BACKGROUND
sorghum  is a c <dig> crop that displays excellent tolerance to both drought and high temperature stresses  <cit> .
sorghum has the highest water use efficiency among major crop plants and is unusually tolerant to low soil fertility, traits essential for survival and productivity in arid and semi-arid areas with limited irrigation capability.
worldwide, sorghum is the 5th most important grain crop, providing food and fodder for the inhabitants of drought-susceptible regions .
recently, sorghum has been demonstrated as a viable bioenergy feedstock  <cit> .
compared with other bioenergy grain crops, sorghum is particularly advantageous because it can be grown profitably on marginal land and therefore, would not remove more fertile land from existing food and fiber production  <cit> .
as a close evolutionary relative of both rice and maize, sorghum research contributes directly to a better understanding of the structure, function, and evolution of cereal genomes  <cit> .
being unusually tolerant to drought and high temperature stresses, sorghum also serves as a repository of genes that have the potential to improve stress tolerance in other crops.
recent progress in sorghum genomic studies has generated a series of important tools that can be used to identify favorable genes or alleles for further enhancement of resistance to abiotic stresses and for improved biofuels-related traits.
for example, well-established genetic, physical, and cytological maps facilitate the mapping and identification of genes responsible for important agronomic traits  <cit> .
syntenic alignment of the sorghum genome with those of maize, rice, and other cereals provides important insight into genome evolution  <cit> .
furthermore, the construction of cdna microarrays provides a platform for high throughput gene discovery  <cit> .
an important milestone was the recent completion and posting of the genomic sequence of an inbred sorghum line, btx <dig> .
utilization of these important genomic, genetic, and biotechnological resources such as targeting induced local lesions in genomes  will undoubtedly speed-up the elucidation of sorghum gene function and the identification of candidate genes for improving sorghum germplasm.
tilling was first developed in arabidopsis thaliana  <cit>  and has been successfully applied to identify knockout mutations and provide allelic mutations in target genes from pathogenic bacteria  <cit> , animals, and plants  <cit> .
the application of tilling to animal species includes caenorhabditis elegans  <cit> , zebrafish  <cit> , and drosophila  <cit> .
the application of tilling to plant species includes arabidopsis  <cit> , barley  <cit> , lotus  <cit> , wheat  <cit> , maize  <cit> , populus  <cit> , rice  <cit> , pea  <cit> , and soybean  <cit> .
the technique has proven to be valuable in characterizing the function of target genes  <cit> .
the challenge for researchers is to decipher the function of sorghum genes, particularly those that are unique to the species.
unfortunately, many of the reverse genetic tools, such as t-dna tagging and transposon-tagging are still not available in sorghum.
although active transposon elements have been identified in sorghum, no viable transposon-tagged population has been established  <cit> .
rnai has emerged as an effective gene knockout/knockdown tool for many but, has yet to be applied to sorghum.
development of rnai technology requires genetic transformation and very little work on transformation protocols have been undertaken in sorghum due to the concern that transgenes may transmit to wild related species.
in contrast to transgenic methods, chemical mutagenesis can be applied to most species including sorghum.
chemical mutagenesis does not require gene transfer and is therefore not subject to biosafety and extensive regulatory concerns  <cit> .
chemical mutagenesis has been used in sorghum breeding previously; unfortunately, these mutagenized populations were not annotated and preserved  <cit> .
here, we report the generation of an ethyl methanesulfonate -mutagenized sorghum mutant population, phenotyping of mutant lines in the population, tilling analysis of a subset of mutant lines using four target genes, and verification of the observed mutations with the resultant phenotypes.
RESULTS
generation of mutant population
chemical mutagen, ems, was used to generate the mutagenized population because of its high rate of success in many plant species  <cit> .
previously published reports demonstrate that it is a beneficial strategy to try a range of concentrations of the chemical mutagen being applied to evaluate the toxicity and sterility on germinal tissue before preparing large mutant populations  <cit> .
therefore, the first step was to test the effect of ems on sorghum germination.
a range of concentrations from  <dig>  to  <dig> %  ems was applied and evaluated.
the m <dig> plants were scored for germination, reduced plant height, frequency of leaf chimeras, and fertility of m <dig> panicles.
no significant reduction in the germination rate was observed in any of the applied concentrations of ems from  <dig> % to  <dig> %.
however, seedling growth was severely stunted at concentrations of  <dig> % or higher.
even at  <dig> %, most m <dig> plants exhibited wrinkled leaves.
over 10% of the m <dig> plants treated with  <dig> % ems had at least one leaf with chimeric white stripes.
based on this result,  <dig> %,  <dig> %,  <dig> %,  <dig> %, and  <dig> % ems were used to generate the mutagenized sorghum populations following the scheme outlined in figure  <dig>
in  <dig>  about ~ <dig>  sorghum seeds were treated with ems  and field planted at  <dig>  seeds per hectare.
in a previous study, an examination of resulting m <dig> plants using four hyperpolymorphic sorghum simple sequence repeat  markers, xtxp <dig>  xtxp <dig>  xtxp <dig>  and xtxp <dig>  showed that over 30% of the m <dig> plants were the result of cross pollination from unknown sources .
in order to prevent cross fertilization with other sorghum varieties growing in the region, each healthy panicle was bagged before anthesis.
there was an inverse correlation between m <dig> plant fertility and mutagen dosage .
typically, lowering the treated dosage will decrease the overall mutation rate.
therefore, the key for determining the optimal mutagen dosage is to maximize mutational density while minimizing lethality and aneuploidy  <cit> .
seed setting was greatly affected by ems treatment.
untreated btx <dig> plants set about  <dig> g  per panicle.
after ems treatment, not only the number of seeds per panicle was greatly reduced but the number of sorghum panicles that set seeds at all also decreased with increasing concentrations of ems treatment.
at a  <dig> % concentration of ems, about 41% of the bagged panicles set seeds .
on the other hand, an applied concentration of  <dig> % ems produced only a few panicles that set seeds .
moreover, the seeds from the  <dig> % treated m <dig> plants had poor germination rates and very few lines produced healthy plants that set seeds in the next generation.
overall, a concentration of  <dig> % ems was the highest dosage at which treated seeds developed into healthy m <dig> plants  and produced viable m2seeds.
since the ems concentrations used to generate this mutagenized sorghum population was relatively low compared with that used in other plants  <cit> , only the m <dig> seeds from  <dig> % ems treated plants were used to conduct the pilot tilling study.
to maximize the utility of the limited m <dig> seeds,  <dig>  of the m <dig> seeds from each m <dig> panicle were planted as a head row.
three panicles from each row were bagged before anthesis.
to prevent redundancy of mutations, only one fertile plant from each m <dig> head row were selected to produce m <dig> seeds.
the plants were barcoded and leaf tissues were sampled for dna preparation.
a total of  <dig>  m <dig> lines were produced in the field.
an additional  <dig> m <dig> lines were produced in the greenhouse following a similar procedure as noted in figure  <dig>
phenotyping of the mutagenized population
among the  <dig>  m <dig> families planted in the field,  <dig>  families had good stand .
throughout the growing season these families were repeatedly evaluated for phenotypes distinctive from wild-type btx <dig>  the phenotypes were organized into seedling phenotypes, tillering types, leaf necrosis, senescence, panicle shapes, and seed size .
in the field, all m <dig> families were inspected closely for novel morphological traits compared to the wild type.
every m <dig> family segregated for at least one distinctive mutant phenotype and many lines segregated for multiple mutant phenotypes at an approximately 1: <dig> ratio .
for example, one line  was homozygous for brown midrib  but, heterozygous for male sterility and dwarfing.
many lines that segregate for multiple phenotypes can be found online at .
*phenotypes were named according to rooney  <cit>  wherever possible; new phenotypes are named using a three letter code according to  <cit> .
the observed phenotypes are listed in table  <dig> arranged in order from the highest to lowest frequency.
albinism, usually used as an indicator for mutation frequency  <cit> , occurred in  <dig> % of the head rows in the m <dig> generation.
about 17% of the rows segregated for "dwarf" or "semi-dwarf" plants, the most frequently observed phenotype characterized by reduced height but, largely normal leaf size and stem thickness.
over 6% of the rows segregated for "tiny plants", which differed from "dwarf plants" by reduction in the size of all organs, such as small leaves and thin stems .
leaf lesions, ranging from tiny yellow spots to large blotches of necrosis, were frequently observed .
in addition to these frequent mutant features, a number of other mutant phenotypes with altered agronomic traits  were also observed.
a selection of the mutant phenotypes observed in the field is presented in figure  <dig>  further information on the phenotypes observed from the forward genetic screen can be found online at .
the assortment and frequency of the various mutant phenotypes observed in the population suggested that it will be a useful resource for screening informative sorghum mutants for forward and reverse genetic studies.
identification of mutations by tilling
eight-fold pools of genomic dna from leaf tissues of m <dig> plants were used for tilling.
four gene targets were selected based on their potential contribution to bioenergy, nutrition, and agronomic performance for high throughput tilling .
a total of five mutations were detected from  <dig>  mb of dna analyzed by tilling in four gene targets .
the overall mutation density was calculated to be ~1/ <dig> kb for the ems generated mutant population .
this was calculated by dividing the total number of mutations revealed by tilling by the total base pairs screened, which includes the sum of the total length of the four amplicon sizes × the total number of individuals screened .
previous studies have reported the difficulty of tracking mutations on the ends of the fragment   <cit> ; therefore,  <dig> bp was subtracted off each amplicon.
*for calculation of the mutation rate, sequences were removed  <dig> bp from each end due to the base ambiguity.
ems mutagenesis is reported to typically produce g:c to a:t transition mutations because it alkylates g residues  <cit>  and these alkylated g residues base pair with t instead of the conventional base pairing with c  <cit> .
three of the mutations uncovered via tilling were g:c to a:t transitions as expected.
the remaining two mutations detected were a:t to g:c transitions which have also been reported as occurring in other tilling studies; however, generally these mutations occur at a much lower frequency than the predominant g:c to a:t mutation  <cit> .
of the five mutations identified, one of the mutant lines was determined to be homozygous  and the other four were heterozygous.
heterozygosity was evaluated by examining the sequence data and by forming a heteroduplex on a single individual sample, applying the mismatch endonuclease cel i, separating products on a denaturing acrylamide gel, and verifying the presence or absence of the predetermined cleaved fragment.
all five mutations detected by tilling were reexamined by morphological observation and re-sequenced to verify the results.
the genetic data were consistent with morphological observations.
no mutations were detected for one of the targets, 1-aminocyclopropane-1-carboxylate oxidase .
a graph of the target genes marking the location of the induced polymorphisms is shown in figure  <dig>  a parsesnp graph was not produced for aco <dig> or myoinositol kinase  <dig>  because no induced mutation was identified in the population or information was not available on the predicted gene structure.
the mutations revealed via tilling from the gene targets caffeic acid o-methytransferase  and phytochrome a  were all determined to be missense mutations.
based on the pssm difference and sift score, the mutation revealed in comt gene for individual p7h <dig> is predicted to be damaging to the protein.
this induced mutation in p7h <dig> altered the codon from a hydrophobic glycine amino acid to a hydrophilic serine amino acid , which may affect the protein structure.
no silent, splice site, or nonsense mutations were identified in this pilot study.
however, since there is no information available on the predicted exon/intron borders  for mik <dig>  except some similarity to deposited annotated maize genes, the mutations found in this target could represent any of the various mutation classes.
verification of identified genotype with observed phenotype
five mutations were identified by tilling of  <dig> mutant lines using four target genes.
two mutant lines  were identified in the target gene comt, containing missense mutations at different positions in exon  <dig>  alterations in the comt gene are associated with bm or bmr mutations in maize and sorghum, respectively, characterized by a brown midrib with reduced lignin content and increased digestibility  <cit> .
the two mutant lines  identified in this study also showed a similar brown midrib phenotype  comparable to the previously reported sorghum bmr mutants that have nonsense mutations in exon  <dig> and exon  <dig> of comt, produced by chemical mutagen diethylsulphonate .
sequencing of individual m <dig> plants from p5a <dig> and p7h <dig> indicated that the comt locus in p5a <dig> mutant was homozygous; whereas the comt locus in p7h <dig> mutant was heterozygous.
in the m3generation, the individuals from the mutant line of p7h <dig> were still segregating for the brown midrib phenotype at an approximately 1: <dig> ratio, while p5a <dig> was homozygous for the mutation but segregated for plant height and male sterility.
p5a <dig> and p7h <dig> for the comt locus are allelic mutants; however, given the morphological traits of p5a <dig>  it may contain additional, unlinked mutations.
the mutation in line p5a <dig>  is predicted by pssm and sift scores to be less damaging to the protein function than the mutation in p7h <dig> , both lines displayed brown midribs in leaves and light brown color in stem-cross sections .
histological staining for lignin  showed that both lines had reduced lignin in stem-cross sections when compared to wild-type btx <dig> .
all data to date are consistent with the bmr phenotype of the p5a <dig> and p7h <dig> lines being a direct result of the mutations within the comt gene.
discussion
a tilling population is developed in sorghum using chemical mutagen, ems.
the success of chemical mutagenesis in plants depends on maximizing both the seed set of the mutagenized plants and the frequency of induced mutations.
an adequate mutation frequency is required to keep the number of pcr reactions associated with tilling to a manageable number.
in till et al.
, the authors argued that efficient tilling requires a population with mutation frequency of ≥  <dig> mutation/ <dig> kb to ensure that at least one mutation is found per gel run in the li-cor dna analyzer.
achieving this goal requires a high concentration of mutagen or special treatments.
for example, treatment with  <dig> % ems produced rice mutant populations that did not meet this threshold level in mutation density  <cit> .
adequate mutation frequency was, however, achieved through treatment of developing rice zygotes on the panicle immediately after pollination  <cit> .
there are many factors that may affect the induced mutation rate, such as mutagen concentration, length of mutagen application, wash length, treated organs , and chosen ecotype  of the target species.
seed structure can vary from species to species, which may also influence mutagen efficacy.
for example, rice seeds have hulls and the applied chemical needs to penetrate the hulls first and then interact with the embryo to be effective.
this may be one reason why rice seeds need a higher dosage for mutagenesis than arabidopsis and sorghum.
the concentration of  <dig> % ems was used in our pilot tilling study as sorghum appears to be very sensitive to ems treatment.
only about 40% of the m <dig> plants set seeds even at  <dig> % ems concentration .
at  <dig> %, the commonly used concentration in arabidopsis, very few m <dig> sorghum plants set seeds.
moreover, the seed from fertile m1plants had poor germination rates .
the highest concentration of ems that produced an acceptable number of fertile plants was  <dig> %.
at this dose, the population had a mutational density of ~1/ <dig> kb, which is close to the recommended target mutational density .
the estimated mutation density was lower than typically reported for arabidopsis   <cit> , slightly higher than barley  <cit>  or rice   <cit> , but similar to the mutation density reported in soybean 1/ <dig> kb  <cit>  and pea 1/ <dig> kb  <cit> .
ems is reported to have a mutational bias for 5'-pug-3' sites or a middle g base in a stretch of three or more g bases  <cit> .
therefore, tilling gene targets with an elevated g/c content may yield higher mutational densities.
the mutational density may change after additional screening of other genes and/or more mutant lines from the population or changing the pooling strategy.
an 8-fold pooling strategy was employed in the current study.
lowering the pooling fold  may increase the sensitivity of mutation detection in sorghum.
if a suitable mutation density can not be reached with ems, other chemical mutagens may need to be explored.
several other factors also impact the establishment of useful tilling populations in sorghum.
cross pollination must be vigorously controlled to produce a high quality mutagenized population.
under normal growth conditions, sorghum is predominantly self-fertilized with a cross-fertilization rate ranging from 5–10%  <cit> .
after ems-mutagenesis, cross-fertilization increased dramatically.
a previous sorghum mutagenesis attempt was unsuccessful when cloth bags  failed to prevent cross pollination.
in this attempt, cross pollination was prevented by covering the panicles of each mutant generation with rain-proof paper pollination bags  before anthesis.
this approach effectively minimized cross pollination.
corn earworm and birds also posed serious threats to the limited seed set in m <dig> plants during the grain-filling period.
the paper bag was injected with pesticide to control corn earworm and it was often necessary to put cloth bags over the paper ones to prevent bird damage.
the resulting mutagenized sorghum population has an adequate mutation density and low cross-fertilization, providing a useful community resource for functional analysis of sorghum genes.
the variety of visible phenotypes observed in the mutant population is a good indicator of the depth of the genetic lesions, strongly suggesting that the mutant population is altered for multiple traits of agronomic importance.
three allelic mutants  for the comt locus were previously identified in a sorghum line of p <dig> .
our two comt mutants  in the btx <dig> background are phenotypically similar to the previously identified bmr mutants, but are the result of missense mutations within different codons.
the previous bmr mutations have proven to be useful in improving the digestibility of sorghum plants as forage  <cit> .
since the bmr mutations reduce lignin content or improve saccharification of the sorghum stalk, varieties with bmr mutations may also serve as improved bioenergy feedstock for cellulosic ethanol production  <cit> .
CONCLUSIONS
a mutant population in sorghum has been generated using ems-mutagenesis.
phenotyping performed in the field, combined with tilling of four target genes, demonstrated that chemical mutagenesis is an effective approach to generate mutants with altered agronomic traits for genetic studies and to predict the gene function through identification of an allelic series by tilling.
overall, our results demonstrated that the current sorghum mutant population is an adequate resource for tilling.
as sequence for more target genes becomes available through the nearly completed sorghum sequence project, the gene function for more genes can be elucidated by tilling of this and additional newly developed mutant populations.
because of the utility of high quality chemical mutagenized populations in sorghum genomic studies, and the inherent difficulties in developing them, it may take a community effort to effectively establish populations with a sufficient number of accessions for tilling and/or other reverse genetic approaches.
nevertheless, this sorghum mutant population will be a valuable resource to isolate mutants for many other traits.
the resource can be accessed for research through contacting the author at zhanguo.xin@ars.usda.gov.
open field day will be held each year for public touring the mutant plots and selection of interested mutants.
scientists who need to select mutants at certain development stage can also be accommodated.
some mutant lines with adequate supply of seeds have already been distributed to a number of sorghum researchers.

 sorghum is a c crop that displays excellent tolerance to both drought and high temperature stresses. sorghum has the highest water use efficiency among major crop plants and is unusually tolerant to low soil fertility, traits essential for survival and productivity in arid and semi-arid areas with limited irrigation capability. worldwide, sorghum is the 5th most important grain crop, providing food and fodder for the inhabitants of drought-susceptible regions. recently, sorghum has been demonstrated as a viable bioenergy feedstock. compared with other bioenergy grain crops, sorghum is particularly advantageous because it can be grown profitably on marginal land and therefore, would not remove more fertile land from existing food and fiber production. as a close evolutionary relative of both rice and maize, sorghum research contributes directly to a better understanding of the structure, function, and evolution of cereal genomes. being unusually tolerant to drought and high temperature stresses, sorghum also serves as a repository of genes that have the potential to improve stress tolerance in other crops. recent progress in sorghum genomic studies has generated a series of important tools that can be used to identify favorable genes or alleles for further enhancement of resistance to abiotic stresses and for improved biofuels-related traits. for example, well-established genetic, physical, and cytological maps facilitate the mapping and identification of genes responsible for important agronomic traits. syntenic alignment of the sorghum genome with those of maize, rice, and other cereals provides important insight into genome evolution. furthermore, the construction of cdna microarrays provides a platform for high throughput gene discovery. an important milestone was the recent completion and posting of the genomic sequence of an inbred sorghum line, btx. utilization of these important genomic, genetic, and biotechnological resources such as targeting induced local lesions in genomes will undoubtedly speed-up the elucidation of sorghum gene function and the identification of candidate genes for improving sorghum germplasm. tilling was first developed in arabidopsis thaliana and has been successfully applied to identify knockout mutations and provide allelic mutations in target genes from pathogenic bacteria, animals, and plants. the application of tilling to animal species includes caenorhabditis elegans, zebrafish, and drosophila. the application of tilling to plant species includes arabidopsis, barley, lotus, wheat, maize, populus, rice, pea, and soybean. the technique has proven to be valuable in characterizing the function of target genes. the challenge for researchers is to decipher the function of sorghum genes, particularly those that are unique to the species. unfortunately, many of the reverse genetic tools, such as t-dna tagging and transposon-tagging are still not available in sorghum. although active transposon elements have been identified in sorghum, no viable transposon-tagged population has been established. rnai has emerged as an effective gene knockout/knockdown tool for many but, has yet to be applied to sorghum. development of rnai technology requires genetic transformation and very little work on transformation protocols have been undertaken in sorghum due to the concern that transgenes may transmit to wild related species. in contrast to transgenic methods, chemical mutagenesis can be applied to most species including sorghum. chemical mutagenesis does not require gene transfer and is therefore not subject to biosafety and extensive regulatory concerns. chemical mutagenesis has been used in sorghum breeding previously; unfortunately, these mutagenized populations were not annotated and preserved. here, we report the generation of an ethyl methanesulfonate -mutagenized sorghum mutant population, phenotyping of mutant lines in the population, tilling analysis of a subset of mutant lines using four target genes, and verification of the observed mutations with the resultant phenotypes.  generation of mutant population chemical mutagen, ems, was used to generate the mutagenized population because of its high rate of success in many plant species. previously published reports demonstrate that it is a beneficial strategy to try a range of concentrations of the chemical mutagen being applied to evaluate the toxicity and sterility on germinal tissue before preparing large mutant populations. therefore, the first step was to test the effect of ems on sorghum germination. a range of concentrations from to % ems was applied and evaluated. the m plants were scored for germination, reduced plant height, frequency of leaf chimeras, and fertility of m panicles. no significant reduction in the germination rate was observed in any of the applied concentrations of ems from % to %. however, seedling growth was severely stunted at concentrations of % or higher. even at %, most m plants exhibited wrinkled leaves. over 10% of the m plants treated with % ems had at least one leaf with chimeric white stripes. based on this result, %, %, %, %, and % ems were used to generate the mutagenized sorghum populations following the scheme outlined in figure in about ~ sorghum seeds were treated with ems and field planted at seeds per hectare. in a previous study, an examination of resulting m plants using four hyperpolymorphic sorghum simple sequence repeat markers, xtxp xtxp xtxp and xtxp showed that over 30% of the m plants were the result of cross pollination from unknown sources. in order to prevent cross fertilization with other sorghum varieties growing in the region, each healthy panicle was bagged before anthesis. there was an inverse correlation between m plant fertility and mutagen dosage. typically, lowering the treated dosage will decrease the overall mutation rate. therefore, the key for determining the optimal mutagen dosage is to maximize mutational density while minimizing lethality and aneuploidy. seed setting was greatly affected by ems treatment. untreated btx plants set about g per panicle. after ems treatment, not only the number of seeds per panicle was greatly reduced but the number of sorghum panicles that set seeds at all also decreased with increasing concentrations of ems treatment. at a % concentration of ems, about 41% of the bagged panicles set seeds. on the other hand, an applied concentration of % ems produced only a few panicles that set seeds. moreover, the seeds from the % treated m plants had poor germination rates and very few lines produced healthy plants that set seeds in the next generation. overall, a concentration of % ems was the highest dosage at which treated seeds developed into healthy m plants and produced viable m2seeds. since the ems concentrations used to generate this mutagenized sorghum population was relatively low compared with that used in other plants, only the m seeds from % ems treated plants were used to conduct the pilot tilling study. to maximize the utility of the limited m seeds, of the m seeds from each m panicle were planted as a head row. three panicles from each row were bagged before anthesis. to prevent redundancy of mutations, only one fertile plant from each m head row were selected to produce m seeds. the plants were barcoded and leaf tissues were sampled for dna preparation. a total of m lines were produced in the field. an additional m lines were produced in the greenhouse following a similar procedure as noted in figure phenotyping of the mutagenized population among the m families planted in the field, families had good stand. throughout the growing season these families were repeatedly evaluated for phenotypes distinctive from wild-type btx the phenotypes were organized into seedling phenotypes, tillering types, leaf necrosis, senescence, panicle shapes, and seed size. in the field, all m families were inspected closely for novel morphological traits compared to the wild type. every m family segregated for at least one distinctive mutant phenotype and many lines segregated for multiple mutant phenotypes at an approximately 1: ratio. for example, one line was homozygous for brown midrib but, heterozygous for male sterility and dwarfing. many lines that segregate for multiple phenotypes can be found online at. *phenotypes were named according to rooney wherever possible; new phenotypes are named using a three letter code according to. the observed phenotypes are listed in table arranged in order from the highest to lowest frequency. albinism, usually used as an indicator for mutation frequency, occurred in % of the head rows in the m generation. about 17% of the rows segregated for "dwarf" or "semi-dwarf" plants, the most frequently observed phenotype characterized by reduced height but, largely normal leaf size and stem thickness. over 6% of the rows segregated for "tiny plants", which differed from "dwarf plants" by reduction in the size of all organs, such as small leaves and thin stems. leaf lesions, ranging from tiny yellow spots to large blotches of necrosis, were frequently observed. in addition to these frequent mutant features, a number of other mutant phenotypes with altered agronomic traits were also observed. a selection of the mutant phenotypes observed in the field is presented in figure further information on the phenotypes observed from the forward genetic screen can be found online at. the assortment and frequency of the various mutant phenotypes observed in the population suggested that it will be a useful resource for screening informative sorghum mutants for forward and reverse genetic studies. identification of mutations by tilling eight-fold pools of genomic dna from leaf tissues of m plants were used for tilling. four gene targets were selected based on their potential contribution to bioenergy, nutrition, and agronomic performance for high throughput tilling. a total of five mutations were detected from mb of dna analyzed by tilling in four gene targets. the overall mutation density was calculated to be ~1/ kb for the ems generated mutant population. this was calculated by dividing the total number of mutations revealed by tilling by the total base pairs screened, which includes the sum of the total length of the four amplicon sizes × the total number of individuals screened. previous studies have reported the difficulty of tracking mutations on the ends of the fragment; therefore, bp was subtracted off each amplicon. *for calculation of the mutation rate, sequences were removed bp from each end due to the base ambiguity. ems mutagenesis is reported to typically produce g:c to a:t transition mutations because it alkylates g residues and these alkylated g residues base pair with t instead of the conventional base pairing with c. three of the mutations uncovered via tilling were g:c to a:t transitions as expected. the remaining two mutations detected were a:t to g:c transitions which have also been reported as occurring in other tilling studies; however, generally these mutations occur at a much lower frequency than the predominant g:c to a:t mutation. of the five mutations identified, one of the mutant lines was determined to be homozygous and the other four were heterozygous. heterozygosity was evaluated by examining the sequence data and by forming a heteroduplex on a single individual sample, applying the mismatch endonuclease cel i, separating products on a denaturing acrylamide gel, and verifying the presence or absence of the predetermined cleaved fragment. all five mutations detected by tilling were reexamined by morphological observation and re-sequenced to verify the results. the genetic data were consistent with morphological observations. no mutations were detected for one of the targets, 1-aminocyclopropane-1-carboxylate oxidase. a graph of the target genes marking the location of the induced polymorphisms is shown in figure a parsesnp graph was not produced for aco or myoinositol kinase because no induced mutation was identified in the population or information was not available on the predicted gene structure. the mutations revealed via tilling from the gene targets caffeic acid o-methytransferase and phytochrome a were all determined to be missense mutations. based on the pssm difference and sift score, the mutation revealed in comt gene for individual p7h is predicted to be damaging to the protein. this induced mutation in p7h altered the codon from a hydrophobic glycine amino acid to a hydrophilic serine amino acid, which may affect the protein structure. no silent, splice site, or nonsense mutations were identified in this pilot study. however, since there is no information available on the predicted exon/intron borders for mik except some similarity to deposited annotated maize genes, the mutations found in this target could represent any of the various mutation classes. verification of identified genotype with observed phenotype five mutations were identified by tilling of mutant lines using four target genes. two mutant lines were identified in the target gene comt, containing missense mutations at different positions in exon alterations in the comt gene are associated with bm or bmr mutations in maize and sorghum, respectively, characterized by a brown midrib with reduced lignin content and increased digestibility. the two mutant lines identified in this study also showed a similar brown midrib phenotype comparable to the previously reported sorghum bmr mutants that have nonsense mutations in exon and exon of comt, produced by chemical mutagen diethylsulphonate. sequencing of individual m plants from p5a and p7h indicated that the comt locus in p5a mutant was homozygous; whereas the comt locus in p7h mutant was heterozygous. in the m3generation, the individuals from the mutant line of p7h were still segregating for the brown midrib phenotype at an approximately 1: ratio, while p5a was homozygous for the mutation but segregated for plant height and male sterility. p5a and p7h for the comt locus are allelic mutants; however, given the morphological traits of p5a it may contain additional, unlinked mutations. the mutation in line p5a is predicted by pssm and sift scores to be less damaging to the protein function than the mutation in p7h, both lines displayed brown midribs in leaves and light brown color in stem-cross sections. histological staining for lignin showed that both lines had reduced lignin in stem-cross sections when compared to wild-type btx. all data to date are consistent with the bmr phenotype of the p5a and p7h lines being a direct result of the mutations within the comt gene. discussion a tilling population is developed in sorghum using chemical mutagen, ems. the success of chemical mutagenesis in plants depends on maximizing both the seed set of the mutagenized plants and the frequency of induced mutations. an adequate mutation frequency is required to keep the number of pcr reactions associated with tilling to a manageable number. in till et al., the authors argued that efficient tilling requires a population with mutation frequency of ≥ mutation/ kb to ensure that at least one mutation is found per gel run in the li-cor dna analyzer. achieving this goal requires a high concentration of mutagen or special treatments. for example, treatment with % ems produced rice mutant populations that did not meet this threshold level in mutation density. adequate mutation frequency was, however, achieved through treatment of developing rice zygotes on the panicle immediately after pollination. there are many factors that may affect the induced mutation rate, such as mutagen concentration, length of mutagen application, wash length, treated organs, and chosen ecotype of the target species. seed structure can vary from species to species, which may also influence mutagen efficacy. for example, rice seeds have hulls and the applied chemical needs to penetrate the hulls first and then interact with the embryo to be effective. this may be one reason why rice seeds need a higher dosage for mutagenesis than arabidopsis and sorghum. the concentration of % ems was used in our pilot tilling study as sorghum appears to be very sensitive to ems treatment. only about 40% of the m plants set seeds even at % ems concentration. at %, the commonly used concentration in arabidopsis, very few m sorghum plants set seeds. moreover, the seed from fertile m1plants had poor germination rates. the highest concentration of ems that produced an acceptable number of fertile plants was %. at this dose, the population had a mutational density of ~1/ kb, which is close to the recommended target mutational density. the estimated mutation density was lower than typically reported for arabidopsis, slightly higher than barley or rice, but similar to the mutation density reported in soybean 1/ kb and pea 1/ kb. ems is reported to have a mutational bias for 5'-pug-3' sites or a middle g base in a stretch of three or more g bases. therefore, tilling gene targets with an elevated g/c content may yield higher mutational densities. the mutational density may change after additional screening of other genes and/or more mutant lines from the population or changing the pooling strategy. an 8-fold pooling strategy was employed in the current study. lowering the pooling fold may increase the sensitivity of mutation detection in sorghum. if a suitable mutation density can not be reached with ems, other chemical mutagens may need to be explored. several other factors also impact the establishment of useful tilling populations in sorghum. cross pollination must be vigorously controlled to produce a high quality mutagenized population. under normal growth conditions, sorghum is predominantly self-fertilized with a cross-fertilization rate ranging from 5–10%. after ems-mutagenesis, cross-fertilization increased dramatically. a previous sorghum mutagenesis attempt was unsuccessful when cloth bags failed to prevent cross pollination. in this attempt, cross pollination was prevented by covering the panicles of each mutant generation with rain-proof paper pollination bags before anthesis. this approach effectively minimized cross pollination. corn earworm and birds also posed serious threats to the limited seed set in m plants during the grain-filling period. the paper bag was injected with pesticide to control corn earworm and it was often necessary to put cloth bags over the paper ones to prevent bird damage. the resulting mutagenized sorghum population has an adequate mutation density and low cross-fertilization, providing a useful community resource for functional analysis of sorghum genes. the variety of visible phenotypes observed in the mutant population is a good indicator of the depth of the genetic lesions, strongly suggesting that the mutant population is altered for multiple traits of agronomic importance. three allelic mutants for the comt locus were previously identified in a sorghum line of p. our two comt mutants in the btx background are phenotypically similar to the previously identified bmr mutants, but are the result of missense mutations within different codons. the previous bmr mutations have proven to be useful in improving the digestibility of sorghum plants as forage. since the bmr mutations reduce lignin content or improve saccharification of the sorghum stalk, varieties with bmr mutations may also serve as improved bioenergy feedstock for cellulosic ethanol production.  a mutant population in sorghum has been generated using ems-mutagenesis. phenotyping performed in the field, combined with tilling of four target genes, demonstrated that chemical mutagenesis is an effective approach to generate mutants with altered agronomic traits for genetic studies and to predict the gene function through identification of an allelic series by tilling. overall, our results demonstrated that the current sorghum mutant population is an adequate resource for tilling. as sequence for more target genes becomes available through the nearly completed sorghum sequence project, the gene function for more genes can be elucidated by tilling of this and additional newly developed mutant populations. because of the utility of high quality chemical mutagenized populations in sorghum genomic studies, and the inherent difficulties in developing them, it may take a community effort to effectively establish populations with a sufficient number of accessions for tilling and/or other reverse genetic approaches. nevertheless, this sorghum mutant population will be a valuable resource to isolate mutants for many other traits. the resource can be accessed for research through contacting the author at zhanguo.xin@ars.usda.gov. open field day will be held each year for public touring the mutant plots and selection of interested mutants. scientists who need to select mutants at certain development stage can also be accommodated. some mutant lines with adequate supply of seeds have already been distributed to a number of sorghum researchers. 
however, the availability of genetic resources, specifically mutant lines, is limited. a subset of mutant lines was analyzed by tilling using four target genes. two of the mutations identified by tilling and verified by sequencing were detected in the gene encoding caffeic acid o-methyltransferase in two independent mutant lines. the two mutant lines segregated for the expected brown midrib phenotype, a trait associated with altered lignin content and increased digestibility. moreover, tilling has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the ems-induced mutant lines. the diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from tilling indicate that this mutant population represents a useful resource for members of the sorghum research community. sorghum is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. the recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. numerous phenotypes with altered morphological and agronomic traits were observed from m and m lines in the field. a total of five mutations were identified resulting in a calculated mutation density of 1/ kb. a sorghum mutant population consisting of lines was generated from the inbred line btx by treatment with the chemical agent ethyl methanesulfonate.
 sorghum is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. the recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. however, the availability of genetic resources, specifically mutant lines, is limited. chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. induced mutations in novel genes of interest can be efficiently assessed using the technique known as targeting induced local lesion in genomes.  a sorghum mutant population consisting of lines was generated from the inbred line btx by treatment with the chemical agent ethyl methanesulfonate. numerous phenotypes with altered morphological and agronomic traits were observed from m and m lines in the field. a subset of mutant lines was analyzed by tilling using four target genes. a total of five mutations were identified resulting in a calculated mutation density of 1/ kb. two of the mutations identified by tilling and verified by sequencing were detected in the gene encoding caffeic acid o-methyltransferase in two independent mutant lines. the two mutant lines segregated for the expected brown midrib phenotype, a trait associated with altered lignin content and increased digestibility.  tilling as a reverse genetic approach has been successfully applied to sorghum. the diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from tilling indicate that this mutant population represents a useful resource for members of the sorghum research community. moreover, tilling has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the ems-induced mutant lines. 