BACKGROUND
ohno described gene duplications and the resulting changes in selective pressure as providing the opportunity for duplicates to evolve new functions in  <dig>  <cit> .
since then it has become clear that a significant proportion of genes that make up a genome are not entirely unique from one another but are part of larger families of related genes.
genome evolution, at least in part, proceeds by duplications of individual genes  <cit> , genomic segments  <cit> , or even whole genomes  <cit> .
the accumulation of different mutations in duplicates  subsequently leads to either loss of function for one , altered function , or a new function .
the study of the molecular processes by which functional innovation occurs interests not only evolutionary biologists, but protein engineers and agricultural biologists.
a clearer understanding of the extent to which gene families contribute to the selected traits in our most important crop species will help guide decisions regarding future improvements.
many analyses of the divergence between paralogs involve the estimation of the dn/ds ratio, the number of nonsynonymous base substitutions per nonsynonymous site versus the number of synonymous base substitutions per synonymous site  <cit> .
a dn/ds ratio <  <dig> indicates purifying or negative selection  that tends to keep amino acid sequences the same if changes are deleterious.
a ratio equal to  <dig> indicates changes that are neutral to fitness, while a dn/ds ratio >  <dig> would indicate adaptive or positive selection presumably because natural selection favors the amino acid changes.
most methods of estimating dn/ds are approximate because many codons may differ in more than one position and therefore could have evolved by different but equally parsimonious paths.
other factors can also make detection of purifying versus adaptive selection difficult, namely codon bias, the fact that selection pressures are not uniform along an evolutionary lineage, and selection is not equal among functional domains of a gene  <cit> .
force et al.
proposed a ddc  model whereby complementary deleterious mutations in regulatory elements between duplicate genes partitions the original function resulting in different sub-functions  <cit> .
papp et al.
found that the number of shared regulatory elements between duplicated genes in yeast decreases with evolutionary time  <cit> .
duplicate genes were identified as symmetrical best hits  of encoded proteins .
the age of the duplicates was estimated by the accumulation of synonymous substitutions in the coding regions.
if synonymous substitutions were accompanied by an equal number of nonsynonymous  or by a greater number of nonsynonymous substitutions  then one would not expect that the proteins would maintain enough similarity to be symbets.
it follows that if subfunctionalization is to occur by changes in regulatory elements then some degree of purifying selection should maintain the protein function.
otherwise, the adaptive selection on the regulatory elements would be eliminated by loss of original function in the protein.
coding regions of paralogs that have subfunctionalized via changes to regulatory elements should exhibit a bias toward synonymous substitutions.
in plants, a significantly greater proportion of genes belong to gene families than in animals or other major taxa  <cit> .
either gene duplication events have been more common in plants, or more duplicates have been retained during the evolutionary history of plants.
in either case, subfunctionalization via changes in regulatory sequences following duplication appears to be an especially common mechanism of innovative evolution in plants  <cit> .
if this is the case, there should exist a significant number of gene families that can be identified by a bias toward synonymous substitutions when contigs are assembled from a significantly large database of ests .
ests are generated when large numbers of randomly selected cdna clones from various tissues, genotypes, developmental stages, or treatments are partially sequenced.
the greater the number of ests from independently constructed libraries the more information can be derived from in silico analyses.
thirty species currently have over  <dig> k ests in the ncbi database.
in this paper we outline a simple method to identify, from ests, gene families that exhibit purifying selection during subfunctionalization of paralogs.
we also describe several software tools developed to automate many of the time-consuming steps in the identification of these gene family members.
we demonstrate the validity of the method by identifying, from est data alone, a well-characterized gene family of arabidopsis.
and finally, we demonstrate the identification of two previously unidentified gene families in glycine max, one a known plant gene family and the other a putative novel small gene family.
RESULTS
validation of concept
since our goal is not detecting rates of synonymous versus nonsynonymous substitutions we simply tally differences between potential paralog pairs with regard to codon position.
the rationale is that only  <dig> of  <dig> first positions of codons are 2-fold redundant ,  <dig> of  <dig> second positions are redundant, while  <dig> of  <dig> third positions are 2-fold redundant,  <dig> are 3-fold redundant, and  <dig> are 4-fold redundant.
if differences between contigs are evolutionary and subject to negative selection, significantly more differences will occur in the third position and least will occur in the second position.
however, if differences between contigs are artifacts  or the result of alternative splicing, no pattern among codon positions should be exhibited.
if differences appear nonrandom with respect to codon position , and third position differences are more than  <dig> times first position differences, and all differences are distributed as 3rd > 1st > 2nd, then we conclude that the contigs represent different genes.
however, if these criteria are not met we do not conclude that the contigs necessarily represent the same gene.
our goal is only to identify contigs that represent different genes of the same family.
we do not expect that all members of a particular family will be detectable by this method.
if desired other members may be identified with iterative searches using previously identified contigs.
to illustrate that this method can identify members of a gene family with some accuracy using only est data we tested it on a well-characterized gene family  from arabidopsis.
the simests search of a. thaliana dbest using atpal <dig> protein as query  resulted in  <dig> est files  in fasta format.
the ests assembled into nine contigs ranging from  <dig> to  <dig> bases and  <dig> to  <dig> ests each.
assembly parameters were  <dig> base minimum overlap with 100% match, and gap creation and extension penalties set at  <dig>  each.
the few gaps created were never more than one base.
the stringent overlap criteria and gap penalties reflect the fact that we would rather miss a potentially valid contig than generate an invalid one.
most contigs exhibited large open reading frames that extended most of the contig length.
some contigs had shorter overlapping orfs that could be joined by eliminating a single nucleotide gap.
however, no major sequence editing was done to increase the quality of contigs.
twenty-eight pairwise alignments were made and the differences according to codon position reported by scat are show in table  <dig>  the pattern exhibited suggest that atcontig <dig> represents a different pal gene than atcontig <dig>  that is,  <dig> shows nonrandom distribution of differences , third position differences are  <dig> -fold greater than first position, and overall 3rd > 1st > 2nd.
similarly, atcontig <dig> and atcontig <dig> represent different pal genes by these criteria.
the eight contigs assorted into three groups based on their nucleotide substitution pattern with each other contig.
those three groups designated genea, geneb, and genec are represented by atcontig <dig>  atcontig <dig>  and atcontig <dig>  respectively.
atcontig <dig> and  <dig> were included as part of genec in the comparison to real sequences because they exhibited close similarity to atcontig <dig>  the results of the comparison to the actual gene sequences for the pal gene family of arabidopsis are shown in table  <dig>  each contig representing the three gene groups was aligned with each of the four actual pal gene sequences from arabidopsis.
each contig group identified, by greater than 96% similarity, a different member of the pal gene family.
additionally, each contig exhibited less than 86% similarity to other members of the gene family.
atcontig <dig>  represents atpal <dig> , atcontig <dig>  represents atpal <dig> , and atcontig <dig> represents atpal <dig> .
no contigs generated at the parameters specified in the assembly program represented atpal <dig>  sequence comparisons between the four actual atpal genes show that atpal  <dig> is only about 70% similar to the original query gene, atpal <dig>  while atpal <dig> is 90% similar, and atpal <dig> about 80% similar to the original query.
that is, atpal <dig> is the most divergent gene family member relative to our query sequence.
this may explain the reason contigs representing pal <dig> were not generated.
this may indicate a lower limit to the similarity necessary between paralogs for identification by this method.
also, a potential upper limit of 95% similarity may be indicated by the fact that atpal <dig>  the query sequence, and its contig generated from ests are 96% similar.
that is, paralogs of very recent divergence or otherwise highly conserved at the nucleotide level may not be resolved as unique by this method.
an alternative explanation for missing atpal <dig> could be under representation in the est database relative to the other genes.
our stringent contig assembly parameters resulted in a significant number  of est sequences not joining a contig but remaining as singletons.
the overall result from this experiment verifies that it is possible to identify at least some gene family members using est data alone.
cad gene family identification in glycine max
we then turned our attention to the identification of a gene family not yet identified in glycine max.
cinnamyl alcohol dehydrogenase  is an enzyme of lignin biosynthesis, a component of the cell wall, and is encoded by a nine-member gene family in arabidopsis.
the simests search of g. max dbest using atcad protein as query resulted in  <dig> est files in fasta format.
the ests assembled into  <dig> contigs ranging from  <dig> to  <dig> bases and  <dig> to  <dig> ests each.
nine contigs that exhibited nearly full length orfs were processed by scat to yield the results shown in table  <dig>  the results indicate by our criteria that contig <dig> represents a different gene than contig <dig>  i.e.,  <dig> shows nonrandom distribution of differences , third position differences are  <dig> -fold greater than first position, and overall 3rd > 1st > 2nd.
this result alone would constitute an initial putative identification of a gene family not previously described in glycine max.
however, the results generated for contig <dig> versus contig <dig>  and contig <dig> versus contig <dig> meet two of the three criteria and could warrant further investigation as additional members of this gene family.
no significant similarity  indicated by bl2seq between contig <dig> and the others could be interpreted in two ways.
1) the two contigs in question could represent significantly different genes, or 2) the contigs could represent non-overlapping regions of the same gene.
cad is a protein of about  <dig> amino acids in arabidopsis and our contig orfs are  <dig> to  <dig> codons.
therefore, they should all overlap by a minimum of  <dig> codons.
it is possible that those  <dig> codons are dissimilar enough to generate no significant similarity.
however, if option one is correct, then we have potentially identified six members of the cad gene family of soybean, if incorrect, at least two.
clearly, this warrants further experimental analysis in the lab.
next we plan to construct gene specific primers for pcr and isolate these members of the cad gene family from soybean.
additional application of simests and scat using the contig sequences as queries could identify more members of this gene family.
there are hundreds of plant gene families that have been characterized in only a handful of species.
with this method many of those can now be identified in any species for which a significant number of ests exist.
soybean has over  <dig>  ests in dbest and relatively few gene families.
an obvious next step made possible by rapid screening of scat is to search for all known plant gene families not yet identified in soybean.
novel gene family identification in glycine max
est clustering can be a simple and effective method for identifying gene families.
unigene, the most widely available clustered set, uses a build procedure that begins by clustering mrna sequences.
the ests are then joined to existing mrna clusters.
new clusters are created for ests that do not join an mrna cluster.
since there are  <dig>  clusters in build # <dig> and only  <dig> contain mrna sequences, most clusters are est-only.
unigene also indicates for each cluster lacking an identifying mrna a similarity measure to other known genes, either strongly similar to, moderately similar to, or weakly similar to.
however, several clusters are designated only "transcribed locus" because the ests show no significant similarity to any known genes.
with the speed of screening provided by scat we decided to examine these clusters specifically for evidence that some may contain est sequences from multiple gene family members.
previous analysis of mrna-containing unigene clusters revealed that many clusters contain mrna sequences from two or more members of a gene family  <cit> .
it follows that many of the est-only clusters could also contain sequences from multiple genes.
the first indication of such a case was found in unigene cluster gma.
<dig> containing  <dig> ests.
assembly resulted in two contigs of  <dig> and  <dig> bases, each with two significant open reading frames .
results from pairwise alignments using scat are shown in table  <dig>  contig1a and 1b represent the two orfs on the same contig.
since bl2seq will automatically compare reverse complements during alignment results for contig1a versus contig2a and contig1a and contig2b are identical and exhibit a reverse pattern to contig1b versus contig2a and 2b.
the results for the latter comparisons exhibit the characteristic pattern of third position bias, however they only border on meeting the three criteria.
that is,  <dig> shows nonrandom distribution of differences , third position differences are  <dig> -fold greater than first position, and overall 3rd > 1st = 2nd.
however, these results also indicate a gap by the numbers in parentheses, and that gap is exactly three nucleotides, one in each position of a codon.
examination of the alignment shows that they are consecutive and represent a single codon indel .
this additional information taken with the pattern causes us to identify this as a potential gene family for further experimental analysis in the lab.
without even starting with a query it is possible to identify potential gene families from other groups of sequence-similar ests.
the next phase being pursued by our group is the rapid screening by scat of the largest est-only unigene clusters.
CONCLUSIONS
gene families in plants that exhibit purifying selection between members can be identified quickly and easily from est databases by integrating software tools simests pcat, or scat with commonly available contig assembly and orf identification programs.
this is illustrated by the successful identification of  <dig> members of a well-characterized small gene family in arabidopsis using est data alone.
two additional approaches with unisolated gene families in glycine max also produced positive results.
orthologous sequences can be used to identify at least some members of previously uncharacterized gene families, and new gene families can be discovered.
with this method many gene families can now be identified in any plant species for which a significant number of ests exist.

 ohno described gene duplications and the resulting changes in selective pressure as providing the opportunity for duplicates to evolve new functions in. since then it has become clear that a significant proportion of genes that make up a genome are not entirely unique from one another but are part of larger families of related genes. genome evolution, at least in part, proceeds by duplications of individual genes, genomic segments, or even whole genomes. the accumulation of different mutations in duplicates subsequently leads to either loss of function for one, altered function, or a new function. the study of the molecular processes by which functional innovation occurs interests not only evolutionary biologists, but protein engineers and agricultural biologists. a clearer understanding of the extent to which gene families contribute to the selected traits in our most important crop species will help guide decisions regarding future improvements. many analyses of the divergence between paralogs involve the estimation of the dn/ds ratio, the number of nonsynonymous base substitutions per nonsynonymous site versus the number of synonymous base substitutions per synonymous site. a dn/ds ratio < indicates purifying or negative selection that tends to keep amino acid sequences the same if changes are deleterious. a ratio equal to indicates changes that are neutral to fitness, while a dn/ds ratio > would indicate adaptive or positive selection presumably because natural selection favors the amino acid changes. most methods of estimating dn/ds are approximate because many codons may differ in more than one position and therefore could have evolved by different but equally parsimonious paths. other factors can also make detection of purifying versus adaptive selection difficult, namely codon bias, the fact that selection pressures are not uniform along an evolutionary lineage, and selection is not equal among functional domains of a gene. force et al. proposed a ddc model whereby complementary deleterious mutations in regulatory elements between duplicate genes partitions the original function resulting in different sub-functions. papp et al. found that the number of shared regulatory elements between duplicated genes in yeast decreases with evolutionary time. duplicate genes were identified as symmetrical best hits of encoded proteins. the age of the duplicates was estimated by the accumulation of synonymous substitutions in the coding regions. if synonymous substitutions were accompanied by an equal number of nonsynonymous or by a greater number of nonsynonymous substitutions then one would not expect that the proteins would maintain enough similarity to be symbets. it follows that if subfunctionalization is to occur by changes in regulatory elements then some degree of purifying selection should maintain the protein function. otherwise, the adaptive selection on the regulatory elements would be eliminated by loss of original function in the protein. coding regions of paralogs that have subfunctionalized via changes to regulatory elements should exhibit a bias toward synonymous substitutions. in plants, a significantly greater proportion of genes belong to gene families than in animals or other major taxa. either gene duplication events have been more common in plants, or more duplicates have been retained during the evolutionary history of plants. in either case, subfunctionalization via changes in regulatory sequences following duplication appears to be an especially common mechanism of innovative evolution in plants. if this is the case, there should exist a significant number of gene families that can be identified by a bias toward synonymous substitutions when contigs are assembled from a significantly large database of ests. ests are generated when large numbers of randomly selected cdna clones from various tissues, genotypes, developmental stages, or treatments are partially sequenced. the greater the number of ests from independently constructed libraries the more information can be derived from in silico analyses. thirty species currently have over k ests in the ncbi database. in this paper we outline a simple method to identify, from ests, gene families that exhibit purifying selection during subfunctionalization of paralogs. we also describe several software tools developed to automate many of the time-consuming steps in the identification of these gene family members. we demonstrate the validity of the method by identifying, from est data alone, a well-characterized gene family of arabidopsis. and finally, we demonstrate the identification of two previously unidentified gene families in glycine max, one a known plant gene family and the other a putative novel small gene family.  validation of concept since our goal is not detecting rates of synonymous versus nonsynonymous substitutions we simply tally differences between potential paralog pairs with regard to codon position. the rationale is that only of first positions of codons are 2-fold redundant, of second positions are redundant, while of third positions are 2-fold redundant, are 3-fold redundant, and are 4-fold redundant. if differences between contigs are evolutionary and subject to negative selection, significantly more differences will occur in the third position and least will occur in the second position. however, if differences between contigs are artifacts or the result of alternative splicing, no pattern among codon positions should be exhibited. if differences appear nonrandom with respect to codon position, and third position differences are more than times first position differences, and all differences are distributed as 3rd > 1st > 2nd, then we conclude that the contigs represent different genes. however, if these criteria are not met we do not conclude that the contigs necessarily represent the same gene. our goal is only to identify contigs that represent different genes of the same family. we do not expect that all members of a particular family will be detectable by this method. if desired other members may be identified with iterative searches using previously identified contigs. to illustrate that this method can identify members of a gene family with some accuracy using only est data we tested it on a well-characterized gene family from arabidopsis. the simests search of a. thaliana dbest using atpal protein as query resulted in est files in fasta format. the ests assembled into nine contigs ranging from to bases and to ests each. assembly parameters were base minimum overlap with 100% match, and gap creation and extension penalties set at each. the few gaps created were never more than one base. the stringent overlap criteria and gap penalties reflect the fact that we would rather miss a potentially valid contig than generate an invalid one. most contigs exhibited large open reading frames that extended most of the contig length. some contigs had shorter overlapping orfs that could be joined by eliminating a single nucleotide gap. however, no major sequence editing was done to increase the quality of contigs. twenty-eight pairwise alignments were made and the differences according to codon position reported by scat are show in table the pattern exhibited suggest that atcontig represents a different pal gene than atcontig that is, shows nonrandom distribution of differences, third position differences are -fold greater than first position, and overall 3rd > 1st > 2nd. similarly, atcontig and atcontig represent different pal genes by these criteria. the eight contigs assorted into three groups based on their nucleotide substitution pattern with each other contig. those three groups designated genea, geneb, and genec are represented by atcontig atcontig and atcontig respectively. atcontig and were included as part of genec in the comparison to real sequences because they exhibited close similarity to atcontig the results of the comparison to the actual gene sequences for the pal gene family of arabidopsis are shown in table each contig representing the three gene groups was aligned with each of the four actual pal gene sequences from arabidopsis. each contig group identified, by greater than 96% similarity, a different member of the pal gene family. additionally, each contig exhibited less than 86% similarity to other members of the gene family. atcontig represents atpal, atcontig represents atpal, and atcontig represents atpal. no contigs generated at the parameters specified in the assembly program represented atpal sequence comparisons between the four actual atpal genes show that atpal is only about 70% similar to the original query gene, atpal while atpal is 90% similar, and atpal about 80% similar to the original query. that is, atpal is the most divergent gene family member relative to our query sequence. this may explain the reason contigs representing pal were not generated. this may indicate a lower limit to the similarity necessary between paralogs for identification by this method. also, a potential upper limit of 95% similarity may be indicated by the fact that atpal the query sequence, and its contig generated from ests are 96% similar. that is, paralogs of very recent divergence or otherwise highly conserved at the nucleotide level may not be resolved as unique by this method. an alternative explanation for missing atpal could be under representation in the est database relative to the other genes. our stringent contig assembly parameters resulted in a significant number of est sequences not joining a contig but remaining as singletons. the overall result from this experiment verifies that it is possible to identify at least some gene family members using est data alone. cad gene family identification in glycine max we then turned our attention to the identification of a gene family not yet identified in glycine max. cinnamyl alcohol dehydrogenase is an enzyme of lignin biosynthesis, a component of the cell wall, and is encoded by a nine-member gene family in arabidopsis. the simests search of g. max dbest using atcad protein as query resulted in est files in fasta format. the ests assembled into contigs ranging from to bases and to ests each. nine contigs that exhibited nearly full length orfs were processed by scat to yield the results shown in table the results indicate by our criteria that contig represents a different gene than contig i.e., shows nonrandom distribution of differences, third position differences are -fold greater than first position, and overall 3rd > 1st > 2nd. this result alone would constitute an initial putative identification of a gene family not previously described in glycine max. however, the results generated for contig versus contig and contig versus contig meet two of the three criteria and could warrant further investigation as additional members of this gene family. no significant similarity indicated by bl2seq between contig and the others could be interpreted in two ways. 1) the two contigs in question could represent significantly different genes, or 2) the contigs could represent non-overlapping regions of the same gene. cad is a protein of about amino acids in arabidopsis and our contig orfs are to codons. therefore, they should all overlap by a minimum of codons. it is possible that those codons are dissimilar enough to generate no significant similarity. however, if option one is correct, then we have potentially identified six members of the cad gene family of soybean, if incorrect, at least two. clearly, this warrants further experimental analysis in the lab. next we plan to construct gene specific primers for pcr and isolate these members of the cad gene family from soybean. additional application of simests and scat using the contig sequences as queries could identify more members of this gene family. there are hundreds of plant gene families that have been characterized in only a handful of species. with this method many of those can now be identified in any species for which a significant number of ests exist. soybean has over ests in dbest and relatively few gene families. an obvious next step made possible by rapid screening of scat is to search for all known plant gene families not yet identified in soybean. novel gene family identification in glycine max est clustering can be a simple and effective method for identifying gene families. unigene, the most widely available clustered set, uses a build procedure that begins by clustering mrna sequences. the ests are then joined to existing mrna clusters. new clusters are created for ests that do not join an mrna cluster. since there are clusters in build # and only contain mrna sequences, most clusters are est-only. unigene also indicates for each cluster lacking an identifying mrna a similarity measure to other known genes, either strongly similar to, moderately similar to, or weakly similar to. however, several clusters are designated only "transcribed locus" because the ests show no significant similarity to any known genes. with the speed of screening provided by scat we decided to examine these clusters specifically for evidence that some may contain est sequences from multiple gene family members. previous analysis of mrna-containing unigene clusters revealed that many clusters contain mrna sequences from two or more members of a gene family. it follows that many of the est-only clusters could also contain sequences from multiple genes. the first indication of such a case was found in unigene cluster gma. containing ests. assembly resulted in two contigs of and bases, each with two significant open reading frames. results from pairwise alignments using scat are shown in table contig1a and 1b represent the two orfs on the same contig. since bl2seq will automatically compare reverse complements during alignment results for contig1a versus contig2a and contig1a and contig2b are identical and exhibit a reverse pattern to contig1b versus contig2a and 2b. the results for the latter comparisons exhibit the characteristic pattern of third position bias, however they only border on meeting the three criteria. that is, shows nonrandom distribution of differences, third position differences are -fold greater than first position, and overall 3rd > 1st = 2nd. however, these results also indicate a gap by the numbers in parentheses, and that gap is exactly three nucleotides, one in each position of a codon. examination of the alignment shows that they are consecutive and represent a single codon indel. this additional information taken with the pattern causes us to identify this as a potential gene family for further experimental analysis in the lab. without even starting with a query it is possible to identify potential gene families from other groups of sequence-similar ests. the next phase being pursued by our group is the rapid screening by scat of the largest est-only unigene clusters.  gene families in plants that exhibit purifying selection between members can be identified quickly and easily from est databases by integrating software tools simests pcat, or scat with commonly available contig assembly and orf identification programs. this is illustrated by the successful identification of members of a well-characterized small gene family in arabidopsis using est data alone. two additional approaches with unisolated gene families in glycine max also produced positive results. orthologous sequences can be used to identify at least some members of previously uncharacterized gene families, and new gene families can be discovered. with this method many gene families can now be identified in any plant species for which a significant number of ests exist. 
exhaustive searches for all members of a known gene family as well as the identification of new gene families has become increasingly important. gene families in plants, particularly that subset where purifying selection has occurred in the coding region, can be identified quickly and easily by integrating our software tools and commonly available contig assembly and orf identification programs. in this report we 1) describe the software tools developed to automate the gene family identification, 2) demonstrate the validity of the method by correctly identifying of pal gene family members from arabidopsis using est data alone, 3) identify to cad gene family members from glycine max, and 4) identify members of a putative glycine max gene family previously unidentified in any plant species. such negative selection can be detected by a bias toward synonymous over nonsynonymous substitutions. gene duplication events have played a significant role in genome evolution, particularly in plants. subfunctionalization via changes in regulatory sequences following duplication appears to be a common mechanism of evolution in plants and can be accompanied by purifying selection on the coding region. we have simplified the process and significantly shortened the time required by condensing many steps into a few scripts or programs to rapidly identify putative gene family members beginning with a single query sequence.
 gene duplication events have played a significant role in genome evolution, particularly in plants. exhaustive searches for all members of a known gene family as well as the identification of new gene families has become increasingly important. subfunctionalization via changes in regulatory sequences following duplication appears to be a common mechanism of evolution in plants and can be accompanied by purifying selection on the coding region. such negative selection can be detected by a bias toward synonymous over nonsynonymous substitutions. however, the process of identifying this bias requires many steps usually employing several different software programs. we have simplified the process and significantly shortened the time required by condensing many steps into a few scripts or programs to rapidly identify putative gene family members beginning with a single query sequence.  in this report we 1) describe the software tools developed to automate the gene family identification, 2) demonstrate the validity of the method by correctly identifying of pal gene family members from arabidopsis using est data alone, 3) identify to cad gene family members from glycine max, and 4) identify members of a putative glycine max gene family previously unidentified in any plant species.  gene families in plants, particularly that subset where purifying selection has occurred in the coding region, can be identified quickly and easily by integrating our software tools and commonly available contig assembly and orf identification programs. 2– march third annual midsouth computational biology and bioinformatics society conference. bioinformatics: a calculated discovery baton rouge, la, usa 