wheat is the most widely grown crop in the world with massive importance for human nutrition . cultivated wheat ( triticum aestivum ) is an allohexaploid species with three homoeologous genomes ( a , b , d ) , each comprising seven pairs of chromosomes . all three genomes are very large , so that together they contain about 30 as much dna as rice and 6 as much as the human genome . due to the technical difficulties , the complete genome sequence of wheat will not be available for several years at the earliest . however , there is a rich resource of wheat ests of which there are 850  000 and a further 500  000 from other triticeae species in dbest [ ( 1 ) ; january 2007 ] . these can be mapped to the genes of rice as the most closely related fully sequenced genome ( 2 ) . in this way , all the ests derived from the same transcript can be grouped and linked to information on the orthologues in rice and arabidopsis . this procedure thus facilitates the application of knowledge gained in model species , particularly arabidopsis , to wheat crops . the aim of wheat estimated transcript server ( whets ) is to flexibly allow the user to assemble triticeae ests mapped to rice genes in this way and provide access to the results in a convenient form . a common way to exploit ests however , because whets assembles related sequences in real time , the user can adjust the set of ests to be used , alter the stringency setting and view the affect of the changes on the assembly . also , by anchoring the ests to rice loci , non - contiguous ests representing the same genes are automatically treated a part of the same set . the three very similar homoeologues of wheat genes are frequently all expressed ( 4 ) , but assembly programs do not normally separate these so they are grouped together in contigs . these homoeologous sequences are best identified by analysis of shared snps such as can be achieved with snpserver ( 5 ) which uses autosnp ( 6 ) algorithms to separate alleles or homoeologues . a similar approach is included in whets to provide a best estimate of homoeologue - specific sequence . aligning the triticeae ests to rice has the additional advantage of being able to infer likely intron position which can be used to derive allelic markers , an approach taken in the usda wheat snp database ( http://wheat.pw.usda.gov/snp/new/index.shtml ) . part of the aim of whets is therefore to bring together the useful features of unigene , snpserver and usda snp database into one site tailored specifically for wheat transcripts . however , it also has features unavailable elsewhere , such as the ability to display triticeae est distribution corresponding to a set of rice loci and the option to filter sequences according to library source tissue . whets has a relational database containing sequences and annotation for all triticeae est and high quality cdna ( hq - cdna ) sequences from dbest , coding sequences , annotation and intron positions for rice from the institute for genome research ( tigr ) rice pseudo molecule release 4 ( 7 ) , and annotation for each locus from the arabidopsis information resource ( tair ) version 6 ( 8) ( figure 1 ) . est sequences are first masked for vector contamination using the cross_match program ( 9 ) . the whets database also contains the results of a blast ( 10 ) similarity searches : blastp of all the tigr rice protein sequences against all the tair arabidopsis proteins , and blastn of the triticeae ests against the tigr rice cds . these tables contain the top scoring hits and any lower scoring hits with longer aligned regions , thus defining many - to - many relationships between arabidopsis and rice genes and between rice genes and triticeae sequences . the database is updated weekly by automated scripts which compare the contents with triticeae entries in genbank using entrez utilities ( 11 ) . new sequences are subjected to a blastn search against the rice sequences and the sequences , and blast results are added to the whets database ( figure 1 ) . users can start directly by supplying these as input , or they can start with a set of arabidopsis agi numbers or triticeae accession numbers . the user can then select filter settings for species , tissue and sequence type ( est or hq - cdna ) . whets will then display the number and accessions of all the matching triticeae sequences for each locus . the user can then select the locus for which they wish to obtain sequences for in the main part of whets . when a single rice locus is selected , the user can again filter for species , tissue and sequence type . the sequences which pass this filter are assembled using the cap3 program ( 12 ) , and the resulting contigs are passed to an algorithm which analyses shared snps . if the contigs are found to contain groups which share more snps ( i.e. base differences from the consensus ) than a user - defined cut - off ( default five snps per kb ) , these are split off into separate contigs . the cap3 step tends to assemble paralogues which match the same rice locus into separate contigs , whereas the snp analysis step is designed to separate homoeologues . conversely , in situations where there are relatively few ests it can be useful to assemble the sequences from wheat and related species with low stringency . whets also assembles the hq - cdna sequences where present using a much higher base quality setting for the cap3 program than used for ests . this has the effect that the consensus sequence of any contig will normally be the same as any hq - cdna within it . after assembly , the rice cds is aligned to the contigs consensus and singlet sequences with blast and the results displayed using a modified version of a perl script from the korf et al . links are supplied which open windows detailing all species , tissue , sequence type and cultivar of the constituent sequences . the main output for user downloading is a fasta file containing the rice template cds , contigs consensus and singlet sequences . additional details , such as intron positions are supplied in the descriptor fields of this file . also available are other files , such as ace format files for each of the contigs containing all the information on the constituent sequences and their alignment , and a spreadsheet - compatible file containing details of all snps used to split contigs . whets is implemented in mysql ( http://www.mysql.com/ ) and perl using some bioperl ( 14 ) modules . more details on allocation of blast hits within the whets database , strand of est used and the algorithm for separating contigs into putative homoeologues are available in the supplementary data . whets has a relational database containing sequences and annotation for all triticeae est and high quality cdna ( hq - cdna ) sequences from dbest , coding sequences , annotation and intron positions for rice from the institute for genome research ( tigr ) rice pseudo molecule release 4 ( 7 ) , and annotation for each locus from the arabidopsis information resource ( tair ) version 6 ( 8) ( figure 1 ) . est sequences are first masked for vector contamination using the cross_match program ( 9 ) . the whets database also contains the results of a blast ( 10 ) similarity searches : blastp of all the tigr rice protein sequences against all the tair arabidopsis proteins , and blastn of the triticeae ests against the tigr rice cds . these tables contain the top scoring hits and any lower scoring hits with longer aligned regions , thus defining many - to - many relationships between arabidopsis and rice genes and between rice genes and triticeae sequences . the database is updated weekly by automated scripts which compare the contents with triticeae entries in genbank using entrez utilities ( 11 ) . new sequences are subjected to a blastn search against the rice sequences and the sequences , and blast results are added to the whets database ( figure 1 ) . users can start directly by supplying these as input , or they can start with a set of arabidopsis agi numbers or triticeae accession numbers . the user can then select filter settings for species , tissue and sequence type ( est or hq - cdna ) . whets will then display the number and accessions of all the matching triticeae sequences for each locus . the user can then select the locus for which they wish to obtain sequences for in the main part of whets . when a single rice locus is selected , the user can again filter for species , tissue and sequence type . the sequences which pass this filter are assembled using the cap3 program ( 12 ) , and the resulting contigs are passed to an algorithm which analyses shared snps . if the contigs are found to contain groups which share more snps ( i.e. base differences from the consensus ) than a user - defined cut - off ( default five snps per kb ) , these are split off into separate contigs . the cap3 step tends to assemble paralogues which match the same rice locus into separate contigs , whereas the snp analysis step is designed to separate homoeologues . conversely , in situations where there are relatively few ests it can be useful to assemble the sequences from wheat and related species with low stringency . whets also assembles the hq - cdna sequences where present using a much higher base quality setting for the cap3 program than used for ests . this has the effect that the consensus sequence of any contig will normally be the same as any hq - cdna within it . after assembly , the rice cds is aligned to the contigs consensus and singlet sequences with blast and the results displayed using a modified version of a perl script from the korf et al . links are supplied which open windows detailing all species , tissue , sequence type and cultivar of the constituent sequences . the main output for user downloading is a fasta file containing the rice template cds , contigs consensus and singlet sequences . additional details , such as intron positions are supplied in the descriptor fields of this file . also available are other files , such as ace format files for each of the contigs containing all the information on the constituent sequences and their alignment , and a spreadsheet - compatible file containing details of all snps used to split contigs . whets is implemented in mysql ( http://www.mysql.com/ ) and perl using some bioperl ( 14 ) modules . more details on allocation of blast hits within the whets database , strand of est used and the algorithm for separating contigs into putative homoeologues are available in the supplementary data . to test that whets correctly separates homoeologues , we examined the well - characterized waxy locus , which encodes granule - bound starch synthase i. the three homoeologous forms are all sequenced , as are several allelic variants of these . as there are only a total of 2  715 wheat hq - cdna sequences available , the normal use of whets is only with ests . we , therefore , ran whets with the orthologous rice locus os06g04200 setting the filter to use ests and wheat sequences only . figure 2 shows the output and how the resulting contigs match with the known homoeologues . from ests alone , whets correctly identifies the homoeologues and indicates the existence of a splice variant of the b homoeologue with a deletion in its 5 utr . also shown ( figure 3 ) is the additional window detailing constituent sequences of one of the contigs . the black line at the top corresponds to the rice gene cds with intron position and size indicated as red vertical lines with triangles . the rows below show these hits , with blast hsps for contigs and singlets shown as lines coloured according to percentage identity , and coordinates aligned to the rice template . the cap3 step gives three contigs ; one of these ( contig 1 ) is then divided into five new contigs by the snp analysis step ( contigs 1.1 , 1.2 , etc . ) the genome of origin ( a , b , d ) has been added to the screenshot according to 100% identity matches of the contig consensus to the known - homoeologue transcript sequences ( exons of accessions ab019622 , ab019623 and ab019624 ) . contigs 1.4 and 1.5 appear to be splice variants with an indel in the 5utr . figure 3.display window showing details for a contig which is opened by clicking on contig 1.1  link in figure 2 . the black line at the top corresponds to the rice gene cds with intron position and size indicated as red vertical lines with triangles . the rows below show these hits , with blast hsps for contigs and singlets shown as lines coloured according to percentage identity , and coordinates aligned to the rice template . the cap3 step gives three contigs ; one of these ( contig 1 ) is then divided into five new contigs by the snp analysis step ( contigs 1.1 , 1.2 , etc . ) the genome of origin ( a , b , d ) has been added to the screenshot according to 100% identity matches of the contig consensus to the known - homoeologue transcript sequences ( exons of accessions ab019622 , ab019623 and ab019624 ) . contigs 1.4 and 1.5 appear to be splice variants with an indel in the 5utr . display window showing details for a contig which is opened by clicking on contig 1.1  link in figure 2 . whets is designed to be a practical tool for wheat biologists to rapidly get the best estimate of transcript sequence for a target gene , supplemented with information on tissue distribution and likely gene structure . it is particularly aimed at producing wheat sequences from which to design pcr primers for cdna templates .
wheat biologists face particular problems because of the lack of genomic sequence and the three homoeologous genomes which give rise to three very similar forms for many transcripts . 
 however , over 1.3 million available public - domain triticeae ests ( of which 850  000 are wheat ) and the full rice genomic sequence can be used to estimate likely transcript sequences present in any wheat cdna sample to which pcr primers may then be designed . 
 wheat estimated transcript server ( whets ) is designed to do this in a convenient form , and to provide information on the number of matching est and high quality cdna ( hq - cdna ) sequences , tissue distribution and likely intron position inferred from rice . 
 triticeae est and hq - cdna sequences are mapped onto rice loci and stored in a database . 
 the user selects a rice locus ( directly or via arabidopsis ) and the matching triticeae sequences are assembled according to user - defined filter and stringency settings . 
 assembly is achieved initially with the cap3 program and then with a single nucleotide polymorphism ( snp)-analysis algorithm designed to separate homoeologues . 
 alignment of the resulting contigs and singlets against the rice template sequence is then displayed . 
 sequences and assembly details are available for download in fasta and ace formats , respectively . 
 whets is accessible at http://www4.rothamsted.bbsrc.ac.uk/whets .