BACKGROUND
much of the world's protein and oil comes from soybean , and it is the major source of seed meal used in animal feed.
in fact, soybean contains more protein than any other ordinary food source, including meat, cheese and fish  <cit> .
it grows in a variety of temperate climates, and has the added benefit of improving soil quality by fixing nitrogen.
except for corn, more soybean is grown in the usa than any other single crop.
unfortunately, despite the importance of soybean, genetic tools for investigation of gene function and crop improvement have been difficult to develop.
although soybean can be transformed with either agrobacterium tumefaciens or a. rhizogenes, neither system is ideal.
the efficiency of a. tumefaciens transformation is typically low  <cit>  and is genotype specific  <cit> .
currently, the most successful combination of genotypes, chemical enhancers and selection, yields transformation efficiencies of up to 16%  <cit> .
a. rhizogenes root transformation has higher efficiency  and seems to be genotype independent, but is not heritable  <cit> .
particle bombardment can also be used to obtain transformants with variable success rates  <cit> , but can also introduce multiple copies that may recombine or result in co-suppression  <cit> .
often the goal is to obtain a knockout to better understand gene function.
however, gene disruption by induction of transposon insertion has not yet been successful.
rnai has produced knockdowns in some cases  <cit> , but still relies on transformation.
additionally, all of these methods require time-consuming tissue culture steps that are not compatible with high-throughput generation of mutants, and still can produce chimeric transformants that may not pass the trait on to the next generation.
in contrast to transgenic methods, chemical mutagenesis can be applied to most species, even those that lack well-developed genetic tools.
chemical mutagenesis has several other benefits.
no tissue culture is required, and the induced changes are stable and heritable so that the succeeding generations will not be chimeric.
because chemical mutagenesis induces single nucleotide changes, it can provide an allelic series in a gene target in addition to knockouts.
importantly, lines carrying induced mutations are not transgenic, and are therefore not associated with any regulatory restrictions.
chemical mutagenesis has been successfully used for many phenotypic screens in soybean, yielding mutants in traits such as ethylene sensitivity and nodulation  <cit> .
the combination of chemical mutagenesis with screening for induced changes in a gene target of interest is a powerful technique for obtaining an allelic series that can be used to study gene function or crop improvement.
tilling  is a high-throughput reverse genetic method to obtain allelic series from a chemically mutagenized population .
a chosen target is amplified from pooled dnas using fluorescently labeled pcr primers.
following amplification, the pcr products are denatured and re-annealed.
if a mutation is present in the pooled dna, a heteroduplex will form.
a single-strand specific nuclease found in celery juice extract  is used to cleave a strand of the heteroduplex, and the products are electrophoresed on a denaturing acrylamide gel  <cit> .
mutations are detected by the observation of cleaved bands.
we have established a popular tilling service for arabidopsis thaliana, where we have identified over  <dig> mutations in more than  <dig> targets during the past five years  <cit> .
tilling has also been successfully applied to maize, barley and wheat, despite their having much larger genomes than arabidopsis  <cit> .
here we extend tilling to four chemically mutagenized soybean populations and describe a generally applicable strategy for eliminating amplification of multiple products from the closely related homeologs or paralogs in the soybean genome.
RESULTS
mutation discovery in mutagenized soybean populations
four mutagenized soybean populations in two genetic backgrounds were constructed for tilling, referred to as a, b, c, and d .
the chemical mutagens nmu and ems have been shown to induce mutations in previous phenotypic screens of soybean  <cit> .
genomic dna was isolated from leaf tissue and samples were normalized prior to pooling eight-fold for screening.
each population was screened independently with the same primers .
we discovered  <dig> mutations:  <dig> in a,  <dig> in b,  <dig> in c, and  <dig> in d .
two individual lines, one from the a population and the other from c, had more than one base change detected in an amplicon.
because these changes were homozygous and not the expected g/c to a/t ems-induced transitions, we considered the individual lines to be likely cultivar contaminants, and we excluded them from the analysis.
mutation density was estimated as the total number of mutations divided by the total number of base pairs screened .
for each target,  <dig> bp is subtracted from the amplicon size to adjust for the  <dig> bp regions at the top and bottom of tilling gel images that are difficult to analyze  <cit> .
the a and d populations showed similar mutation densities .
mutation density in the population designated c was ~1/ <dig> kb and ~1/ <dig> kb in the b population.
the c and d populations had the same distribution of mutations with 4% truncation mutations, 44–45% missense, and 51–52% silent mutations.
the distribution in the b population was 8% truncation, 33% missense, and 58% silent mutations.
the a population deviated significantly from these mutation distributions in that no truncations were found, 66% missense and 34% silent mutations were found .
however, none of the distributions of mutations were significantly different than the expected distribution calculated from ems-induced changes in the targets .
in the a and c ems-treated populations, as well as the nmu-treated d population, ~90% of base changes were g/c to a/t transitions .
in the ems-treated b population, 75% of base changes were g/c to a/t transitions.
however, the frequency of g/c to a/t transitions is not statistically significantly different between the b population and the other three populations.
each ems-treated population contained an individual with a t to a transversion.
the nmu population contained  <dig> individuals with g to t transversions.
because it is well established that ems mutagenesis induces g/c to a/t transitions, the most conservative estimation of mutation density would only consider such base changes to be induced mutations.
in that case, the mutation densities become ~1/ <dig> kb in a, ~1/ <dig> kb in b, and ~1/ <dig> kb in c.
elimination of near-duplicate amplicons
three primer sets were initially tested for amplification of a specific target by observation of a single band of the expected size on an agarose gel.
although all three primer sets yielded a single band on an agarose gel, only one set  produced good quality tilling gels as determined by adequate quantities of single stranded full-length pcr product and by the detection of a low number of cleaved bands likely to represent induced mutations based on expected densities of chemically induced mutations in plants.
amplification products from the other two primer sets resulted in tilling gels with multiple cleaved fragments in every lane, suggesting that more than one target was being amplified and digested.
following this observation, subsequent primers were tested by agarose gel analysis and sequencing.
of  <dig> primer sets tested,  <dig> primer sets amplified more than one target.
given the high proportion of tested primer sets that amplified more than one target, we wondered whether we could screen for mutations in these targets by eliminating extra templates in the genomic dna.
for example, amplification and cje digestion with the gmrhg <dig> primers resulted in multiple bands in every pool .
the multiple bands were still observed when tilling assays were performed on unpooled dnas , and multiple heterozygous sites were detected upon sequencing individuals , consistent with the hypothesis that the primers amplified more than one target.
two sequences were obtained upon cloning the gmrhg <dig> pcr product; one sequence corresponded to the gmrhg <dig> target and the other sequence  contained the polymorphisms observed when sequencing the gmrhg <dig> pcr product.
we wondered whether an alternative to extensive primer testing would be to eliminate amplification of extraneous targets from the genomic dna.
to remove a target from tilling assays, sequence information was used to choose a restriction enzyme that cut once within the extraneous target .
the restriction-digested dna was purified by centrifugation through sephadex spin columns prior to performing the tilling assay.
digestion of the template eliminated amplification of the additional target  and allowed the identification of  <dig> more mutant alleles .
discussion
to determine whether soybean is suitable for high-throughput mutation discovery, we screened seven targets in four mutagenized populations and discovered a total of  <dig> induced mutations.
the a and d populations had the highest mutation frequencies, followed by the c and b populations.
given the sequences of the seven targets, the distribution of mutations was as expected.
the majority of induced mutations were g/c to a/t transitions.
we also found we could discover additional mutations by digesting the template dna to eliminate an extraneous amplicon that was hampering mutation identification.
both ems and nmu mutagenesis of soybean seed resulted in populations with mutation frequencies that are feasible for use in a high-throughput tilling operation.
the mutation frequencies in these soybean populations were higher than those reported for barley and maize  <cit> , and except for the b population, are similar or higher than what we have found in our arabidopsis populations.
although the b population was treated with the same concentration of ems as the a population, the resulting mutation frequency was lower.
it is possible that the genetic background could have an effect on the efficiency or toxicity of the mutagen, as has been observed in rice  <cit> , but differences due to other environmental or experimental conditions cannot be ruled out.
the b and c populations are from the same genetic background, but the b population was mutagenized with a 20% lower concentration of ems and as a result has approximately half the mutation density as the c population.
we have noted that treatment of arabidopsis seed batches with the same concentration of mutagen can vary in mutation frequency from experiment to experiment, probably because of the effect of environmental conditions on the plant response.
so it is expected that mutagenesis experiments performed at different locations with different mutagen concentrations may result in very different mutation frequencies.
because soybean is considered a paleopolyploid, it is possible that the mutation frequency could be increased even further without adverse affects due to the genetic redundancy provided by the largely duplicated gene set.
for example, allotetraploid and allohexaploid wheat populations have been developed with mutations frequencies of 1/ <dig> and 1/ <dig> kb, respectively  <cit> .
however, while visible mutations were more frequently observed when the nmu concentration was increased to  <dig>  mm, the proportion of treated seeds that germinated and grew was reduced twofold .
hence, more severe mutation protocols can increase mutation frequency, but they also reduce the recovery of viable seeds dramatically.
in arabidopsis, maize, and wheat, more than 99% of ems-induced mutations are g/c to a/t transitions  <cit> .
in contrast, the percentage in rice, barley, and drosophila ranges from 70–84%  <cit> .
in the ems-treated soybean populations, the percentage of g/c to a/t transitions was in the range of these previously published frequencies .
based on studies in e. coli and mouse  <cit> , nmu is also believed to induce primarily g/c to a/t transitions, but few reports are available for plants.
here we find that 90% of mutations induced by nmu were g/c to a/t transitions.
our study also addressed a problem caused by near identical copies of genes, such as the homeologous sets found in polyploid species or members of gene families.
the incompletely sequenced genome makes it difficult to define primers specific for a single gene, so that amplification of multiple products becomes a significant issue for a high-throughput soybean tilling service.
pre-testing unlabeled primers by amplifying dna followed by agarose gel electrophoresis and sequencing should reduce the number of primer sets chosen for tilling that amplify more than one target.
we found that pre-testing was successful for soybean targets which are known to be members of gene families .
the maize tilling service, which faces a similar problem, has successfully implemented such pre-testing in a high-throughput manner  <cit> .
in our study, we found that only ~40% of soybean primers passed pre-testing and of those, only 60% produced high quality tilling data.
our observation that amplification of multiple products derived from homeologous templates reduces the ability to detect mutations agrees with that of slade and colleagues  <cit> .
clearly, robust amplification of a single target will be a requirement for future soybean tilling.
sequence information from homeologous or paralogous genes could be used to direct primer design toward less conserved regions.
in cases where a primer set that only amplifies one target cannot be identified, it is possible to use sequence information gathered while testing the primers to find a restriction enzyme that digests only one homeolog or paralog thus eliminating amplification from the corresponding template dna.
restriction digestion adds an extra step and requires larger amounts of template dna.
the step, however, can easily be done in a high throughput manner by digesting templates in 96- or 384-well format prior to pcr and even the additional amount of dna required would allow at least  <dig> genes to be screened with the present dna yield .
legumes have unique biological and agronomic characteristics that cannot be investigated in either arabidopsis or maize model systems.
a tilling service is currently available for lotus japonicus  <cit> .
while much knowledge will be gained using l. japonicus as a model system for legume gene function, the application of that knowledge to modification of soybean traits remains difficult.
given the limits of other functional genomics approaches in soybean  <cit> , a tilling service could provide allelic series in genes of scientific or agronomic importance.
individual mutations may not result in phenotypic changes due to the redundant nature of the soybean genome.
however, the high mutation frequency combined with the ability to screen individual targets allows one to screen homeologs or gene family members individually and then combine the mutant alleles through breeding.
this would greatly facilitate progress in the study and breeding of soybean and other polyploids in which the efficiency of mutation breeding might otherwise be low.
one public service is already operational  <cit> , and others may be developed in the future.
CONCLUSIONS
we have successfully extended the tilling method to four chemically mutagenized soybean populations in two genetic backgrounds.
the substantial mutation density suggests that soybean should be an amenable subject for a high-throughput tilling service.
we have also developed a strategy that could be generally applied to eliminate amplification of multiple products from the soybean genome and it can easily be fit into a high-throughput pipeline.

 much of the world's protein and oil comes from soybean, and it is the major source of seed meal used in animal feed. in fact, soybean contains more protein than any other ordinary food source, including meat, cheese and fish. it grows in a variety of temperate climates, and has the added benefit of improving soil quality by fixing nitrogen. except for corn, more soybean is grown in the usa than any other single crop. unfortunately, despite the importance of soybean, genetic tools for investigation of gene function and crop improvement have been difficult to develop. although soybean can be transformed with either agrobacterium tumefaciens or a. rhizogenes, neither system is ideal. the efficiency of a. tumefaciens transformation is typically low and is genotype specific. currently, the most successful combination of genotypes, chemical enhancers and selection, yields transformation efficiencies of up to 16%. a. rhizogenes root transformation has higher efficiency and seems to be genotype independent, but is not heritable. particle bombardment can also be used to obtain transformants with variable success rates, but can also introduce multiple copies that may recombine or result in co-suppression. often the goal is to obtain a knockout to better understand gene function. however, gene disruption by induction of transposon insertion has not yet been successful. rnai has produced knockdowns in some cases, but still relies on transformation. additionally, all of these methods require time-consuming tissue culture steps that are not compatible with high-throughput generation of mutants, and still can produce chimeric transformants that may not pass the trait on to the next generation. in contrast to transgenic methods, chemical mutagenesis can be applied to most species, even those that lack well-developed genetic tools. chemical mutagenesis has several other benefits. no tissue culture is required, and the induced changes are stable and heritable so that the succeeding generations will not be chimeric. because chemical mutagenesis induces single nucleotide changes, it can provide an allelic series in a gene target in addition to knockouts. importantly, lines carrying induced mutations are not transgenic, and are therefore not associated with any regulatory restrictions. chemical mutagenesis has been successfully used for many phenotypic screens in soybean, yielding mutants in traits such as ethylene sensitivity and nodulation. the combination of chemical mutagenesis with screening for induced changes in a gene target of interest is a powerful technique for obtaining an allelic series that can be used to study gene function or crop improvement. tilling is a high-throughput reverse genetic method to obtain allelic series from a chemically mutagenized population. a chosen target is amplified from pooled dnas using fluorescently labeled pcr primers. following amplification, the pcr products are denatured and re-annealed. if a mutation is present in the pooled dna, a heteroduplex will form. a single-strand specific nuclease found in celery juice extract is used to cleave a strand of the heteroduplex, and the products are electrophoresed on a denaturing acrylamide gel. mutations are detected by the observation of cleaved bands. we have established a popular tilling service for arabidopsis thaliana, where we have identified over mutations in more than targets during the past five years. tilling has also been successfully applied to maize, barley and wheat, despite their having much larger genomes than arabidopsis. here we extend tilling to four chemically mutagenized soybean populations and describe a generally applicable strategy for eliminating amplification of multiple products from the closely related homeologs or paralogs in the soybean genome.  mutation discovery in mutagenized soybean populations four mutagenized soybean populations in two genetic backgrounds were constructed for tilling, referred to as a, b, c, and d. the chemical mutagens nmu and ems have been shown to induce mutations in previous phenotypic screens of soybean. genomic dna was isolated from leaf tissue and samples were normalized prior to pooling eight-fold for screening. each population was screened independently with the same primers. we discovered mutations: in a, in b, in c, and in d. two individual lines, one from the a population and the other from c, had more than one base change detected in an amplicon. because these changes were homozygous and not the expected g/c to a/t ems-induced transitions, we considered the individual lines to be likely cultivar contaminants, and we excluded them from the analysis. mutation density was estimated as the total number of mutations divided by the total number of base pairs screened. for each target, bp is subtracted from the amplicon size to adjust for the bp regions at the top and bottom of tilling gel images that are difficult to analyze. the a and d populations showed similar mutation densities. mutation density in the population designated c was ~1/ kb and ~1/ kb in the b population. the c and d populations had the same distribution of mutations with 4% truncation mutations, 44–45% missense, and 51–52% silent mutations. the distribution in the b population was 8% truncation, 33% missense, and 58% silent mutations. the a population deviated significantly from these mutation distributions in that no truncations were found, 66% missense and 34% silent mutations were found. however, none of the distributions of mutations were significantly different than the expected distribution calculated from ems-induced changes in the targets. in the a and c ems-treated populations, as well as the nmu-treated d population, ~90% of base changes were g/c to a/t transitions. in the ems-treated b population, 75% of base changes were g/c to a/t transitions. however, the frequency of g/c to a/t transitions is not statistically significantly different between the b population and the other three populations. each ems-treated population contained an individual with a t to a transversion. the nmu population contained individuals with g to t transversions. because it is well established that ems mutagenesis induces g/c to a/t transitions, the most conservative estimation of mutation density would only consider such base changes to be induced mutations. in that case, the mutation densities become ~1/ kb in a, ~1/ kb in b, and ~1/ kb in c. elimination of near-duplicate amplicons three primer sets were initially tested for amplification of a specific target by observation of a single band of the expected size on an agarose gel. although all three primer sets yielded a single band on an agarose gel, only one set produced good quality tilling gels as determined by adequate quantities of single stranded full-length pcr product and by the detection of a low number of cleaved bands likely to represent induced mutations based on expected densities of chemically induced mutations in plants. amplification products from the other two primer sets resulted in tilling gels with multiple cleaved fragments in every lane, suggesting that more than one target was being amplified and digested. following this observation, subsequent primers were tested by agarose gel analysis and sequencing. of primer sets tested, primer sets amplified more than one target. given the high proportion of tested primer sets that amplified more than one target, we wondered whether we could screen for mutations in these targets by eliminating extra templates in the genomic dna. for example, amplification and cje digestion with the gmrhg primers resulted in multiple bands in every pool. the multiple bands were still observed when tilling assays were performed on unpooled dnas, and multiple heterozygous sites were detected upon sequencing individuals, consistent with the hypothesis that the primers amplified more than one target. two sequences were obtained upon cloning the gmrhg pcr product; one sequence corresponded to the gmrhg target and the other sequence contained the polymorphisms observed when sequencing the gmrhg pcr product. we wondered whether an alternative to extensive primer testing would be to eliminate amplification of extraneous targets from the genomic dna. to remove a target from tilling assays, sequence information was used to choose a restriction enzyme that cut once within the extraneous target. the restriction-digested dna was purified by centrifugation through sephadex spin columns prior to performing the tilling assay. digestion of the template eliminated amplification of the additional target and allowed the identification of more mutant alleles. discussion to determine whether soybean is suitable for high-throughput mutation discovery, we screened seven targets in four mutagenized populations and discovered a total of induced mutations. the a and d populations had the highest mutation frequencies, followed by the c and b populations. given the sequences of the seven targets, the distribution of mutations was as expected. the majority of induced mutations were g/c to a/t transitions. we also found we could discover additional mutations by digesting the template dna to eliminate an extraneous amplicon that was hampering mutation identification. both ems and nmu mutagenesis of soybean seed resulted in populations with mutation frequencies that are feasible for use in a high-throughput tilling operation. the mutation frequencies in these soybean populations were higher than those reported for barley and maize, and except for the b population, are similar or higher than what we have found in our arabidopsis populations. although the b population was treated with the same concentration of ems as the a population, the resulting mutation frequency was lower. it is possible that the genetic background could have an effect on the efficiency or toxicity of the mutagen, as has been observed in rice, but differences due to other environmental or experimental conditions cannot be ruled out. the b and c populations are from the same genetic background, but the b population was mutagenized with a 20% lower concentration of ems and as a result has approximately half the mutation density as the c population. we have noted that treatment of arabidopsis seed batches with the same concentration of mutagen can vary in mutation frequency from experiment to experiment, probably because of the effect of environmental conditions on the plant response. so it is expected that mutagenesis experiments performed at different locations with different mutagen concentrations may result in very different mutation frequencies. because soybean is considered a paleopolyploid, it is possible that the mutation frequency could be increased even further without adverse affects due to the genetic redundancy provided by the largely duplicated gene set. for example, allotetraploid and allohexaploid wheat populations have been developed with mutations frequencies of 1/ and 1/ kb, respectively. however, while visible mutations were more frequently observed when the nmu concentration was increased to mm, the proportion of treated seeds that germinated and grew was reduced twofold. hence, more severe mutation protocols can increase mutation frequency, but they also reduce the recovery of viable seeds dramatically. in arabidopsis, maize, and wheat, more than 99% of ems-induced mutations are g/c to a/t transitions. in contrast, the percentage in rice, barley, and drosophila ranges from 70–84%. in the ems-treated soybean populations, the percentage of g/c to a/t transitions was in the range of these previously published frequencies. based on studies in e. coli and mouse, nmu is also believed to induce primarily g/c to a/t transitions, but few reports are available for plants. here we find that 90% of mutations induced by nmu were g/c to a/t transitions. our study also addressed a problem caused by near identical copies of genes, such as the homeologous sets found in polyploid species or members of gene families. the incompletely sequenced genome makes it difficult to define primers specific for a single gene, so that amplification of multiple products becomes a significant issue for a high-throughput soybean tilling service. pre-testing unlabeled primers by amplifying dna followed by agarose gel electrophoresis and sequencing should reduce the number of primer sets chosen for tilling that amplify more than one target. we found that pre-testing was successful for soybean targets which are known to be members of gene families. the maize tilling service, which faces a similar problem, has successfully implemented such pre-testing in a high-throughput manner. in our study, we found that only ~40% of soybean primers passed pre-testing and of those, only 60% produced high quality tilling data. our observation that amplification of multiple products derived from homeologous templates reduces the ability to detect mutations agrees with that of slade and colleagues. clearly, robust amplification of a single target will be a requirement for future soybean tilling. sequence information from homeologous or paralogous genes could be used to direct primer design toward less conserved regions. in cases where a primer set that only amplifies one target cannot be identified, it is possible to use sequence information gathered while testing the primers to find a restriction enzyme that digests only one homeolog or paralog thus eliminating amplification from the corresponding template dna. restriction digestion adds an extra step and requires larger amounts of template dna. the step, however, can easily be done in a high throughput manner by digesting templates in 96- or 384-well format prior to pcr and even the additional amount of dna required would allow at least genes to be screened with the present dna yield. legumes have unique biological and agronomic characteristics that cannot be investigated in either arabidopsis or maize model systems. a tilling service is currently available for lotus japonicus. while much knowledge will be gained using l. japonicus as a model system for legume gene function, the application of that knowledge to modification of soybean traits remains difficult. given the limits of other functional genomics approaches in soybean, a tilling service could provide allelic series in genes of scientific or agronomic importance. individual mutations may not result in phenotypic changes due to the redundant nature of the soybean genome. however, the high mutation frequency combined with the ability to screen individual targets allows one to screen homeologs or gene family members individually and then combine the mutant alleles through breeding. this would greatly facilitate progress in the study and breeding of soybean and other polyploids in which the efficiency of mutation breeding might otherwise be low. one public service is already operational, and others may be developed in the future.  we have successfully extended the tilling method to four chemically mutagenized soybean populations in two genetic backgrounds. the substantial mutation density suggests that soybean should be an amenable subject for a high-throughput tilling service. we have also developed a strategy that could be generally applied to eliminate amplification of multiple products from the soybean genome and it can easily be fit into a high-throughput pipeline. 
to address this problem, we removed an extraneous target by pretreating genomic dna with a restriction enzyme. the development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome. the nmu-treated population and one ems mutagenized population had similar mutation density, while another ems population had a mutation density of ~1/ kb. the remaining population had a mutation density of ~1/ kb. we have applied tilling to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate and one with n-nitroso-n-methylurea. digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template. nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as targeting induced local lesions in genomes.
 soybean is an important nitrogen-fixing crop that provides much of the world's protein and oil. however, the available tools for investigation of soybean gene function are limited. nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as targeting induced local lesions in genomes. we have applied tilling to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate and one with n-nitroso-n-methylurea.  we screened seven targets in each population and discovered a total of induced mutations. the nmu-treated population and one ems mutagenized population had similar mutation density, while another ems population had a mutation density of ~1/ kb. the remaining population had a mutation density of ~1/ kb. because of soybean's polyploid history, pcr amplification of multiple targets could impede mutation discovery. indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. to address this problem, we removed an extraneous target by pretreating genomic dna with a restriction enzyme. digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.  the development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome. 