BACKGROUND
an important aspect of functional genomics is understanding the phenotypic consequence of mutations in all genes in a genome.
a comprehensive collection of gene knockouts allows a defined set of mutations to be systematically studied for more efficient association of genes to functions .
multiple approaches have been used to develop comprehensive knockout resources.
biological differences between organisms make specific technologies such as homologous recombination, rna interference, or insertional mutagenesis more useful in generating a resource for an individual species.
in plants, a comprehensive knockout collection was generated for arabidopsis thaliana via mutagenesis with insertion tags  <cit> .
genomic dna flanking each tag was systematically amplified and sequenced from each mutant.
these flanking sequence tags  index each mutant to the genome and are accessible through the signal t-dna express database, which links the mutant stocks to genome annotations  <cit> .
a similar fst approach was used to develop a rice functional genomics resource based on insertional mutagenesis populations  <cit> .
the current rice collections have more than  <dig>  insertion lines with associated fsts that are integrated at the orygenesdb database  <cit> .
maize is comparable to rice as a model grass species for genome studies.
similar to rice, the maize genome is now being sequenced with a minimal tiling path strategy  <cit> .
there are also extensive gene-enriched sequences that are estimated to include partial sequence from 95% of maize genes with smaller introns  <cit> .
in contrast to rice, maize is a monoecious plant, and maize has a shorter life cycle.
these biological characteristics facilitate genetic analysis.
also, maize inbreds are highly polymorphic.
robust pcr markers, genetic maps, and recombinant inbred lines have been developed that aid quantitative trait studies and positional cloning .
a comprehensive knockout collection of maize mutants would complement the existing genome resources to make functional genomics studies in maize simple and rapid.
there are multiple insertion-tagged maize populations that were generated with either activator  or mutator  transposons .
there are > <dig>  mutagenized lines among the combined mu populations  <cit> .
these mu lines are expected to have more than  <dig>  million independent insertions, because mu transposons accumulate to high copy numbers within individual plants .
moreover, mu elements show a bias for inserting into or near transcribed regions of the genome and are associated with a high rate of forward mutagenesis  <cit> .
this high mutation frequency makes mu mutagenesis attractive for generating knockout resources, but the high-copy nature of mu elements presents a challenge in isolating individual insertion sites for sequencing.
single plants have multiple germinal insertions that represent both progenitor mutations and mutations unique to the individual.
in addition, active transposon systems can generate somatic insertions that fail to segregate in subsequent generations.
due to these challenges, most mu populations have been developed to conduct reverse genetics screens for only one gene at a time  <cit> .
fsts have been generated from two mu populations.
fernandes et al.
<cit>  identified  <dig>  non-redundant fsts using a transgenic mu <dig> element that was engineered for plasmid rescue of genomic flanking sequences.
the plasmids were isolated from pools of actively transposing plants and many of the fsts are from somatic insertions.
a key challenge to sequencing fsts from pools of actively transposing plants is identifying germinal insertion sites and associating the germinal insertions to individual seed stocks.
fernandes et al.
<cit>  sequenced from two dimensional grids.
recovery of the same fst from both row and column pools of plants was used to associate  <dig> of the plasmid-rescue insertion sites to individual plants.
in contrast, fsts from the uniformmu population were generated with both a strategy to associate each fst to individual lines and to select for germinal insertions  <cit> .
uniformmu is a mu population that is introgressed into the color-converted w <dig> inbred.
previously, we showed that uniformmu is a robust forward genetics resource for identifying visible mutant phenotypes  <cit> .
uniformmu fsts were isolated by amplifying native transposon insertion sites with a modified tail-pcr protocol, mutail  <cit> .
mutail products were cloned from individual lines into small libraries.
sequencing randomly selected clones, similar to an est strategy, recovers 67%–75% of the insertion sites from each line.
with this sequencing strategy, each fst corresponds to an individual plant or maize ear.
the uniformmu fsts also derive from plants in which the mu transposons were stabilized by selecting against somatic transposition using the bronze1-mum <dig>  mutation  <cit> .
the selection against somatic activity predicts that all of the fsts should be from germinal insertion sites.
database searches with  <dig>  non-redundant uniformmu fsts indicated a bias for insertions in or near transcribed regions of the genome <cit> .
moreover, in silico mapping of the fsts indicated that uniformmu transposon insertions are distributed randomly throughout the maize chromosomes suggesting the transposon mutagenesis gave rise to mutations on a genome-wide basis.
these properties predict that uniformmu fsts will be a resource of sequence-indexed mutations that is simple to use for reverse genetics applications.
here we test the utility of uniformmu fsts for reverse genetics.
we assayed a sample of fsts for germinal segregation.
we focused on fsts that were predominantly recovered from individual uniformmu lines.
fsts found only in one plant have the highest possibility of representing somatic insertions and provide the strongest test for whether somatic insertions exist within the uniformmu fsts.
our data indicate that 89% of the uniformmu fsts can be confirmed as germinal insertions by designing only one or two locus-specific primers for pcr validation.
the most common problems for the 11% of insertions that were not validated as germinal insertions were design errors in the locus-specific primers due to low quality sequence data or human error.
one of the fsts co-segregates with a seed mutant that suggests an important role for chloroplast-localized oxidative pentose phosphate pathway enzymes in grain-fill, embryogenesis, and plant development.
these results demonstrate that the strategy for sequencing uniformmu fsts creates effective reverse genetics resources for characterizing gene knockouts in maize.
RESULTS
a sample of  <dig> sequence-indexed insertion sites from the uniformmu population were tested for germinal inheritance.
the uniformmu fsts represent novel insertion sites from the individual lines as well as progenitor sequences that are found throughout the population.
the fsts initially were clustered by both blastcluster analysis  <cit>  and cap <dig> assembly  <cit> to identify non-redundant sequences.
a total of  <dig>  fsts unique to individual uniformmu seed mutants and  <dig> fsts found in two to three mutants were identified with these clustering methods.
we selected  <dig> fsts found in individual mutants,  <dig> fsts found in two mutants, and  <dig> fsts found in multiple uniformmu lines for pcr validation.
the  <dig> low redundancy insertions found in 1– <dig> mutants were recovered from  <dig> of the lines analyzed by mutail sequencing.
thus, the  <dig> insertions sites tested were a significant sample of both uniformmu lines and mutail fsts.
the fsts selected for pcr validation were compared to the maize assembled genomic islands  database as well as the tigr zea mays gene index  to determine the position and identity of the insertion sites  <cit> .
both the magi and zmgi databases are enriched for maize gene sequences.
the insertions were categorized as being within exons, introns, promoters, or unknown sequences .
sixty-three percent of the insertion sites are found within  <dig> bp of transcribed sequences as defined by either a match of greater than 95% sequence identity between the fst and a zmgi sequence or a match to a magi sequence that shows sequence identity to a zmgi sequence .
the remaining  <dig> fsts either matched 1– <dig> magi sequences that contained no est support for a transcribed sequence or did not show a significant match to magi or zmgi sequences.
the fsts were also analyzed for the presence of the conserved mu terminal inverted repeat .
mutail-pcr products are amplified with  <dig> bp of mutir sequence downstream of the transposon specific primer  <cit> .
presence of the mutir sequence identifies the precise insertion site for a fst .
mutir sequences were present in  <dig> fsts and absent in the remaining  <dig> insertions sites selected for validation.
mutir sequences are expected to be absent from a proportion of fsts, because the mutail products were cloned in random orientations and are frequently larger than single-pass sequence reads.
locus-specific pcr primers were designed based on the location of the mutir and magi sequence matches.
we used three strategies to design the primers .
first, left and/or right primers were designed when the precise insertion site was placed within a magi sequence.
the left primer was based on the magi sequence, and the right primer was designed using an alignment of the magi and fst.
second, a single right primer was designed when the magi showed partial overlap with the fst.
these primers were designed using either an alignment between the fst and magi or were designed with the fst alone.
third, a presumptive right primer was designed for the  <dig> fsts that did not have mutir sequences based on the hypothesis that these mutail products were not completely sequenced.
the locus-specific primers were used in combination with mutir primers to amplify the insertion sites from individual mutant lines.
the primers were tested with the initial dna template used to generate the fst, as well as with siblings or progeny of the individuals used for templates.
figure  <dig> shows examples of pcr validation assays for  <dig> fsts.
in these examples, the plant used for mutail sequencing came from a backcross of an individual uniformmu line to the w <dig> inbred.
germinal insertions are expected to be found in all siblings or to segregate in a 1: <dig> ratio, depending on whether the progenitor plant was homozygous or heterozygous for individual insertion sites.
overall, we confirmed  <dig> insertion sites from the sample of  <dig> fsts, using an average of  <dig>  locus-specific primers per fst .
in many cases, two primers for an individual fst were tested simultaneously and both of these primers amplified the mu insertion site .
for  <dig> of the insertion sites, a single primer was sufficient to confirm and track the insertion.
importantly, all  <dig> mu insertions confirmed in these assays showed germinal segregation.
overall, our data indicate that the vast majority of these sequence-indexed mutants are simple to recover from the uniformmu population.
there were two noticeable trends in the fsts that failed to amplify in the pcr assays .
first, the locus-specific primers for five insertion sites  were not designed to anneal at the insertion site.
three of these fsts included repetitive or chimeric sequences that identified magi sequences with only a partial sequence match to the fst.
in these cases, the specific primers were designed from magi sequences that were not part of the fst.
for bc <dig> and bc <dig>  the locus-specific primers were designed to amplify from the non-mutir end of the insertion site sequences.
the second trend was observed for a group of four other fsts .
the locus-specific primers for these insertions showed base pair differences between the most recent magi or fst consensus sequence suggesting that the primers were originally designed from lower quality sequence reads or assemblies.
mu-specific products were not observed from a final group of three insertions sites  for unknown reasons.
overall, 75% of the insertion sites that failed to be confirmed had errors in the locus-specific primer design, suggesting that sequence errors or human errors were the primary causes for difficulties in confirming an insertion site.
the pcr validation assays also identified an insertion site, bc <dig>  that co-segregates with a rough endosperm  seed mutant phenotype.
the bc <dig> insertion disrupts the open reading frame of a predicted 6-phosphogluconate dehydrogenase  gene at the pgd <dig> locus, and we named this insertion allele pgd3-umu <dig>  there are three known loci in maize for 6pgdh enzymes: pgd <dig>  pgd <dig>  and pgd <dig>  <cit> .
pgd <dig> maps to the short arm of chromosome  <dig> in bin  <dig>   <cit> .
we designed both left and right locus-specific primers to develop a co-dominant pcr assay for this insertion .
co-segregation between the pgd3-umu <dig> insertion site and the rgh*-00s-005- <dig> mutant was observed in a test of  <dig> progeny from backcrosses to w <dig>  as well as  <dig> homozygous rgh/rgh mutant individuals from a segregating self-pollination .
we expanded the linkage analysis to test a total of  <dig> meiotic products and observed no recombination between pgd3-umu <dig> and the rgh mutant .
these data indicate that the map distance between pgd3-umu <dig> and the rgh phenotype is less than  <dig>  cm.
this tight linkage raises the hypothesis that the rgh mutant phenotype may represent the pgd3-umu <dig> knockout phenotype, and we characterized the rgh*-00s-005- <dig> mutant further.
the linked rgh mutant shows highly reduced grain-fill, along with a characteristic etching or pitting at the endosperm surface .
longitudinal hand sections of mature mutant kernels showed both reduced endosperm development as well as embryo defects, with most mutants failing to develop embryonic roots or leaves .
rare embryos with a visually apparent shoot-root axis were observed in homozygous mutants.
a small fraction of these seeds were able to germinate when cultured prior to seed desiccation.
these mutant escapes developed into morphologically normal seedlings and plants .
however, the plants showed a pale green leaf phenotype that was less severe near leaf veins.
the homozygous rgh individuals were also confirmed as homozygous for the pgd3-umu <dig> allele using the locus-specific pcr assay .
self or sibling pollinations of these mutant plants showed all mutant seed, which confirmed that the plants were homozygous rgh individuals and not the result of hetero-fertilization .
6pgdh enzyme activity was measured from  <dig> days after pollination  endosperm extracts of w <dig>  rgh*-00s-005- <dig> homozygous mutants as well as single and double pgd1; pgd <dig> mutants .
the w <dig> sample showed two major isozyme activities.
the faster migrating activity corresponds to three bands: pgd <dig> homodimers, pgd1/pgd <dig> heterodimers, and pgd <dig> homodimers  <cit> .
a subset of these bands are absent in the pgd <dig> and pgd <dig> single mutants, and all three bands are lost in pgd1; pgd <dig> double mutant samples.
the rgh mutant sample lacks the slower migrating activity.
serial dilutions of the w <dig> extract suggest that the rgh mutant endosperm has <5% normal activity for this 6pgdh isozyme .
we conclude the slower migrating 6pgdh isozyme is likely to be pgd <dig> as no reduction of this isozyme activity was observed in the other pgd mutants.
these data suggest that the fst for pgd3-umu <dig> identified an enzymatic knockout.
6pgdh enzymes are found in both the cytosol and chloroplast in plants .
to predict the likely subcellular localization of the pgd <dig> protein, a complete pgd <dig> locus was assembled from est and magi sequences.
the predicted peptide for the pgd <dig> locus contains an n-terminal extension that is absent from the predicted pgd <dig> and pgd <dig> proteins in a multiple sequence alignment .
pgd <dig> clusters with a known chloroplast-localized 6pgdh protein from spinach in a clustalw sequence similarity tree .
furthermore, the pgd <dig> protein is predicted to be targeted to the chloroplast by the protein localization programs: targetp, psort, and predotar  <cit> .
these sequence analyses suggest that the pgd3-umu <dig> allele disrupts a chloroplast-localized 6pgdh in maize.
this prediction is consistent with previous observations that the remaining 6pgdh activity in pgd1; pgd <dig> double mutants is entirely plastid-localized  <cit> .
discussion
the current maize knockout resources have enough insertions to disrupt every gene within the genome.
fsts will make these maize mutants far more available to the research community, and sequencing mutail products is a high throughput approach to generate maize fsts.
however, fsts alone do not make a reverse genetics resource easy to use.
the tags need to have low entry barriers for tracking an insertion site of interest as well as for obtaining the line or strain that corresponds to the insertion site.
fsts from the uniformmu population are predicted to have low entry barriers due to the genetic markers included in the population and the high-throughput sequencing strategy used to generate the fsts  <cit> .
the bz1-mum <dig> mutant was used to select against somatic activity, and here we have shown that the vast majority of the resulting fsts segregate as germinal insertions.
we did not observe any evidence of somatic insertions.
somatic insertions would be expected to amplify from the dna template used to generate the fsts, but not to amplify from any other related individuals.
in the cases where we could not confirm an insertion, the insertion site did not amplify from the template dna used to generate the fsts.
a secondary analysis of the specific primers indicated that three factors led to a small fraction of insertion sites that are more difficult to track.
first, differences between available maize genomic sequences and the fsts caused the precise insertion site to be mis-identified for  <dig> % of the fsts.
these mis-identifications led to errors in locus-specific primer design.
the sequence differences are likely to result from sequence errors, incomplete maize genomic sequence, and sequence divergence between the w <dig> inbred background used to develop uniformmu and the b <dig> inbred chosen for genomic sequencing.
comparative genomic sequence studies of maize inbreds suggests that there are significant differences in gene content and repetitive elements between inbreds  <cit> .
a second factor that contributed to insertion sites that were not confirmed as germinal was human error in identifying the tir-genomic dna junction.
these errors led to primer design errors for  <dig> % of the fsts.
finally, pcr or sample tracking errors occur at a low frequency in any high-throughput sequencing project.
we speculate that a small amount of cross-contamination between sequencing libraries can explain the remaining  <dig> % of insertion sites that were not confirmed.
importantly, 89% of the fsts were readily confirmed as germinal insertions by locus-specific pcr.
furthermore, the mutail fsts were generated from individual plants or lines that were successfully bulked, and each sequence read name corresponds directly to an individual.
these characteristics make it simple to identify the seed stocks for each fst.
combined, the characteristics of the mutail fsts and the uniformmu population make a robust resource for reverse genetics.
the pgd3-umu <dig> insertion site that co-segregates with a seed mutant phenotype illustrates the utility of the uniformmu fst resource for generating hypotheses about gene function.
the pgd3-umu <dig> insertion is in a predicted chloroplast-localized 6pgdh enzyme in the oxidative section of the pentose phosphate pathway .
the linked rgh*-00s-005- <dig> seed mutant lacks a 6pgdh isozyme activity suggesting pgd3-umu <dig> causes an enzymatic knockout.
these linkage and biochemical data suggest the hypothesis that pgd <dig> has an essential role in seed development.
consistent with this hypothesis, the oppp has been shown to be involved in hexose cycling and starch synthesis in developing seeds  <cit> .
also, previous studies with maize pgd <dig> and pgd <dig> mutants argued that the plastidic enzymes for the oxidative section of the oppp can compensate for loss of 6pgdh in the cytosol  <cit> .
moreover, a study in maize root tips found that the oxidative section of the oppp to be most active in plastids  <cit> .
combined these data are consistent with plastidic oppp enzymes being required for seed development.
however, linkage data alone cannot prove that the rgh phenotype is a result of the pgd3-umu <dig> mutation.
additional experiments are necessary to test this hypothesis such as identifying multiple mutant alleles of pgd <dig>  alternatively, a pgd <dig> transgene could be developed to test for complementation of the 6pgdh enzyme defect and the rgh*-00s-005- <dig> phenotype.
the maize genetics community has multiple resources to assist in identifying alleles or in generating transgenics for individual genes of interest .
the pgd3-umu <dig> example also illustrates the complementary nature of having reverse genetics resources in multiple plant species.
the arabidopsis genome contains two copies of genes predicted to encode plastid-localized 6pgdh enzymes and one gene predicted to encode a cytosolic 6pgdh .
each of these genes have multiple sequence-indexed mutations that disrupt exon sequences and are likely to cause null alleles  <cit> .
gene redundancy in arabidopsis may be one reason why mutant phenotypes associated with plastid-localized 6pgdh have not been reported previously.
if plastidic 6pgdh enzymes are essential in plants, a double mutant for knockouts of arabidopsis orthologs, at1g <dig> and at5g <dig>  would be expected to show a visible phenotype.
the rice genome has a single locus that is predicted to encode plastid-localized 6pgdh, os11g <dig>  similar to maize.
however, the current rice knockout resources do not have fsts that disrupt this locus  <cit> .
the rice ortholog for cytosolic 6pgdh, os06g <dig>  has three insertions, but these insertions map to promoter and 3' utr regions and may not be null alleles.
thus, the maize mutants in 6pgdh enzymes suggest an agricultural relevance for the plastid-localized oppp that would not be as simple to identify using the current arabidopsis or rice resources.
CONCLUSIONS
the pgd3-umu <dig> allele is just one example of the current collection of  <dig>  non-redundant insertion sites sequenced in uniformmu.
we conclude from our analysis that the vast majority of the uniformmu insertion sites are germinal.
importantly, multiple studies have shown that mu insertions are heavily biased for transcribed regions of the genome  <cit> .
in addition, the uniformmu population is in an inbred background that simplifies phenotypic analysis  <cit> .
combined, these features provide a robust, genome-wide resource for reverse genetics in maize.
further sequencing of uniformmu fsts would enhance this resource by expanding it towards a comprehensive knockout collection.

 an important aspect of functional genomics is understanding the phenotypic consequence of mutations in all genes in a genome. a comprehensive collection of gene knockouts allows a defined set of mutations to be systematically studied for more efficient association of genes to functions. multiple approaches have been used to develop comprehensive knockout resources. biological differences between organisms make specific technologies such as homologous recombination, rna interference, or insertional mutagenesis more useful in generating a resource for an individual species. in plants, a comprehensive knockout collection was generated for arabidopsis thaliana via mutagenesis with insertion tags. genomic dna flanking each tag was systematically amplified and sequenced from each mutant. these flanking sequence tags index each mutant to the genome and are accessible through the signal t-dna express database, which links the mutant stocks to genome annotations. a similar fst approach was used to develop a rice functional genomics resource based on insertional mutagenesis populations. the current rice collections have more than insertion lines with associated fsts that are integrated at the orygenesdb database. maize is comparable to rice as a model grass species for genome studies. similar to rice, the maize genome is now being sequenced with a minimal tiling path strategy. there are also extensive gene-enriched sequences that are estimated to include partial sequence from 95% of maize genes with smaller introns. in contrast to rice, maize is a monoecious plant, and maize has a shorter life cycle. these biological characteristics facilitate genetic analysis. also, maize inbreds are highly polymorphic. robust pcr markers, genetic maps, and recombinant inbred lines have been developed that aid quantitative trait studies and positional cloning. a comprehensive knockout collection of maize mutants would complement the existing genome resources to make functional genomics studies in maize simple and rapid. there are multiple insertion-tagged maize populations that were generated with either activator or mutator transposons. there are > mutagenized lines among the combined mu populations. these mu lines are expected to have more than million independent insertions, because mu transposons accumulate to high copy numbers within individual plants. moreover, mu elements show a bias for inserting into or near transcribed regions of the genome and are associated with a high rate of forward mutagenesis. this high mutation frequency makes mu mutagenesis attractive for generating knockout resources, but the high-copy nature of mu elements presents a challenge in isolating individual insertion sites for sequencing. single plants have multiple germinal insertions that represent both progenitor mutations and mutations unique to the individual. in addition, active transposon systems can generate somatic insertions that fail to segregate in subsequent generations. due to these challenges, most mu populations have been developed to conduct reverse genetics screens for only one gene at a time. fsts have been generated from two mu populations. fernandes et al. identified non-redundant fsts using a transgenic mu element that was engineered for plasmid rescue of genomic flanking sequences. the plasmids were isolated from pools of actively transposing plants and many of the fsts are from somatic insertions. a key challenge to sequencing fsts from pools of actively transposing plants is identifying germinal insertion sites and associating the germinal insertions to individual seed stocks. fernandes et al. sequenced from two dimensional grids. recovery of the same fst from both row and column pools of plants was used to associate of the plasmid-rescue insertion sites to individual plants. in contrast, fsts from the uniformmu population were generated with both a strategy to associate each fst to individual lines and to select for germinal insertions. uniformmu is a mu population that is introgressed into the color-converted w inbred. previously, we showed that uniformmu is a robust forward genetics resource for identifying visible mutant phenotypes. uniformmu fsts were isolated by amplifying native transposon insertion sites with a modified tail-pcr protocol, mutail. mutail products were cloned from individual lines into small libraries. sequencing randomly selected clones, similar to an est strategy, recovers 67%–75% of the insertion sites from each line. with this sequencing strategy, each fst corresponds to an individual plant or maize ear. the uniformmu fsts also derive from plants in which the mu transposons were stabilized by selecting against somatic transposition using the bronze1-mum mutation. the selection against somatic activity predicts that all of the fsts should be from germinal insertion sites. database searches with non-redundant uniformmu fsts indicated a bias for insertions in or near transcribed regions of the genome. moreover, in silico mapping of the fsts indicated that uniformmu transposon insertions are distributed randomly throughout the maize chromosomes suggesting the transposon mutagenesis gave rise to mutations on a genome-wide basis. these properties predict that uniformmu fsts will be a resource of sequence-indexed mutations that is simple to use for reverse genetics applications. here we test the utility of uniformmu fsts for reverse genetics. we assayed a sample of fsts for germinal segregation. we focused on fsts that were predominantly recovered from individual uniformmu lines. fsts found only in one plant have the highest possibility of representing somatic insertions and provide the strongest test for whether somatic insertions exist within the uniformmu fsts. our data indicate that 89% of the uniformmu fsts can be confirmed as germinal insertions by designing only one or two locus-specific primers for pcr validation. the most common problems for the 11% of insertions that were not validated as germinal insertions were design errors in the locus-specific primers due to low quality sequence data or human error. one of the fsts co-segregates with a seed mutant that suggests an important role for chloroplast-localized oxidative pentose phosphate pathway enzymes in grain-fill, embryogenesis, and plant development. these results demonstrate that the strategy for sequencing uniformmu fsts creates effective reverse genetics resources for characterizing gene knockouts in maize.  a sample of sequence-indexed insertion sites from the uniformmu population were tested for germinal inheritance. the uniformmu fsts represent novel insertion sites from the individual lines as well as progenitor sequences that are found throughout the population. the fsts initially were clustered by both blastcluster analysis and cap assembly to identify non-redundant sequences. a total of fsts unique to individual uniformmu seed mutants and fsts found in two to three mutants were identified with these clustering methods. we selected fsts found in individual mutants, fsts found in two mutants, and fsts found in multiple uniformmu lines for pcr validation. the low redundancy insertions found in 1– mutants were recovered from of the lines analyzed by mutail sequencing. thus, the insertions sites tested were a significant sample of both uniformmu lines and mutail fsts. the fsts selected for pcr validation were compared to the maize assembled genomic islands database as well as the tigr zea mays gene index to determine the position and identity of the insertion sites. both the magi and zmgi databases are enriched for maize gene sequences. the insertions were categorized as being within exons, introns, promoters, or unknown sequences. sixty-three percent of the insertion sites are found within bp of transcribed sequences as defined by either a match of greater than 95% sequence identity between the fst and a zmgi sequence or a match to a magi sequence that shows sequence identity to a zmgi sequence. the remaining fsts either matched 1– magi sequences that contained no est support for a transcribed sequence or did not show a significant match to magi or zmgi sequences. the fsts were also analyzed for the presence of the conserved mu terminal inverted repeat. mutail-pcr products are amplified with bp of mutir sequence downstream of the transposon specific primer. presence of the mutir sequence identifies the precise insertion site for a fst. mutir sequences were present in fsts and absent in the remaining insertions sites selected for validation. mutir sequences are expected to be absent from a proportion of fsts, because the mutail products were cloned in random orientations and are frequently larger than single-pass sequence reads. locus-specific pcr primers were designed based on the location of the mutir and magi sequence matches. we used three strategies to design the primers. first, left and/or right primers were designed when the precise insertion site was placed within a magi sequence. the left primer was based on the magi sequence, and the right primer was designed using an alignment of the magi and fst. second, a single right primer was designed when the magi showed partial overlap with the fst. these primers were designed using either an alignment between the fst and magi or were designed with the fst alone. third, a presumptive right primer was designed for the fsts that did not have mutir sequences based on the hypothesis that these mutail products were not completely sequenced. the locus-specific primers were used in combination with mutir primers to amplify the insertion sites from individual mutant lines. the primers were tested with the initial dna template used to generate the fst, as well as with siblings or progeny of the individuals used for templates. figure shows examples of pcr validation assays for fsts. in these examples, the plant used for mutail sequencing came from a backcross of an individual uniformmu line to the w inbred. germinal insertions are expected to be found in all siblings or to segregate in a 1: ratio, depending on whether the progenitor plant was homozygous or heterozygous for individual insertion sites. overall, we confirmed insertion sites from the sample of fsts, using an average of locus-specific primers per fst. in many cases, two primers for an individual fst were tested simultaneously and both of these primers amplified the mu insertion site. for of the insertion sites, a single primer was sufficient to confirm and track the insertion. importantly, all mu insertions confirmed in these assays showed germinal segregation. overall, our data indicate that the vast majority of these sequence-indexed mutants are simple to recover from the uniformmu population. there were two noticeable trends in the fsts that failed to amplify in the pcr assays. first, the locus-specific primers for five insertion sites were not designed to anneal at the insertion site. three of these fsts included repetitive or chimeric sequences that identified magi sequences with only a partial sequence match to the fst. in these cases, the specific primers were designed from magi sequences that were not part of the fst. for bc and bc the locus-specific primers were designed to amplify from the non-mutir end of the insertion site sequences. the second trend was observed for a group of four other fsts. the locus-specific primers for these insertions showed base pair differences between the most recent magi or fst consensus sequence suggesting that the primers were originally designed from lower quality sequence reads or assemblies. mu-specific products were not observed from a final group of three insertions sites for unknown reasons. overall, 75% of the insertion sites that failed to be confirmed had errors in the locus-specific primer design, suggesting that sequence errors or human errors were the primary causes for difficulties in confirming an insertion site. the pcr validation assays also identified an insertion site, bc that co-segregates with a rough endosperm seed mutant phenotype. the bc insertion disrupts the open reading frame of a predicted 6-phosphogluconate dehydrogenase gene at the pgd locus, and we named this insertion allele pgd3-umu there are three known loci in maize for 6pgdh enzymes: pgd pgd and pgd. pgd maps to the short arm of chromosome in bin. we designed both left and right locus-specific primers to develop a co-dominant pcr assay for this insertion. co-segregation between the pgd3-umu insertion site and the rgh*-00s-005- mutant was observed in a test of progeny from backcrosses to w as well as homozygous rgh/rgh mutant individuals from a segregating self-pollination. we expanded the linkage analysis to test a total of meiotic products and observed no recombination between pgd3-umu and the rgh mutant. these data indicate that the map distance between pgd3-umu and the rgh phenotype is less than cm. this tight linkage raises the hypothesis that the rgh mutant phenotype may represent the pgd3-umu knockout phenotype, and we characterized the rgh*-00s-005- mutant further. the linked rgh mutant shows highly reduced grain-fill, along with a characteristic etching or pitting at the endosperm surface. longitudinal hand sections of mature mutant kernels showed both reduced endosperm development as well as embryo defects, with most mutants failing to develop embryonic roots or leaves. rare embryos with a visually apparent shoot-root axis were observed in homozygous mutants. a small fraction of these seeds were able to germinate when cultured prior to seed desiccation. these mutant escapes developed into morphologically normal seedlings and plants. however, the plants showed a pale green leaf phenotype that was less severe near leaf veins. the homozygous rgh individuals were also confirmed as homozygous for the pgd3-umu allele using the locus-specific pcr assay. self or sibling pollinations of these mutant plants showed all mutant seed, which confirmed that the plants were homozygous rgh individuals and not the result of hetero-fertilization. 6pgdh enzyme activity was measured from days after pollination endosperm extracts of w rgh*-00s-005- homozygous mutants as well as single and double pgd1; pgd mutants. the w sample showed two major isozyme activities. the faster migrating activity corresponds to three bands: pgd homodimers, pgd1/pgd heterodimers, and pgd homodimers. a subset of these bands are absent in the pgd and pgd single mutants, and all three bands are lost in pgd1; pgd double mutant samples. the rgh mutant sample lacks the slower migrating activity. serial dilutions of the w extract suggest that the rgh mutant endosperm has <5% normal activity for this 6pgdh isozyme. we conclude the slower migrating 6pgdh isozyme is likely to be pgd as no reduction of this isozyme activity was observed in the other pgd mutants. these data suggest that the fst for pgd3-umu identified an enzymatic knockout. 6pgdh enzymes are found in both the cytosol and chloroplast in plants. to predict the likely subcellular localization of the pgd protein, a complete pgd locus was assembled from est and magi sequences. the predicted peptide for the pgd locus contains an n-terminal extension that is absent from the predicted pgd and pgd proteins in a multiple sequence alignment. pgd clusters with a known chloroplast-localized 6pgdh protein from spinach in a clustalw sequence similarity tree. furthermore, the pgd protein is predicted to be targeted to the chloroplast by the protein localization programs: targetp, psort, and predotar. these sequence analyses suggest that the pgd3-umu allele disrupts a chloroplast-localized 6pgdh in maize. this prediction is consistent with previous observations that the remaining 6pgdh activity in pgd1; pgd double mutants is entirely plastid-localized. discussion the current maize knockout resources have enough insertions to disrupt every gene within the genome. fsts will make these maize mutants far more available to the research community, and sequencing mutail products is a high throughput approach to generate maize fsts. however, fsts alone do not make a reverse genetics resource easy to use. the tags need to have low entry barriers for tracking an insertion site of interest as well as for obtaining the line or strain that corresponds to the insertion site. fsts from the uniformmu population are predicted to have low entry barriers due to the genetic markers included in the population and the high-throughput sequencing strategy used to generate the fsts. the bz1-mum mutant was used to select against somatic activity, and here we have shown that the vast majority of the resulting fsts segregate as germinal insertions. we did not observe any evidence of somatic insertions. somatic insertions would be expected to amplify from the dna template used to generate the fsts, but not to amplify from any other related individuals. in the cases where we could not confirm an insertion, the insertion site did not amplify from the template dna used to generate the fsts. a secondary analysis of the specific primers indicated that three factors led to a small fraction of insertion sites that are more difficult to track. first, differences between available maize genomic sequences and the fsts caused the precise insertion site to be mis-identified for % of the fsts. these mis-identifications led to errors in locus-specific primer design. the sequence differences are likely to result from sequence errors, incomplete maize genomic sequence, and sequence divergence between the w inbred background used to develop uniformmu and the b inbred chosen for genomic sequencing. comparative genomic sequence studies of maize inbreds suggests that there are significant differences in gene content and repetitive elements between inbreds. a second factor that contributed to insertion sites that were not confirmed as germinal was human error in identifying the tir-genomic dna junction. these errors led to primer design errors for % of the fsts. finally, pcr or sample tracking errors occur at a low frequency in any high-throughput sequencing project. we speculate that a small amount of cross-contamination between sequencing libraries can explain the remaining % of insertion sites that were not confirmed. importantly, 89% of the fsts were readily confirmed as germinal insertions by locus-specific pcr. furthermore, the mutail fsts were generated from individual plants or lines that were successfully bulked, and each sequence read name corresponds directly to an individual. these characteristics make it simple to identify the seed stocks for each fst. combined, the characteristics of the mutail fsts and the uniformmu population make a robust resource for reverse genetics. the pgd3-umu insertion site that co-segregates with a seed mutant phenotype illustrates the utility of the uniformmu fst resource for generating hypotheses about gene function. the pgd3-umu insertion is in a predicted chloroplast-localized 6pgdh enzyme in the oxidative section of the pentose phosphate pathway. the linked rgh*-00s-005- seed mutant lacks a 6pgdh isozyme activity suggesting pgd3-umu causes an enzymatic knockout. these linkage and biochemical data suggest the hypothesis that pgd has an essential role in seed development. consistent with this hypothesis, the oppp has been shown to be involved in hexose cycling and starch synthesis in developing seeds. also, previous studies with maize pgd and pgd mutants argued that the plastidic enzymes for the oxidative section of the oppp can compensate for loss of 6pgdh in the cytosol. moreover, a study in maize root tips found that the oxidative section of the oppp to be most active in plastids. combined these data are consistent with plastidic oppp enzymes being required for seed development. however, linkage data alone cannot prove that the rgh phenotype is a result of the pgd3-umu mutation. additional experiments are necessary to test this hypothesis such as identifying multiple mutant alleles of pgd alternatively, a pgd transgene could be developed to test for complementation of the 6pgdh enzyme defect and the rgh*-00s-005- phenotype. the maize genetics community has multiple resources to assist in identifying alleles or in generating transgenics for individual genes of interest. the pgd3-umu example also illustrates the complementary nature of having reverse genetics resources in multiple plant species. the arabidopsis genome contains two copies of genes predicted to encode plastid-localized 6pgdh enzymes and one gene predicted to encode a cytosolic 6pgdh. each of these genes have multiple sequence-indexed mutations that disrupt exon sequences and are likely to cause null alleles. gene redundancy in arabidopsis may be one reason why mutant phenotypes associated with plastid-localized 6pgdh have not been reported previously. if plastidic 6pgdh enzymes are essential in plants, a double mutant for knockouts of arabidopsis orthologs, at1g and at5g would be expected to show a visible phenotype. the rice genome has a single locus that is predicted to encode plastid-localized 6pgdh, os11g similar to maize. however, the current rice knockout resources do not have fsts that disrupt this locus. the rice ortholog for cytosolic 6pgdh, os06g has three insertions, but these insertions map to promoter and 3' utr regions and may not be null alleles. thus, the maize mutants in 6pgdh enzymes suggest an agricultural relevance for the plastid-localized oppp that would not be as simple to identify using the current arabidopsis or rice resources.  the pgd3-umu allele is just one example of the current collection of non-redundant insertion sites sequenced in uniformmu. we conclude from our analysis that the vast majority of the uniformmu insertion sites are germinal. importantly, multiple studies have shown that mu insertions are heavily biased for transcribed regions of the genome. in addition, the uniformmu population is in an inbred background that simplifies phenotypic analysis. combined, these features provide a robust, genome-wide resource for reverse genetics in maize. further sequencing of uniformmu fsts would enhance this resource by expanding it towards a comprehensive knockout collection. 
transposon insertion sites from uniformmu fsts were tested for inheritance by locus-specific pcr. we found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. we show that fsts from the uniformmu population identify stable, germinal insertion sites in maize. we conclude from these data that the current collection of non-redundant insertion sites from uniformmu provide a genome-wide resource for reverse genetics. transposons can also generate somatic insertions that fail to segregate in subsequent generations. gene knockouts are a critical resource for functional genomics. the locus-specific pcr assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. in arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic dna flanking insertion mutants.
 gene knockouts are a critical resource for functional genomics. in arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic dna flanking insertion mutants. these flanking sequence tags map each mutant to a specific locus within the genome. in maize, fsts have been generated using dna transposons. transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. transposons can also generate somatic insertions that fail to segregate in subsequent generations.  transposon insertion sites from uniformmu fsts were tested for inheritance by locus-specific pcr. we confirmed 89% of the fsts to be germinal transposon insertions. we found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. the locus-specific pcr assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. the mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.  we show that fsts from the uniformmu population identify stable, germinal insertion sites in maize. moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. we conclude from these data that the current collection of non-redundant insertion sites from uniformmu provide a genome-wide resource for reverse genetics. 