BACKGROUND
maize  is an incredibly important cereal crop grown widely throughout the world.
increased demand for maize owing to population growth and biofuel production, and the impacts of climate change on maize production will ratchet up the pressure for increased and more sustainable maize production.
since the 1980s, molecular markers have been widely used in maize genetics and breeding.
high-density molecular markers in applied and basic research are advantageous and necessary for map-based cloning and genome-wide association study.
in contrast to single nucleotide polymorphisms , insertions and deletions  are the second most common type of polymorphism.
in a previous study, mills reported that indels are distributed throughout the human genome at an average density of one indel per  <dig>  kb  <cit> .
several studies have suggested numerous indels that may cause human disease  <cit> .
one of the most common genetic diseases in humans, cystic fibrosis, is frequently caused by various indels in coding regions of the cftr gene  <cit> .
the genetic diseases of tuberous sclerosis, rett syndrome, and hemophilia b are also caused by small indels .
similarly, indels can alter the phenotype of plants .
consequently, indels have been increasingly recognized as an important source of molecular markers, and indel markers have been successfully used for several genetic studies in crops  <cit> .
the maize genome was completed in  <dig> using traditional sanger sequencing technology,  <cit> .
however, the subsequent development of next-generation sequencing  technology has generated an enormous amount of short reads that science is scrambling to analyze.
ngs technology has also drastically reduced the time and cost requirements of sequencing, which has enabled the re-sequencing of a large number of genomes.
this has provided for the possibility of large-scale genetic variation surveys, where many individuals within a single species have been sequenced.
examples include  <dig> silkworm samples  <cit> ,  <dig> soybean samples  <cit> ,  <dig> sorghum samples  <cit> , and  <dig> rice samples .
as of  <dig>  more than  <dig> maize inbred line and landrace genomes had been completely re-sequenced and published .
rapid bioinformatics developments have introduced various software programs designed to identify indels, including dindel  <cit> , varscan  <cit> , gatk  <cit> , and samtool’s mpileup  <cit> .
the first step in most of these programs is to map reads directly to a reference sequence.
accurate indel calls from reads are challenging for a couple of reasons.
first, reads covering indels are generally more difficult to map correctly to reference genomes, especially ones containing large indel events.
moreover, incorrect alignments at the nucleotide level lead to an incorrect placement of gaps in the alignments.
schuler proposed a pcr amplification computer simulation concept known as the electronic polymerase chain reaction , which has been used widely in various aspects of biology, including the chromosomal localization of dna sequences, genomic sequencing, pcr primer design, and gene cloning  <cit> .
unlike traditional methods of sequence alignment, e-pcr programs search sequence databases using fragments similar in length to actual pcr primers that would target both ends of a sequence, instead of using a full-length sequence.
coupled with these developments in bioinformatics, ngs data enables the discovery of genome-wide indel polymorphisms by comparing the whole genome sequences of individuals within a species.
lai et al.
re-sequenced a group of six elite maize inbred lines, and uncovered more than  <dig>  polymorphic indels with sizes smaller than six bp  <cit> .
despite such progresses, information regarding indel polymorphism, size, and alleles is still inadequate, particularly considering the indel polymorphism levels observed in large populations.
we developed a set of highly polymorphic indel markers with large size differences and high-density occurrence using the ngs reads of  <dig> maize inbred lines and landraces along with one b <dig> maize reference genome  <cit> , which was used as the template.
furthermore, we identified and analyzed these genome-wide indel polymorphisms among the populations employed using an e-pcr strategy, with the aim of enhancing the efficiency of maize genetic research and molecular marker assisted breeding.
RESULTS
identification and distribution of unique primers in the maize genome
we designed a total of  <dig> , <dig> pairs of e-pcr primers using the maize b <dig> genome as a template.
of these,  <dig> , <dig> pairs are located in intergenic regions, and  <dig> , <dig> pairs are located in genic region, accounting for  <dig>  % and  <dig>  % of the total, respectively .
we mapped  <dig> , <dig> of these primer pairs to unique genomic regions,  <dig>  % of the total.
of these,  <dig> , <dig> pairs are located in intergenic regions, and  <dig> , <dig> pairs are located in genic regions, accounting for  <dig>  % and  <dig>  % of the unique primer pairs, respectively.
chromosome  <dig> contains the maximum number of unique primers , whereas chromosome  <dig> has the least .
this implies that the number of primers located on a particular chromosome can be positively correlated with chromosome length.
the average unique primer density is  <dig>  per kb of dna.
the highest density occurs on chromosome  <dig> , and the lowest on chromosome  <dig> .
the density of unique primers in different genomic regions varies, and follows, in descending order: from  <dig>  kb upstream of to transcription start sites , from transcription end sites to  <dig>  kb downstream of them , code determining sequences , introns, 5′-untranslated regions , 3′-utrs, and intergenic regions.table  <dig> distribution of e-pcr primers and polymorphic indels in different regions of the maize genome
totalh
aall primers on the whole  <dig> chromosomes
bprimers were located in unique genomic region
cpolymorphism primers were primers with length information in  <dig> or more than  <dig> genomes and pic value >0
ddensity was calculated by number/kb
ethis rate was the percentage of unique primer against overall primer
fthis rate was the percentage of unique primer with polymorphisms against unique primer
gthis rate was the percentage of unique primer with polymorphisms greater than or equal to  <dig>  against unique primer
hthere were  <dig> , <dig> primers in total, and the same primer might be divided into different regions and double counting due to the alternative splicing occurring in maize genome
indel variation in the maize genome
the ngs sequencing data from  <dig> maize genomes consisted of  <dig> , <dig>  reads, with an average length of  <dig>  bp, and an average sequencing depth of  <dig> ×.
the sequencing depth of read qi <dig> was the lowest, at  <dig> ×, whereas w64a had the highest sequencing depth, that of  <dig> × .
read  <dig> had the most e-pcr hits, that of  <dig> , <dig>  accounting for  <dig>  % of the total unique primers, whereas qi <dig> had the fewest hits,  <dig> , accounting for  <dig>  % of unique primers.
there were a sum total of  <dig> , <dig>  e-pcr hits, accounting for  <dig>  % of the unique primers .
we were able to locate  <dig>  % of the unique primers  on over  <dig> genomes.
a total of  <dig> , <dig> indels were identified, a rate of  <dig>  % of the unique primers.
chromosome  <dig> contains the maximum number of indels , whereas chromosome  <dig> has the minimum .
the highest indel density occurs on chromosome  <dig>  at  <dig>  per kb; whereas the lowest is on chromosome  <dig>  at  <dig>  per kb; average indel density is  <dig>  indels/mb.
the density of indels in various genomic regions follows, from highest to lowest: tss_up_ <dig> kb, tes_down_ <dig> kb, 5′-utr, 3′-utr, intron, cdss, and intergenic regions.
accordingly,  <dig>  % of the indels are located in genic regions, while  <dig>  % are located in intergenic regions.
the rate of indel polymorphism varies by genomic region and is listed, from highest to lowest, as follows: tss_up_ <dig> kb, 5′-utr, 3′-utr, tes_down_ <dig> kb, introns, intergenic regions, and cdss.
indels in cds regions not only have a low rate of polymorphism, but also have lower polymorphism information content  values;  <dig>  % of indels within cdss have pic values lower than  <dig>  .fig.
<dig> indel pic value distribution in different genomic regions
frequency and distribution of indels in different genomic regions
the number of indels decreased as the pic value increased, and the pic value of  <dig>  indels ranged from  <dig> to  <dig> , accounting for  <dig>  % of all indels.
there were  <dig>  indels with pic values greater than or equal to  <dig> , accounting for  <dig>  % of all indels.
the number of polymorphic allelic indels ranged from  <dig> to  <dig>  with an average of  <dig> ; however, most of the polymorphic indels only had two or three alleles,  <dig>  % and  <dig>  %, respectively.
proportionally, the number of alleles is nearly the same in different genomic regions, although, cds regions have a higher proportion of two allele polymorphisms than do other regions, accounting for  <dig>  % of all two allele indel polymorphisms .
the size difference between the smallest and largest alleles varies from  <dig> to 211 bp in length, with the number decreasing as indel size increases.
indels of 1 bp in length account for 33 % of all indels, and indels of 2 bp length account for  <dig>  %, while sizes smaller than 11 bp account for  <dig>  %.
other than within cds regions, the proportion of indels in other regions all reduces with an increase in the difference in length between polymorphic indel alleles .fig.
<dig> indel allele number distribution for different genomic regions
fig.
<dig> indel size distribution in different genomic regions
primer design for highly polymorphic indel markers with large major allele differences
primer pairs for  <dig>  unique indels, all with pic values greater than or equal to  <dig> , and with major allele differences greater than or equal to 3 bp, were designed from the maize b <dig> genome.
these indel loci primers were designed to generate pcr products with lengths of 60–100 bp, sizes that polyacrylamide gels can resolve .
primer pairs were also designed for  <dig>  unique indels, with pic values greater than or equal to  <dig> , and with major allele differences greater than or equal to 8 bp.
these indel loci primers were designed with pcr product lengths of 150–300 bp, sizes that agarose gels can resolve .
the exact positions of these indel markers in the maize genome, as well as the primer sequences, amplicon lengths, pic values, major allele differences, number of alleles, and number of e-pcr products in the maize genomes evaluated are presented in additional files:  <dig> and  <dig>  this data should prove useful in furthering maize genetic research by facilitating primer design in sequences with indels.
experimental validation of indel accuracy and polymorphism
indel accuracy was experimentally validated between the maize  <dig> genome and the b <dig>  mo <dig>  and zheng <dig> genomes.
with genomic dna from  <dig>  b <dig>  mo <dig>  and zheng <dig> as templates,  <dig> indel loci were pcr-amplified.
of the  <dig> indel loci,  <dig> were readily amplified, and  <dig> of these indel loci were polymorphic between the  <dig> and b <dig> genomes, with an accuracy of  <dig>  %.
indel accuracy between  <dig> and mo <dig> was  <dig>  %, while that between  <dig> and zheng <dig> was  <dig>  % .fig.
<dig> indel accuracy experimental validation.
pcr products from lines  <dig> through  <dig> ; the numbers below the horizontal line indicate the chromosome and locus of the indel.
m: marker dl2000
indel polymorphisms were also experimentally validated.
we selected another  <dig> indel loci for pcr amplification.
the pic value of these indel loci in  <dig> maize genomes ranged from  <dig>  to  <dig> , with an average pic value of  <dig> , and the allele number ranged from  <dig> to  <dig>  with an average of  <dig> .
the pic value of the indel loci in  <dig> maize inbred lines ranged from  <dig>  to  <dig> , with an average of  <dig> , and the allele number ranged from  <dig> to  <dig>  with an average of  <dig>  .fig.
<dig> indel polymorphisms experimental validation.
pcr products from lines  <dig> to  <dig> are y <dig>  jd <dig>  rp <dig>  lh <dig>  5220- <dig>   <dig>  9ht <dig>  f <dig>  sn <dig>  qa <dig>  sc <dig>  9ht <dig>  9lb <dig>  jd <dig>  liao <dig>  mian <dig>  scml <dig>  h <dig>  qi <dig>  and y1035; the numbers below the horizontal line indicate the chromosome and locus of the indel.
m: marker dl2000
discussion
next-generation sequencing technology can produce a huge quantity of dna sequence data, which becomes a powerful tool for the discovery of high-density molecular markers.
a large variety of indel identification software with the main objectives of optimal performance, sensitivity, and specificity is rapidly becoming available  <cit> .
the e-pcr strategy we used identifies indels by aligning flanking sequences to indels rather than mapping complete reads to reference sequences containing indels.
this can effectively reduce the influence of indels on alignment.
the results of this study show that indels can be efficiently and accurately identified using e-pcr in silico assays, which also save considerable time and laboratory costs over using traditional in vivo/vitro approaches.
the development of high-density molecular markers significantly increases the efficiency of map-based cloning and marker-assisted selection.
in this study high-density indel markers, widely distributed across the maize genome, were developed at an average density of one indel per  <dig>  kb, significantly higher than the  <dig>  per kb density developed by lai et al.
<cit>  in their maize study, and the  <dig>  per kb density used by mills et al.
in their human genome study  <cit> .
our indel loci reside largely within intergenic regions , compared with genic regions  .
indel markers within genes are genic or functional markers  <cit> .
functional markers are superior to random dna markers, such as restriction fragment length polymorphism , simple sequence repeat , and amplified fragment length polymorphism , because functional markers are completely linked with trait locus alleles  <cit> .
polymorphism among homologous indels is the basis for developing indel markers.
our experimental results show that the average pic value and allele number of in vitro experimentally developed indel primers are both lower than the in silico primers used in e-pcr.
this is primarily caused by the difference in resolution between agarose/polyacrylamide gels and e-pcr.
we also note that the use of short amplicons reduces the opportunity for the formation of secondary structure, and minimizes length-dependent differential amplification.
pcr products were effectively separated and easily scored after 30 min of electrophoresis on 6 % polyacrylamide gels owing to our use of short amplicons and indel to amplicon size ratios larger than 3 %.
the average rate of indel polymorphism is  <dig>  % across the maize genome, with tss_up_ <dig> kb regions having the highest polymorphism rate, whereas the cds regions have the lowest.
intriguingly, polymorphism levels in genic regions of the maize genome were higher than that in intergenic regions.
this may be caused by differences in complexity between genic and intergenic regions, as intergenic regions in the maize genome are very rich in repeat sequences.
this makes sequence analysis very difficult.
the majority of indels in cds regions had lengths divisible by three, which is a direct result of selection against frameshift mutations .
the accuracy and identification of unique loci are critically important for developing molecular markers.
the predominant error associated with illumina ngs platforms are substitution errors, which have relatively little effect on indel identification  <cit> .
pcr-based experimental validation shows that indel markers have an accuracy of approximately 90 %.
<dig>  % of  <dig> maize inbred lines contained pcr products for those primers we synthesized for polymorphism validation, which also showed that these indel markers were highly effective.
those pcr products inconsistent with e-pcr results may occur due to non-specific amplification.
an increase in read depth per locus can be used to improve the accuracy of indel identification.
moreover, alignment parameters can be set to reduce non-specific amplification by increasing the number of mismatches in the identification of unique loci in the genome, and accuracy can be improved by decreasing the number of mismatches in the analysis of variation between populations.
gel electrophoresis results show that the actual pcr products of indel markers have fewer non-specific bands, which suggests that e-pcr can be a powerful tool for reducing non-specific amplification.
CONCLUSIONS
here we report a large-scale analysis of genome-wide indel polymorphisms among maize populations, including inbred lines from different stages of breeding history and landraces.
most of these populations are the parents of the commercial hybrid and key lines in today’s global germplasm pool.
the indel markers developed in this study provide a simple and efficient tool for any laboratory focusing on map-based gene cloning and molecular marker assisted breeding in maize.

 maize is an incredibly important cereal crop grown widely throughout the world. increased demand for maize owing to population growth and biofuel production, and the impacts of climate change on maize production will ratchet up the pressure for increased and more sustainable maize production. since the 1980s, molecular markers have been widely used in maize genetics and breeding. high-density molecular markers in applied and basic research are advantageous and necessary for map-based cloning and genome-wide association study. in contrast to single nucleotide polymorphisms, insertions and deletions are the second most common type of polymorphism. in a previous study, mills reported that indels are distributed throughout the human genome at an average density of one indel per kb. several studies have suggested numerous indels that may cause human disease. one of the most common genetic diseases in humans, cystic fibrosis, is frequently caused by various indels in coding regions of the cftr gene. the genetic diseases of tuberous sclerosis, rett syndrome, and hemophilia b are also caused by small indels. similarly, indels can alter the phenotype of plants. consequently, indels have been increasingly recognized as an important source of molecular markers, and indel markers have been successfully used for several genetic studies in crops. the maize genome was completed in using traditional sanger sequencing technology,. however, the subsequent development of next-generation sequencing technology has generated an enormous amount of short reads that science is scrambling to analyze. ngs technology has also drastically reduced the time and cost requirements of sequencing, which has enabled the re-sequencing of a large number of genomes. this has provided for the possibility of large-scale genetic variation surveys, where many individuals within a single species have been sequenced. examples include silkworm samples, soybean samples, sorghum samples, and rice samples. as of more than maize inbred line and landrace genomes had been completely re-sequenced and published. rapid bioinformatics developments have introduced various software programs designed to identify indels, including dindel, varscan, gatk, and samtool’s mpileup. the first step in most of these programs is to map reads directly to a reference sequence. accurate indel calls from reads are challenging for a couple of reasons. first, reads covering indels are generally more difficult to map correctly to reference genomes, especially ones containing large indel events. moreover, incorrect alignments at the nucleotide level lead to an incorrect placement of gaps in the alignments. schuler proposed a pcr amplification computer simulation concept known as the electronic polymerase chain reaction, which has been used widely in various aspects of biology, including the chromosomal localization of dna sequences, genomic sequencing, pcr primer design, and gene cloning. unlike traditional methods of sequence alignment, e-pcr programs search sequence databases using fragments similar in length to actual pcr primers that would target both ends of a sequence, instead of using a full-length sequence. coupled with these developments in bioinformatics, ngs data enables the discovery of genome-wide indel polymorphisms by comparing the whole genome sequences of individuals within a species. lai et al. re-sequenced a group of six elite maize inbred lines, and uncovered more than polymorphic indels with sizes smaller than six bp. despite such progresses, information regarding indel polymorphism, size, and alleles is still inadequate, particularly considering the indel polymorphism levels observed in large populations. we developed a set of highly polymorphic indel markers with large size differences and high-density occurrence using the ngs reads of maize inbred lines and landraces along with one b maize reference genome, which was used as the template. furthermore, we identified and analyzed these genome-wide indel polymorphisms among the populations employed using an e-pcr strategy, with the aim of enhancing the efficiency of maize genetic research and molecular marker assisted breeding.  identification and distribution of unique primers in the maize genome we designed a total of, pairs of e-pcr primers using the maize b genome as a template. of these,, pairs are located in intergenic regions, and, pairs are located in genic region, accounting for  % and  % of the total, respectively. we mapped, of these primer pairs to unique genomic regions,  % of the total. of these,, pairs are located in intergenic regions, and, pairs are located in genic regions, accounting for  % and  % of the unique primer pairs, respectively. chromosome contains the maximum number of unique primers, whereas chromosome has the least. this implies that the number of primers located on a particular chromosome can be positively correlated with chromosome length. the average unique primer density is per kb of dna. the highest density occurs on chromosome, and the lowest on chromosome. the density of unique primers in different genomic regions varies, and follows, in descending order: from kb upstream of to transcription start sites, from transcription end sites to kb downstream of them, code determining sequences, introns, 5′-untranslated regions, 3′-utrs, and intergenic regions.table distribution of e-pcr primers and polymorphic indels in different regions of the maize genome totalh aall primers on the whole chromosomes bprimers were located in unique genomic region cpolymorphism primers were primers with length information in or more than genomes and pic value >0 ddensity was calculated by number/kb ethis rate was the percentage of unique primer against overall primer fthis rate was the percentage of unique primer with polymorphisms against unique primer gthis rate was the percentage of unique primer with polymorphisms greater than or equal to against unique primer hthere were, primers in total, and the same primer might be divided into different regions and double counting due to the alternative splicing occurring in maize genome indel variation in the maize genome the ngs sequencing data from maize genomes consisted of, reads, with an average length of  bp, and an average sequencing depth of ×. the sequencing depth of read qi was the lowest, at ×, whereas w64a had the highest sequencing depth, that of ×. read had the most e-pcr hits, that of, accounting for  % of the total unique primers, whereas qi had the fewest hits,, accounting for  % of unique primers. there were a sum total of, e-pcr hits, accounting for  % of the unique primers. we were able to locate  % of the unique primers on over genomes. a total of, indels were identified, a rate of  % of the unique primers. chromosome contains the maximum number of indels, whereas chromosome has the minimum. the highest indel density occurs on chromosome at per kb; whereas the lowest is on chromosome at per kb; average indel density is indels/mb. the density of indels in various genomic regions follows, from highest to lowest: tss_up_ kb, tes_down_ kb, 5′-utr, 3′-utr, intron, cdss, and intergenic regions. accordingly,  % of the indels are located in genic regions, while  % are located in intergenic regions. the rate of indel polymorphism varies by genomic region and is listed, from highest to lowest, as follows: tss_up_ kb, 5′-utr, 3′-utr, tes_down_ kb, introns, intergenic regions, and cdss. indels in cds regions not only have a low rate of polymorphism, but also have lower polymorphism information content values;  % of indels within cdss have pic values lower than.fig. indel pic value distribution in different genomic regions frequency and distribution of indels in different genomic regions the number of indels decreased as the pic value increased, and the pic value of indels ranged from to, accounting for  % of all indels. there were indels with pic values greater than or equal to, accounting for  % of all indels. the number of polymorphic allelic indels ranged from to with an average of; however, most of the polymorphic indels only had two or three alleles,  % and  %, respectively. proportionally, the number of alleles is nearly the same in different genomic regions, although, cds regions have a higher proportion of two allele polymorphisms than do other regions, accounting for  % of all two allele indel polymorphisms. the size difference between the smallest and largest alleles varies from to 211 bp in length, with the number decreasing as indel size increases. indels of 1 bp in length account for 33 % of all indels, and indels of 2 bp length account for  %, while sizes smaller than 11 bp account for  %. other than within cds regions, the proportion of indels in other regions all reduces with an increase in the difference in length between polymorphic indel alleles.fig. indel allele number distribution for different genomic regions fig. indel size distribution in different genomic regions primer design for highly polymorphic indel markers with large major allele differences primer pairs for unique indels, all with pic values greater than or equal to, and with major allele differences greater than or equal to 3 bp, were designed from the maize b genome. these indel loci primers were designed to generate pcr products with lengths of 60–100 bp, sizes that polyacrylamide gels can resolve. primer pairs were also designed for unique indels, with pic values greater than or equal to, and with major allele differences greater than or equal to 8 bp. these indel loci primers were designed with pcr product lengths of 150–300 bp, sizes that agarose gels can resolve. the exact positions of these indel markers in the maize genome, as well as the primer sequences, amplicon lengths, pic values, major allele differences, number of alleles, and number of e-pcr products in the maize genomes evaluated are presented in additional files: and this data should prove useful in furthering maize genetic research by facilitating primer design in sequences with indels. experimental validation of indel accuracy and polymorphism indel accuracy was experimentally validated between the maize genome and the b mo and zheng genomes. with genomic dna from b mo and zheng as templates, indel loci were pcr-amplified. of the indel loci, were readily amplified, and of these indel loci were polymorphic between the and b genomes, with an accuracy of  %. indel accuracy between and mo was  %, while that between and zheng was  %.fig. indel accuracy experimental validation. pcr products from lines through; the numbers below the horizontal line indicate the chromosome and locus of the indel. m: marker dl2000 indel polymorphisms were also experimentally validated. we selected another indel loci for pcr amplification. the pic value of these indel loci in maize genomes ranged from to, with an average pic value of, and the allele number ranged from to with an average of. the pic value of the indel loci in maize inbred lines ranged from to, with an average of, and the allele number ranged from to with an average of.fig. indel polymorphisms experimental validation. pcr products from lines to are y jd rp lh 5220- 9ht f sn qa sc 9ht 9lb jd liao mian scml h qi and y1035; the numbers below the horizontal line indicate the chromosome and locus of the indel. m: marker dl2000 discussion next-generation sequencing technology can produce a huge quantity of dna sequence data, which becomes a powerful tool for the discovery of high-density molecular markers. a large variety of indel identification software with the main objectives of optimal performance, sensitivity, and specificity is rapidly becoming available. the e-pcr strategy we used identifies indels by aligning flanking sequences to indels rather than mapping complete reads to reference sequences containing indels. this can effectively reduce the influence of indels on alignment. the results of this study show that indels can be efficiently and accurately identified using e-pcr in silico assays, which also save considerable time and laboratory costs over using traditional in vivo/vitro approaches. the development of high-density molecular markers significantly increases the efficiency of map-based cloning and marker-assisted selection. in this study high-density indel markers, widely distributed across the maize genome, were developed at an average density of one indel per kb, significantly higher than the per kb density developed by lai et al. in their maize study, and the per kb density used by mills et al. in their human genome study. our indel loci reside largely within intergenic regions, compared with genic regions. indel markers within genes are genic or functional markers. functional markers are superior to random dna markers, such as restriction fragment length polymorphism, simple sequence repeat, and amplified fragment length polymorphism, because functional markers are completely linked with trait locus alleles. polymorphism among homologous indels is the basis for developing indel markers. our experimental results show that the average pic value and allele number of in vitro experimentally developed indel primers are both lower than the in silico primers used in e-pcr. this is primarily caused by the difference in resolution between agarose/polyacrylamide gels and e-pcr. we also note that the use of short amplicons reduces the opportunity for the formation of secondary structure, and minimizes length-dependent differential amplification. pcr products were effectively separated and easily scored after 30 min of electrophoresis on 6 % polyacrylamide gels owing to our use of short amplicons and indel to amplicon size ratios larger than 3 %. the average rate of indel polymorphism is  % across the maize genome, with tss_up_ kb regions having the highest polymorphism rate, whereas the cds regions have the lowest. intriguingly, polymorphism levels in genic regions of the maize genome were higher than that in intergenic regions. this may be caused by differences in complexity between genic and intergenic regions, as intergenic regions in the maize genome are very rich in repeat sequences. this makes sequence analysis very difficult. the majority of indels in cds regions had lengths divisible by three, which is a direct result of selection against frameshift mutations. the accuracy and identification of unique loci are critically important for developing molecular markers. the predominant error associated with illumina ngs platforms are substitution errors, which have relatively little effect on indel identification. pcr-based experimental validation shows that indel markers have an accuracy of approximately 90 %.  % of maize inbred lines contained pcr products for those primers we synthesized for polymorphism validation, which also showed that these indel markers were highly effective. those pcr products inconsistent with e-pcr results may occur due to non-specific amplification. an increase in read depth per locus can be used to improve the accuracy of indel identification. moreover, alignment parameters can be set to reduce non-specific amplification by increasing the number of mismatches in the identification of unique loci in the genome, and accuracy can be improved by decreasing the number of mismatches in the analysis of variation between populations. gel electrophoresis results show that the actual pcr products of indel markers have fewer non-specific bands, which suggests that e-pcr can be a powerful tool for reducing non-specific amplification.  here we report a large-scale analysis of genome-wide indel polymorphisms among maize populations, including inbred lines from different stages of breeding history and landraces. most of these populations are the parents of the commercial hybrid and key lines in today’s global germplasm pool. the indel markers developed in this study provide a simple and efficient tool for any laboratory focusing on map-based gene cloning and molecular marker assisted breeding in maize. 
intriguingly, the level of polymorphism in genic regions of the maize genome was higher than that in intergenic regions. of these highly polymorphic indels, we designed primer pairs for and indels with major allele differences greater than or equal to and 8 bp, respectively, based on the differing resolution capabilities of gel electrophoresis. indels have been increasingly recognized as an important source of molecular markers due to high-density occurrence, cost-effectiveness, and ease of genotyping. of highly polymorphic indel markers, all had polymorphisms with average pic values of. the polymorphic indel markers developed from this study may enhance the efficiency of genetic research and marker-assisted breeding in maize. a total of, unique indels were identified in maize genomes, with an overall density of indels/mbp, and an average allele number of, ranging from to there were indels with polymorphism information content values greater than or equal to, accounting for  % of overall indels. the maize genome is rich in indel polymorphisms. the accuracy of our indel markers was experimentally validated, and among indel markers, average accuracy was approximately 90 %. in addition, we also validated the polymorphism of the indel markers. coupled with developments in bioinformatics, next-generation sequencing platforms enable the discovery of millions of indel polymorphisms by comparing the whole genome sequences of individuals within a species.
 insertions and deletions are the most abundant form of structural variation in all genomes. indels have been increasingly recognized as an important source of molecular markers due to high-density occurrence, cost-effectiveness, and ease of genotyping. coupled with developments in bioinformatics, next-generation sequencing platforms enable the discovery of millions of indel polymorphisms by comparing the whole genome sequences of individuals within a species.  a total of, unique indels were identified in maize genomes, with an overall density of indels/mbp, and an average allele number of, ranging from to there were indels with polymorphism information content values greater than or equal to, accounting for  % of overall indels. of these highly polymorphic indels, we designed primer pairs for and indels with major allele differences greater than or equal to and 8 bp, respectively, based on the differing resolution capabilities of gel electrophoresis. the accuracy of our indel markers was experimentally validated, and among indel markers, average accuracy was approximately 90 %. in addition, we also validated the polymorphism of the indel markers. of highly polymorphic indel markers, all had polymorphisms with average pic values of.  the maize genome is rich in indel polymorphisms. intriguingly, the level of polymorphism in genic regions of the maize genome was higher than that in intergenic regions. the polymorphic indel markers developed from this study may enhance the efficiency of genetic research and marker-assisted breeding in maize. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords maizenext-generation sequencinginsertion and deletionindelmolecular markerpolymorphismissue-copyright-statement© the author 2015 