on their ascending order of physical location  beginning from the short arm telomere to the long arm telomere .
detailed information regarding the physical position of the various gnms markers on the rice chromosomes is provided in additional file  <dig>  the map density of class i gnms markers varied from  <dig> kb  in chromosome  <dig> to  <dig> kb  in chromosome  <dig> with an average of  <dig>  kb .
the maximum map density in rice chromosome  <dig> could be due to its greater physical size, maximum gene density and least transposon association compared to other rice chromosomes  <cit> .
in general, mapped gnms markers showed more concentration on both arms and towards the telomeric ends of all rice chromosomes than in the centromeric regions except for chromosomes  <dig>   <dig> and  <dig> .
this possibly showed correspondence with the higher density of genes on the chromosome arms/telomeric regions of most of the rice chromosomes than in the centromeres  <cit> .
a maximum of  <dig> and a minimum of  <dig> markers were present per  <dig> mb physical bin of rice chromosome .
the low number of markers in some bins could be due to either the absence of euchromatin sequences or least class i microsatellite containing rice genes at these intervals.
the gnms marker based rice physical map would provide invaluable information on the genomic distribution of these markers and thus aid marker selection for many applications in rice breeding and genomics.
amplification efficiency and polymorphic potential of gnms markers
we used  <dig> microsatellite markers designed  from each of the  <dig> genic non-coding sequence components as well as from cds of rice genes to understand their potential to amplify the target sequence and detect polymorphism among a set of  <dig> rice genotypes .
fifty-six of the  <dig> markers gave amplification around 55°c annealing temperature with a success rate of  <dig> % that suggested the utility of genic non-coding sequences  for developing large-scale microsatellite markers in rice.
the remaining  <dig> primers , which were derived from the intronic sequences, did not show amplification in any of the non-aromatic indica and basmati rice genotypes.
however, amplification was observed for these markers in the japonica genotypes from which sequence the primers were designed.
the occurrence of null alleles could be due to insertion/deletions  in the corresponding genomic sequences of indica and japonica  <cit> .
it may also be the result of frequent association of intronic microsatellites with the micropon family of miniature inverted-repeat transposable elements  in rice  <cit> .
thirty  of the  <dig> gnms markers that underwent amplification showed polymorphism .
the number of alleles amplified per locus varied from  <dig> to  <dig> .
thirty polymorphic gnms markers amplified  <dig> alleles with a mean allele number of  <dig> .
most  of the gnms markers showing polymorphism were class i type repeat-motifs.
the polymorphism information content  value ranged from  <dig>  to  <dig>  with an average of  <dig>  .
the extent of polymorphism detected by the gnms markers is comparable to that reported previously with random genomic microsatellite markers in a set of rice genotypes  <cit> .
we detected polymorphism with  <dig>  markers from intronic sequences,  <dig> from promoters ,  <dig> from 5'utrs ,  <dig> from 3'utrs  and  <dig> from cds  .
among the intronic gnms markers, the one based on the ubiquitin gene  showed the maximum pic value  followed by that on ribosomal protein s <dig> .
the higher level of polymorphism we observed for the gnms markers derived from the promoters, utrs and introns are expected due to the presence of the most abundant and polymorphic class of ga- or at-rich dinucleotide microsatellite repeat-motifs in these sequence components.
further, a comparative evaluation of the polymorphic potential of  <dig> gnms markers distributed over  <dig> rice chromosomes with that of  <dig> rice microsatellite   <cit>  series markers in two parental genotypes  of a large mapping population revealed higher efficiency for gnms markers  over rm markers  in detecting parental polymorphism .
the gnms markers, being more informative than the genic coding and random genomic microsatellite markers developed earlier would be of immediate use in efficient large-scale genotyping applications in rice  <cit> .
twenty-six  of the  <dig> gnms markers showed polymorphism between the indica and japonica genotypes, while  <dig>  revealed polymorphism among the indica genotypes.
gnms markers derived from the intronic sequences showed maximum inter sub-specific polymorphism as reported for intron length polymorphisms in rice  <cit> .
sequencing of the amplicons obtained with the  <dig> gnms markers, from the genic non-coding and coding sequence components that showed amplification for all the  <dig> rice genotypes, confirmed the presence of target repeat motifs .
assessment of molecular genetic diversity among domesticated rice cultivar groups
the pair-wise similarity index among the  <dig> rice genotypes, based on the combined profiles of all the  <dig> gnms markers, revealed a broad range from  <dig>  to  <dig>  with an average of  <dig>  .
this level of diversity is much higher than that detected previously  with random genomic microsatellite markers.
our result indicates the greater efficiency of gnms markers, which assayed potentially functional genetic diversity in the rice genome.
the aromatic group  had a relatively higher average similarity  with japonica compared to indica genotypes .
the results of higher evolutionary closeness between japonica and aromatics are consistent with earlier nuclear and chloroplast diversity studies based on microsatellite  <cit>  and single nucleotide polymorphism  <cit>  markers.
the long-grained aromatic cultivars were most divergent as reflected by their broader similarity index .
this could be due to the inclusion of both traditional and improved basmati cultivars in this group.
the higher level of diversity among long-grained aromatics further agreed well with our observations of higher proportion of polymorphic loci in these cultivars.
the relationship among the  <dig> rice genotypes is depicted in an unrooted phylogenetic tree .
it revealed two distinct clusters: one comprising long-grained traditional and elite basmati, short-grained aromatics and japonica genotypes; and another comprising only indica genotypes.
the gnms marker based clustering clearly differentiated all  <dig> rice genotypes from each other and resulted in a definitive grouping with high bootstrap values  that corresponded well with their known phenotypic classification and evolutionary relationships  <cit> .
these gnms markers could be used for establishing distinctness among rice varieties.
evolutionary significance of cnms containing rice promoters
using inter-genomic phylogenetic footprinting we detected  <dig> cnms repeats  in the putative promoter sequences of orthologous genes among  <dig> cereal genomes  and  <dig>  cnms repeats in the promoters of orthologous genes of rice and a. thaliana .
with intra-genomic phylogenetic footprinting we identified  <dig>  cnms markers in the promoters of paralogous rice genes .
the cnms markers identified among the  <dig> cereal genomes included  <dig> in the promoters between orthologous rice and maize genes,  <dig> between rice and barley,  <dig> between rice and wheat and  <dig> between rice and sorghum .
results from intra- and inter-genomic phylogenetic footprinting comparisons showed that  <dig> cnms motifs were conserved in the promoters of both orthologous and paralogous rice genes .
the ct-rich dinucleotide repeat-motifs were the predominant microsatellite classes in the cnms, which is consistent with the characteristics of pyrimidine-rich repeat distribution in the promoter regions of the rice genes as observed in our study.
a low frequency of cnms motifs indicated a relatively rapid evolution of rice promoters having such sequences, which could be due to functional constraints and rapid adaptive changes in the regulatory regions of homologous genes for imparting specific roles in gene regulation.
this may be why most of the identified cnms repeats were located in the orthologous and paralogous genes in the immediate upstream region  of the transcription initiation site with preference for known regulatory binding sites as characterized by place and plantcare .
it is possible that these sequences are involved in gene regulation in response to environmental stimuli  <cit> .
for example, the cnms motif n in the promoters of rice gene cytochrome p <dig>  contained sequences similar to gaga , a known regulatory element, which is involved in light responsive phototransduction regulation in plants.
complementary to n, the cnms motif n contained a different regulatory element, c2c2-gata , controlling similar light responsive gene regulation in the serine/threonine protein kinase gene.
a comparative physical mapping of cnms markers of rice chromosome  <dig> and homeologous chromosomes of  <dig> other cereal species  and one dicot species  detected several collinear regions with complex chromosomal syntenic relationships , which provide clues to the role of identified cnms markers in comparative genomics and in the understanding of evolutionary complexities in cereals and arabidopsis.
modal nucleotide substitutions for the  <dig> orthologous and paralogous rice cnms containing promoters indicated that barley, maize, sorghum and wheat diverged from rice after the rice genomic duplication events  and monocot-dicot speciation  .
the divergence time between rice and each of the  <dig> cereal species was consistent with the separation time  of cereals  <cit> , of which maize  has diverged more recently .
an analysis of modal nucleotide substitutions in promoter regions of  <dig> sequenced cnms loci among the domesticated rice cultivar groups indicated high modal nucleotide substitutions  and thus wider divergence between indica and japonica rice from a common ancestor at  <dig>  mya .
the divergence between long-grained aromatics and japonica was more recent  compared to short-grained aromatics from japonica  and long-grained aromatics from indica .
among the aromatics, the divergence of short- and long-grained types  was in-between the separation time of short- and long-grained aromatics from indica .
our results thus supported the earlier observations about the origin of indica and japonica from an ancestral rice genotype  <cit>  before their domestication about  <dig>  to  <dig>  years ago  <cit> , and the evolutionary closeness between japonica and aromatic varieties  <cit> .
our results from molecular dating of divergence showed that indica is most diverged from others and that aromatics are an intermediate group between indica and japonica subspecies; much closer, however, to japonica than indica.
the higher divergence time between short- and long-grained aromatics than between each of them and japonica was suggestive of the higher allelic diversity within the aromatics than in indica and japonica rice cultivars.
however, earlier studies have observed lower genetic diversity in aromatic rice relative to other cultivar groups  <cit> .
this contrasting result is due to inclusion of poor-yielding traditional basmati varieties, which are the products of selection from land-races and improved high-yielding basmati varieties developed through cross-breeding involving traditional basmati and non-basmati genotypes in the aromatic group in our study.
CONCLUSIONS
we studied relative distribution of microsatellites in different sequence components of protein coding rice genes, designed  <dig>  gnms markers, including  <dig>  hypervariable class i types from the promoter, 5'utr, intronic and 3'utr sequences and determined their occurrence and organization on the  <dig> rice chromosomes.
the class i markers were bin-mapped to guide the selection of markers with genome wide distribution for various genotyping applications in rice.
we demonstrated the utility of gnms markers by their robust pcr amplification efficiency and high potential for detecting polymorphism over genic coding and random genomic microsatellite markers, and thus their immediate use in rice genetics, genomics and breeding.
the unrooted phylogenetic tree constructed based on molecular diversity values of a set of gnms markers in rice genotypes clearly established molecular genetic relationships among the domesticated rice cultivar groups, thereby suggesting their utility in defining varietal identity in commerce.
the orthologous and paralogous cnms markers identified in the rice promoters would be useful for comparative genome mapping and phylogenetic analysis in rice and other members of grass family

 microsatellites or simple sequence repeats are tandemly repeated 1– base-pair nucleotide motifs distributed across the genome in many prokaryotes and eukaryotes. an increasing number of microsatellites have been characterized in protein coding sequences and non-coding untranslated regions of genes for several plant species. alterations in these microsatellite sequences are thought to have significant consequences with regard to gene function. variation in the length of microsatellite motifs in non-coding sequences of genes may affect the process of transcription and translation through slippage, gene silencing and pre-mrna splicing as has been observed for many diseases in humans, including cancers and neuronal disorders. microsatellite markers based on such sequence motifs would be useful as "functional genetic markers" for various applications in genomics and crop breeding. however, the identification and characterization of such microsatellites has been limited in plants. completely sequenced genomes provide scope for designing a large number of gene based microsatellite markers. rice is the first cereal with a completely sequenced genome that has enabled the development of a large number of microsatellite markers. recently, zhang et al. developed microsatellite markers polymorphic between indica and japonica. it is difficult to choose useful and informative microsatellite markers from large marker data-sets for genotyping applications in rice. this can be overcome by constructing a smaller informative microsatellite marker database comprising markers located in potentially functional genic sequences with relatively high polymorphic potential. however, genic microsatellite markers when derived from protein-coding sequences are constrained by purifying selection and thus have less potential for revealing polymorphism particularly at the intra-specific level. in contrast, markers derived from non-coding sequence components are under moderate selection pressure and thus expected to be more polymorphic as genetic markers. previous studies have shown non-random and distinct patterns of microsatellite distribution in non-coding sequence components of rice genes predicted in the completely sequenced rice genome. in view of the excellent genetic attributes and higher informativeness expected for genic non-coding microsatellite markers, development of such markers from the protein coding genes predicted in the rice genome would be of practical significance. a comparative analysis of non-coding sequences, known as phylogenetic footprinting, has provided useful inferences about conserved non-coding microsatellite repeat-containing regulatory sequence elements and their significance in gene regulation in plant-specific pathways. these studies have suggested the use of completely finished and recently annotated rice genomic sequences for intra- and inter-genomic phylogenetic footprinting to detect a large number of paralogous and orthologous cnms motifs, respectively, in the 5' non-coding promoter regions of genes among cereals and arabidopsis thaliana. identification of such cnms motifs would help in understanding the pattern of regulatory or non-coding promoter sequence evolution in plant genomes. we undertook this study to characterize the frequency and relative distribution of gnms repeat-motifs in different sequence components of protein coding rice genes; design primers flanking the gnms repeat-motifs; physically locate the markers on rice chromosomes, and evaluate their efficiency in the assessment of molecular diversity; detect and characterize cnms motifs in the putative promoter regions of rice genes using intra- and inter-genomic phylogenetic footprinting; and evaluate markers for their utility in comparative physical mapping and establishing molecular phylogenetic relationships among different rice cultivar groups.  frequency and relative abundance of gnms we identified perfect gnms in protein coding genes including putative promoter sequences predicted in the rice genome. the density of perfect gnms varied from /mb in chromosome to 450/mb in chromosome with an average of gnms/mb. this included gnms repeats in putative promoters, in 5'utrs, in introns and in 3'utrs. of the promoter-derived gnms repeats, were found in tata box containing promoter sequences. the average density of compound gnms in the rice genes was /mb with maximum density in chromosome and minimum density in chromosome the perfect gnms density in the promoters was maximum in chromosome and minimum in chromosome whereas in the 5'utrs it varied from /mb in chromosome to 1360/mb in chromosome. the intronic gnms density was maximum in chromosome and minimum in chromosome while in the 3'utrs the gnms density ranged from 96/mb in chromosome to /mb in chromosome the average microsatellite density was maximum in the 5'utrs followed by introns, promoters and 3'utrs compared to /mb in the cds. thus, the overall gnms density increased gradually from the upstream of the transcription start sites and reached a peak in the downstream of the tss. subsequently, the density decreased and dropped down in the coding region and showed asymmetrical distribution along the direction of transcription. however, this asymmetry arose because of high gnms density in the introns; about and times higher than that in the cds and 3'utrs, respectively. our results are in contrast to an earlier report from fujimori et al., which suggested gradual lowering of microsatellite density along the direction of transcription based on the analyses of full-length cdna sequences. this may be because we used annotated rice gene sets having utrs, introns and promoter sequences predicted from the completely sequenced rice genome that provided a better evaluation of gnms density in the different sequence components studied. the most frequent occurrence of gnms motifs in the 5'utrs is consistent with earlier observations in rice and arabidopsis genomes. interestingly, the majority of the gnms motifs in the 5'utrs were present in various transcription- and translation-related genes encoding transcription/translation initiation and elongation factors and ribosomal proteins. this suggests a functional significance of the repeat motifs present in the 5' utrs, which needs to be further investigated. nature and distribution of gnms the gc-rich trinucleotide gnms repeat-motifs were the most prevalent class of microsatellites in the regulatory regions, whereas the at-rich trinucleotide repeats were distributed evenly in all the coding and non-coding sequence components. however, the proportion of gc-rich trinucleotide motifs was maximum in the 5'utrs followed by putative promoters, 3'utrs and introns compared to % in the cds. this trend corresponds to gc-rich microsatellites being frequently detected in the regions downstream of tss possibly due to higher gc content at the 5'end of the rice genes. the gc-rich trinucleotide gnms repeat-motifs in the 5'utrs and promoters perhaps serve as binding sites for nuclear proteins that are essential for regulating translation and gene expression, and thus are expected to occur more frequently in these sequences. the high frequency of trinucleotide gnms repeat-motifs in the coding regions could be due to selection against frameshift mutations that limits expansion of non-triplet microsatellites. these results agreed well with earlier observations on the relative abundance of gc-rich trinucleotide repeat-motifs in the expressed sequence tags and unigene sequences of cereal genomes. the dinucleotide and tetranucleotide repeat-motifs were predominant particularly in the intronic and 3'utr sequences. the at-rich dinucleotide repeat-motifs were most in intronic sequences followed by 3'utrs, whereas the proportion of at-rich tetranucleotide repeats was maximum in 3'utrs. the purine-rich dinucleotide microsatellites, such as n, were abundant in 5'utrs followed by promoters compared to 28% in cds. our observations are comparable to those from earlier studies on abundance of ga-rich dinucleotide repeat-motifs in the coding and 5'-end flanking regions and at-rich dinucleotide motifs in the intronic sequences in rice genes. the promoter sequences of rice genes frequently contained pyrimidine-rich microsatellites, especially n dinucleotides, possibly due to their potential role in activation of promoters for transcription initiation. the microsatellite with longer repeat-motifs is expected to be more polymorphic due to high length dependent replication slippage. we identified class i gnms repeat-motifs in the protein coding genes predicted in the rice genome with an overall density of gnms/mb. the density of the class i repeat-motif containing gnms varied from /mb in chromosome to /mb in chromosome whereas its proportion ranged from % in promoters to % in intronic sequences. thus, the potential of microsatellite expansion in the genic non-coding sequences of rice genes is not correlated with the frequency of gc-rich trinucleotide repeat-motifs in these regions. our results revealed non-random and strongly biased distribution of gnms repeat-motifs across the regulatory and non-coding regions of the rice genes. design and physical location of gnms markers forward and reverse primers were designed from the flanking sequences of the identified microsatellite repeat-motifs in each of the four genic non-coding sequence components of rice genes. the structural organization of the various sequence components and their implications for designing different types of gnms markers are shown in additional file primers were designed for genic non-coding sequences – in promoters, in 5'utrs, in introns and in 3'utrs. the primer sequences for hypervariable class i gnms markers distributed across rice chromosomes are provided in additional file. class i markers included in promoters, in 5'utrs, in introns and in 3'utrs. the gnms markers were present in the rice genes that regulate biological and cellular functions. for example, we identified gnms markers including class i types in various disease resistance genes predicted in rice chromosome and gnms markers including class i types in rice chromosome. the gnms markers when genetically associated with the target traits would facilitate gene cloning and marker-assisted breeding, thereby accelerating rice genetic improvement. we determined the distribution of class i gnms markers designed from the genic non-coding sequence components based on their physical location on rice chromosomes. we divided each rice chromosome into mb interval sized physical bins and integrated class i gnms markers present in rice genes based on their ascending order of physical location beginning from the short arm telomere to the long arm telomere. detailed information regarding the physical position of the various gnms markers on the rice chromosomes is provided in additional file the map density of class i gnms markers varied from kb in chromosome to kb in chromosome with an average of kb. the maximum map density in rice chromosome could be due to its greater physical size, maximum gene density and least transposon association compared to other rice chromosomes. in general, mapped gnms markers showed more concentration on both arms and towards the telomeric ends of all rice chromosomes than in the centromeric regions except for chromosomes and. this possibly showed correspondence with the higher density of genes on the chromosome arms/telomeric regions of most of the rice chromosomes than in the centromeres. a maximum of and a minimum of markers were present per mb physical bin of rice chromosome. the low number of markers in some bins could be due to either the absence of euchromatin sequences or least class i microsatellite containing rice genes at these intervals. the gnms marker based rice physical map would provide invaluable information on the genomic distribution of these markers and thus aid marker selection for many applications in rice breeding and genomics. amplification efficiency and polymorphic potential of gnms markers we used microsatellite markers designed from each of the genic non-coding sequence components as well as from cds of rice genes to understand their potential to amplify the target sequence and detect polymorphism among a set of rice genotypes. fifty-six of the markers gave amplification around 55°c annealing temperature with a success rate of % that suggested the utility of genic non-coding sequences for developing large-scale microsatellite markers in rice. the remaining primers, which were derived from the intronic sequences, did not show amplification in any of the non-aromatic indica and basmati rice genotypes. however, amplification was observed for these markers in the japonica genotypes from which sequence the primers were designed. the occurrence of null alleles could be due to insertion/deletions in the corresponding genomic sequences of indica and japonica. it may also be the result of frequent association of intronic microsatellites with the micropon family of miniature inverted-repeat transposable elements in rice. thirty of the gnms markers that underwent amplification showed polymorphism. the number of alleles amplified per locus varied from to. thirty polymorphic gnms markers amplified alleles with a mean allele number of. most of the gnms markers showing polymorphism were class i type repeat-motifs. the polymorphism information content value ranged from to with an average of. the extent of polymorphism detected by the gnms markers is comparable to that reported previously with random genomic microsatellite markers in a set of rice genotypes. we detected polymorphism with markers from intronic sequences, from promoters, from 5'utrs, from 3'utrs and from cds. among the intronic gnms markers, the one based on the ubiquitin gene showed the maximum pic value followed by that on ribosomal protein s. the higher level of polymorphism we observed for the gnms markers derived from the promoters, utrs and introns are expected due to the presence of the most abundant and polymorphic class of ga- or at-rich dinucleotide microsatellite repeat-motifs in these sequence components. further, a comparative evaluation of the polymorphic potential of gnms markers distributed over rice chromosomes with that of rice microsatellite series markers in two parental genotypes of a large mapping population revealed higher efficiency for gnms markers over rm markers in detecting parental polymorphism. the gnms markers, being more informative than the genic coding and random genomic microsatellite markers developed earlier would be of immediate use in efficient large-scale genotyping applications in rice. twenty-six of the gnms markers showed polymorphism between the indica and japonica genotypes, while revealed polymorphism among the indica genotypes. gnms markers derived from the intronic sequences showed maximum inter sub-specific polymorphism as reported for intron length polymorphisms in rice. sequencing of the amplicons obtained with the gnms markers, from the genic non-coding and coding sequence components that showed amplification for all the rice genotypes, confirmed the presence of target repeat motifs. assessment of molecular genetic diversity among domesticated rice cultivar groups the pair-wise similarity index among the rice genotypes, based on the combined profiles of all the gnms markers, revealed a broad range from to with an average of. this level of diversity is much higher than that detected previously with random genomic microsatellite markers. our result indicates the greater efficiency of gnms markers, which assayed potentially functional genetic diversity in the rice genome. the aromatic group had a relatively higher average similarity with japonica compared to indica genotypes. the results of higher evolutionary closeness between japonica and aromatics are consistent with earlier nuclear and chloroplast diversity studies based on microsatellite and single nucleotide polymorphism markers. the long-grained aromatic cultivars were most divergent as reflected by their broader similarity index. this could be due to the inclusion of both traditional and improved basmati cultivars in this group. the higher level of diversity among long-grained aromatics further agreed well with our observations of higher proportion of polymorphic loci in these cultivars. the relationship among the rice genotypes is depicted in an unrooted phylogenetic tree. it revealed two distinct clusters: one comprising long-grained traditional and elite basmati, short-grained aromatics and japonica genotypes; and another comprising only indica genotypes. the gnms marker based clustering clearly differentiated all rice genotypes from each other and resulted in a definitive grouping with high bootstrap values that corresponded well with their known phenotypic classification and evolutionary relationships. these gnms markers could be used for establishing distinctness among rice varieties. evolutionary significance of cnms containing rice promoters using inter-genomic phylogenetic footprinting we detected cnms repeats in the putative promoter sequences of orthologous genes among cereal genomes and cnms repeats in the promoters of orthologous genes of rice and a. thaliana. with intra-genomic phylogenetic footprinting we identified cnms markers in the promoters of paralogous rice genes. the cnms markers identified among the cereal genomes included in the promoters between orthologous rice and maize genes, between rice and barley, between rice and wheat and between rice and sorghum. results from intra- and inter-genomic phylogenetic footprinting comparisons showed that cnms motifs were conserved in the promoters of both orthologous and paralogous rice genes. the ct-rich dinucleotide repeat-motifs were the predominant microsatellite classes in the cnms, which is consistent with the characteristics of pyrimidine-rich repeat distribution in the promoter regions of the rice genes as observed in our study. a low frequency of cnms motifs indicated a relatively rapid evolution of rice promoters having such sequences, which could be due to functional constraints and rapid adaptive changes in the regulatory regions of homologous genes for imparting specific roles in gene regulation. this may be why most of the identified cnms repeats were located in the orthologous and paralogous genes in the immediate upstream region of the transcription initiation site with preference for known regulatory binding sites as characterized by place and plantcare. it is possible that these sequences are involved in gene regulation in response to environmental stimuli. for example, the cnms motif n in the promoters of rice gene cytochrome p contained sequences similar to gaga, a known regulatory element, which is involved in light responsive phototransduction regulation in plants. complementary to n, the cnms motif n contained a different regulatory element, c2c2-gata, controlling similar light responsive gene regulation in the serine/threonine protein kinase gene. a comparative physical mapping of cnms markers of rice chromosome and homeologous chromosomes of other cereal species and one dicot species detected several collinear regions with complex chromosomal syntenic relationships, which provide clues to the role of identified cnms markers in comparative genomics and in the understanding of evolutionary complexities in cereals and arabidopsis. modal nucleotide substitutions for the orthologous and paralogous rice cnms containing promoters indicated that barley, maize, sorghum and wheat diverged from rice after the rice genomic duplication events and monocot-dicot speciation. the divergence time between rice and each of the cereal species was consistent with the separation time of cereals, of which maize has diverged more recently. an analysis of modal nucleotide substitutions in promoter regions of sequenced cnms loci among the domesticated rice cultivar groups indicated high modal nucleotide substitutions and thus wider divergence between indica and japonica rice from a common ancestor at mya. the divergence between long-grained aromatics and japonica was more recent compared to short-grained aromatics from japonica and long-grained aromatics from indica. among the aromatics, the divergence of short- and long-grained types was in-between the separation time of short- and long-grained aromatics from indica. our results thus supported the earlier observations about the origin of indica and japonica from an ancestral rice genotype before their domestication about to years ago, and the evolutionary closeness between japonica and aromatic varieties. our results from molecular dating of divergence showed that indica is most diverged from others and that aromatics are an intermediate group between indica and japonica subspecies; much closer, however, to japonica than indica. the higher divergence time between short- and long-grained aromatics than between each of them and japonica was suggestive of the higher allelic diversity within the aromatics than in indica and japonica rice cultivars. however, earlier studies have observed lower genetic diversity in aromatic rice relative to other cultivar groups. this contrasting result is due to inclusion of poor-yielding traditional basmati varieties, which are the products of selection from land-races and improved high-yielding basmati varieties developed through cross-breeding involving traditional basmati and non-basmati genotypes in the aromatic group in our study.  we studied relative distribution of microsatellites in different sequence components of protein coding rice genes, designed gnms markers, including hypervariable class i types from the promoter, 5'utr, intronic and 3'utr sequences and determined their occurrence and organization on the rice chromosomes. the class i markers were bin-mapped to guide the selection of markers with genome wide distribution for various genotyping applications in rice. we demonstrated the utility of gnms markers by their robust pcr amplification efficiency and high potential for detecting polymorphism over genic coding and random genomic microsatellite markers, and thus their immediate use in rice genetics, genomics and breeding. the unrooted phylogenetic tree constructed based on molecular diversity values of a set of gnms markers in rice genotypes clearly established molecular genetic relationships among the domesticated rice cultivar groups, thereby suggesting their utility in defining varietal identity in commerce. the orthologous and paralogous cnms markers identified in the rice promoters would be useful for comparative genome mapping and phylogenetic analysis in rice and other members of grass family 
the gnms markers were most polymorphic in the intronic region followed by markers in the promoter region and least in the cds. the robust polymerase chain reaction amplification efficiency and high polymorphic potential of gnms markers over genic coding and random genomic microsatellite markers suggest their immediate use in efficient genotyping applications in rice. our analyses showed that gnms markers with their high polymorphic potential would be preferred candidate functional markers in various marker-based applications in rice genetics, genomics and breeding. the cnms markers provided encouraging implications for their use in comparative genome mapping and understanding of evolutionary complexities in rice and other members of grass family. completely sequenced plant genomes provide scope for designing a large number of microsatellite markers, which are useful in various aspects of crop breeding and genetic analysis. the average microsatellite density was maximum in the 5' untranslated regions followed by those in introns, promoters, 3'utrs and minimum in the coding sequences. with the objective of developing genic but non-coding microsatellite markers for the rice genome, we characterized the frequency and relative distribution of microsatellite repeat-motifs in predicted protein coding genes including putative promoter sequences. a set of these markers could assess genetic diversity and establish phylogenetic relationships among domesticated rice cultivar groups. we also demonstrated the usefulness of orthologous and paralogous conserved non-coding microsatellite markers, identified in the putative rice promoter sequences, for comparative physical mapping and understanding of evolutionary and gene regulatory complexities among rice and other members of the grass family. primers were designed for gnms repeats, including hypervariable class i types, which were bin-mapped on the rice genome.
 completely sequenced plant genomes provide scope for designing a large number of microsatellite markers, which are useful in various aspects of crop breeding and genetic analysis. with the objective of developing genic but non-coding microsatellite markers for the rice genome, we characterized the frequency and relative distribution of microsatellite repeat-motifs in predicted protein coding genes including putative promoter sequences.  we identified perfect gnms repeats with densities ranging from /mb in chromosome to 450/mb in chromosome with an average of gnms per mb. the average microsatellite density was maximum in the 5' untranslated regions followed by those in introns, promoters, 3'utrs and minimum in the coding sequences. primers were designed for gnms repeats, including hypervariable class i types, which were bin-mapped on the rice genome. the gnms markers were most polymorphic in the intronic region followed by markers in the promoter region and least in the cds. the robust polymerase chain reaction amplification efficiency and high polymorphic potential of gnms markers over genic coding and random genomic microsatellite markers suggest their immediate use in efficient genotyping applications in rice. a set of these markers could assess genetic diversity and establish phylogenetic relationships among domesticated rice cultivar groups. we also demonstrated the usefulness of orthologous and paralogous conserved non-coding microsatellite markers, identified in the putative rice promoter sequences, for comparative physical mapping and understanding of evolutionary and gene regulatory complexities among rice and other members of the grass family. the divergence between long-grained aromatics and subspecies japonica was estimated to be more recent compared to short-grained aromatics from japonica and long-grained aromatics from subspecies indica.  our analyses showed that gnms markers with their high polymorphic potential would be preferred candidate functional markers in various marker-based applications in rice genetics, genomics and breeding. the cnms markers provided encouraging implications for their use in comparative genome mapping and understanding of evolutionary complexities in rice and other members of grass family. 