quantitative trait locus ( qtl ) analysis is an effective method for locating chromosomal regions harboring genetic variants that affect a continuously distributed , polygenic phenotype ( 1 ) . however , the identification of genes affecting complex traits is one of the most difficult tasks in genetics ( 2 ) . recently , several computational methods , including comparative genomics , combined cross analysis , interval - specific and genome - wide haplotype analysis , have been developed for narrowing animal qtls ( 3 ) . however , no bioinformatics tool for qtl - based candidate gene identification in plants is currently available . rice ( oryza sativa ) and arabidopsis thaliana , model plants for monocotyledonous and dicotyledonous species , respectively , have many quantitative traits of agronomic importance and biological significance . most of these quantitative traits are the results of interactions of multiple genetic variations as well as interactions of genetic variations and environmental factors . in recent years specifically , as of the end of 2005 , more than 7000 qtls controlling various complex traits have been located on different chromosome regions in rice ( ) . in practice , the candidate gene approach has been applied in plant genetics in the past for the characterization and cloning of qtls ( 4 ) . it is complementary to map - based cloning for identifying the genes placed within the qtl intervals . using the sequenced and ( partially ) annotated a.thaliana ( 5 ) and rice genomes ( 68 ) , it now becomes feasible to retrieve genomic sequences , select candidate genes within a qtl interval , and analyze gene expression data [ i.e. expressed sequence tags ( ests ) and microarray gene expression data ] for discovering novel genes in rice and a.thaliana . as a result , the candidate gene approach has been commonly used to select appropriate candidate genes and relevant gene expression data that are possibly associated with particular qtls . with such refined association information among candidate genes , their expression information and qtls , further functional analysis of genes and quantitative trait ( e.g. linkage analysis ) can be effectively carried out . therefore , it is an important task to develop a bioinformatics platform for candidate gene finding that integrates the information of gene expression and qtls . to this end , we have designed and developed an integrated database system , called plantqtl - ge , where ge stands for genes and expressions , for identifying candidate genes and searching for relevant gene expression information from microarray gene expression and est data . this resource provides a novel tool to assist the user to focus on those candidate genes that are restricted to the qtls of interest . in practice , the user could get a list of candidate genes located in the interested qtl region , and then isolate , map , and characterize these genes through future experiments such as differential display analysis . various known functional genes , microarray gene expression data , ests , genetic markers , qtls and candidate genes of rice and arabidopsis were collected from the literature and various public databases , and organized and maintained in an integrated database in mysql ( figure 1 ) . rice qtls were further classified into seven categories and a.thaliana qtls into four categories , according to the trait ontology ( to ) ( ) in gramene database ( 9 ) . then , information of known functional genes related to different trait categories and their corresponding est and microarray data were extracted from literature . the ests were retrieved from ncbi est database ( ) by using relevant keywords of traits and classified based on trait ontology . genetic markers with known genomic positions were retrieved from the marker databases in gramene ( release 19 ) ( ) and tair ( & type = marker ) . gene annotations were retrieved from tigr ( ) for rice and tair ( ) for a.thaliana , respectively . the loci information of a sequence was obtained by aligning it against the genomic sequences using blast with an e - value of 1e-05 . the release of rice nipponbare genomic sequences used in the study is irgsp build 3.0 ( ) and the release of a.thaliana genomic sequences is tigr v5 ( ) . a sketch of the system architecture and data flow of plantqtl - ge , which integrates information of genomic sequences , genes , gene expression data , ests , genetic markers and trait ontology . to help select candidate genes , we used known cis - regulatory elements to annotate candidate genes . in particular , we took all 25 conserved cis - elements from the place plant motif database ( ) which are associated with quantitative traits defined in trait ontology mentioned above . plantqtl - ge contained 1558 and 1896 known genes , 3633 and 9270 microarray data entries , 883  598 and 162  596 ests , 21  523 and 2283 genetic markers , as well as 58  687 and 31  527 annotated genes for rice and arabidopsis , respectively . since est and microarray gene expression data as well as cis - elements have been proven to be very useful for identifying candidate genes of interest ( 10,11 ) , we used them to discover and annotate candidate genes . for the genes within the two genetic markers delineating a qtl of interest , our method executes the following three - step procedure : 
 in step 1 , sequences of length 1000 bp upstream of rice and arabidopsis candidate genes within the interval of a given qtl are retrieved from gramene and tair database , respectively . a motif - scanning method named motif - finder ( 12 ) is then applied to locate the cis - elements in the upstream sequences of targeted genes . the resulting genomic loci and occurrence number of each cis - element in the promoter regions of the genes are stored in a cis - element table in the mysql database.the candidate genes are subsequently annotated using the identified cis - elements , in step 2 . specifically , a gene is annotated as plus ( + ) or minus (  ) with respect to a cis - element if its promoter contains the cis - element or it does not , respectively . such cis - element annotations are stored in the cis - element fields in a gene annotation table.similarly , in step 3 the genes are also annotated with respect to gene expression data ( i.e. est and microarray gene expression data ) which are relevant to the quantitative trait of consideration . a sequence is annotated as plus ( + ) with respect to a gene expression if it matches to the corresponding est or microarray probe , or as minus (  ) , otherwise . the gene expression annotation information is recorded in the gene expression fields of the annotation table . in step 1 , sequences of length 1000 bp upstream of rice and arabidopsis candidate genes within the interval of a given qtl are retrieved from gramene and tair database , respectively . a motif - scanning method named motif - finder ( 12 ) is then applied to locate the cis - elements in the upstream sequences of targeted genes . the resulting genomic loci and occurrence number of each cis - element in the promoter regions of the genes are stored in a cis - element table in the mysql database . the candidate genes are subsequently annotated using the identified cis - elements , in step 2 . specifically , a gene is annotated as plus ( + ) or minus (  ) with respect to a cis - element if its promoter contains the cis - element or it does not , respectively . such cis - element annotations are stored in the cis - element fields in a gene annotation table . similarly , in step 3 the genes are also annotated with respect to gene expression data ( i.e. est and microarray gene expression data ) which are relevant to the quantitative trait of consideration . a sequence is annotated as plus ( + ) with respect to a gene expression if it matches to the corresponding est or microarray probe , or as minus (  ) , otherwise . the gene expression annotation information is recorded in the gene expression fields of the annotation table . we compared the sequences of arabidopsis genes against the genomic sequences of rice using blast with a stringent e - value of 1e-15 . we carried out a reciprocal analysis by comparing the sequences of rice genes against the genomic sequences of a.thaliana with the same stringency level . as a result , 2445 arabidopsis putative genes were mapped onto the rice genome and 4588 rice putative genes were mapped onto the arabidopsis genome . various known functional genes , microarray gene expression data , ests , genetic markers , qtls and candidate genes of rice and arabidopsis were collected from the literature and various public databases , and organized and maintained in an integrated database in mysql ( figure 1 ) . rice qtls were further classified into seven categories and a.thaliana qtls into four categories , according to the trait ontology ( to ) ( ) in gramene database ( 9 ) . then , information of known functional genes related to different trait categories and their corresponding est and microarray data were extracted from literature . the ests were retrieved from ncbi est database ( ) by using relevant keywords of traits and classified based on trait ontology . genetic markers with known genomic positions were retrieved from the marker databases in gramene ( release 19 ) ( ) and tair ( & type = marker ) . gene annotations were retrieved from tigr ( ) for rice and tair ( ) for a.thaliana , respectively . the loci information of a sequence was obtained by aligning it against the genomic sequences using blast with an e - value of 1e-05 . the release of rice nipponbare genomic sequences used in the study is irgsp build 3.0 ( ) and the release of a.thaliana genomic sequences is tigr v5 ( ) . a sketch of the system architecture and data flow of plantqtl - ge , which integrates information of genomic sequences , genes , gene expression data , ests , genetic markers and trait ontology . to help select candidate genes , we used known cis - regulatory elements to annotate candidate genes . in particular , we took all 25 conserved cis - elements from the place plant motif database ( ) which are associated with quantitative traits defined in trait ontology mentioned above . plantqtl - ge contained 1558 and 1896 known genes , 3633 and 9270 microarray data entries , 883  598 and 162  596 ests , 21  523 and 2283 genetic markers , as well as 58  687 and 31  527 annotated genes for rice and arabidopsis , respectively . since est and microarray gene expression data as well as cis - elements have been proven to be very useful for identifying candidate genes of interest ( 10,11 ) , we used them to discover and annotate candidate genes . for the genes within the two genetic markers delineating a qtl of interest , our method executes the following three - step procedure : 
 in step 1 , sequences of length 1000 bp upstream of rice and arabidopsis candidate genes within the interval of a given qtl are retrieved from gramene and tair database , respectively . a motif - scanning method named motif - finder ( 12 ) is then applied to locate the cis - elements in the upstream sequences of targeted genes . the resulting genomic loci and occurrence number of each cis - element in the promoter regions of the genes are stored in a cis - element table in the mysql database.the candidate genes are subsequently annotated using the identified cis - elements , in step 2 . specifically , a gene is annotated as plus ( + ) or minus (  ) with respect to a cis - element if its promoter contains the cis - element or it does not , respectively . such cis - element annotations are stored in the cis - element fields in a gene annotation table.similarly , in step 3 the genes are also annotated with respect to gene expression data ( i.e. est and microarray gene expression data ) which are relevant to the quantitative trait of consideration . a sequence is annotated as plus ( + ) with respect to a gene expression if it matches to the corresponding est or microarray probe , or as minus (  ) , otherwise . the gene expression annotation information is recorded in the gene expression fields of the annotation table . in step 1 , sequences of length 1000 bp upstream of rice and arabidopsis candidate genes within the interval of a given qtl are retrieved from gramene and tair database , respectively . a motif - scanning method named motif - finder ( 12 ) is then applied to locate the cis - elements in the upstream sequences of targeted genes . the resulting genomic loci and occurrence number of each cis - element in the promoter regions of the genes are stored in a cis - element table in the mysql database . the candidate genes are subsequently annotated using the identified cis - elements , in step 2 . specifically , a gene is annotated as plus ( + ) or minus (  ) with respect to a cis - element if its promoter contains the cis - element or it does not , respectively . such cis - element annotations are stored in the cis - element fields in a gene annotation table . similarly , in step 3 the genes are also annotated with respect to gene expression data ( i.e. est and microarray gene expression data ) which are relevant to the quantitative trait of consideration . a sequence is annotated as plus ( + ) with respect to a gene expression if it matches to the corresponding est or microarray probe , or as minus (  ) , otherwise . the gene expression annotation information is recorded in the gene expression fields of the annotation table . we compared the sequences of arabidopsis genes against the genomic sequences of rice using blast with a stringent e - value of 1e-15 . we carried out a reciprocal analysis by comparing the sequences of rice genes against the genomic sequences of a.thaliana with the same stringency level . as a result , 2445 arabidopsis putative genes were mapped onto the rice genome and 4588 rice putative genes were mapped onto the arabidopsis genome . it has a web - based query interface , supported by a mysql database with enhanced perl scripts . the interface , shown in figure 2a , allows the user to search information about candidate genes and relevant ests and microarray gene expression data within a qtl interval in rice or a.thaliana . the following steps should be followed to retrieve genetic information associated with a qtl of interest 
 select a plant species . currently , rice and a.thaliana are available.select a query option , which can be linked genetic markers flanking the targeted qtl interval or chromosomal positions in the selected genome , and then input two markers or the start and stop genomic positions of the markers.select a trait category and one or more keywords on traits related to the targeted qtl specified by the user.select an output type , which can be a combination of known genes , microarray data , ests , candidate genes , and similar putative genes in the other species . select a query option , which can be linked genetic markers flanking the targeted qtl interval or chromosomal positions in the selected genome , and then input two markers or the start and stop genomic positions of the markers . select a trait category and one or more keywords on traits related to the targeted qtl specified by the user . select an output type , which can be a combination of known genes , microarray data , ests , candidate genes , and similar putative genes in the other species . ( a ) the interface of plantqtl - ge ; ( b ) a snapshot of the result of searching for genetic elements related to a qtl between markers rm526 and rm525 , which controls dry - weight of grains . the result of a query includes all selected genes , microarray and/or ests within the targeted qtl region . the entities in the output are also hot linked in that further detailed information of the entities can be obtained by following the corresponding hyperlinks . a snapshot of a case study of searching for genetic elements related to a qtl between markers rm526 and rm525 , which controls dry - weight of grains ( an abiotic related trait ) ( 13 ) , is shown in figure 2b . the complete output of this example is available in the supplementary material in our website ( ) . we will update plantqtl - ge database bimonthly to accommodate new information of genes ( from the entrez - nucleotide database , ) , microarray data ( from public databases and literature ) and ests ( from the est database , ) . to do so , we have written and tested a perl script based on the ncbi eutils tools ( ) . we also plan to expand plantqtl - ge system to support more plant species , such as populus trichocarpa ( ) whose whole genome sequence is now available . in addition , we will enhance the cis - element annotation by including motifs identified by a motif - finding method , such as wordspy ( 14 ) , on all genes responsive to biotic and abiotic stresses .
we have designed and implemented a web - based database system , called plantqtl - ge , to facilitate quantitatine traits locus ( qtl ) based candidate gene identification and gene function analysis . 
 we collected a large number of genes , gene expression information in microarray data and expressed sequence tags ( ests ) and genetic markers from multiple sources of oryza sativa and arabidopsis thaliana . 
 the system integrates these diverse data sources and has a uniform web interface for easy access . 
 it supports qtl queries specifying qtl marker intervals or genomic loci , and displays , on rice or arabidopsis genome , known genes , microarray data , ests and candidate genes and similar putative genes in the other plant . 
 candidate genes in qtl intervals are further annotated based on matching ests , microarray gene expression data and cis - elements in regulatory sequences . 
 the system is freely available at .