BACKGROUND
genomic sequencing of crop species has shed light on causative relationships between sequence polymorphisms and traits of agronomic interest.
ongoing efforts in maize qtl  mapping have identified genetic intervals whose underlying genes variably contribute to interesting phenotypes such as oil content  <cit> , root architecture  <cit> , and pest resistance  <cit> .
while many trait variations  result from amino acid differences  <cit> , gene expression differences can also result in observable phenotypes  <cit> .
considering the burgeoning fields of epigenetics and transcriptomics, analysis of gene expression regulation is playing an important role in understanding gene interactions that lead to traits of interest.
the concept of "genetical genomics"  <cit>  was proposed with the advance of high throughput gene expression profiling technologies.
in traditional qtl analyses, linkage mapping leads to the detection of genomic regions which are associated with phenotypic variations within a population.
genetical genomics employs this same approach, except that the phenotypes are levels in gene expression resulting in the detection of expression qtl .
eqtl do not necessarily result from sequence polymorphisms proximal to the gene being measured  but could result from differences in genes unlinked to the target.
in these cases, the eqtl function in a trans-acting manner.
the field of genetical genomics has allowed eqtl analysis within mapping populations in a multitude of species of plants  <cit> , yeast  <cit> , and mammals  <cit> .
an arabidopsis study  <cit>  describes that while there are more trans-acting factors within the genome, cis-acting elements are more significant and therefore stronger in regulatory ability than those acting in trans.
the results suggest a generalization that while multiple trans-acting factors can each weakly contribute to the total expression regulation of a given gene, a single cis-acting variation plays a far greater role.
the mapping and positional cloning of a trans-acting eqtl may reveal an expression regulator such as a transcription factor or small regulatory rna.
while many eqtl have been identified, few trans-acting factors have been cloned.
yvert et al.
<cit>  mapped and cloned two yeast trans-eqtl that regulate genes known to be involved in pathways regulating pheromone response and daughter cell separation following budding.
interestingly, neither of the causative genes functions as expression regulators such as transcription factors nor through other expected mechanisms.
however, their work suggests that the continued high-throughput eqtl studies will identify novel genes to better understand the regulation of known biochemical pathways.
master regulators such as leafy, an arabidopsis regulator of at least seven components involved in reproductive development  <cit> , were previously only identifiable through mutagenesis.
genome-wide trans-eqtl analyses may identify more master regulators that function to control several components of a single pathway.
we report a genome wide eqtl analysis in a highly utilized maize mapping population.
we successfully identify both cis-acting and trans-acting genetic elements that cooperate to regulate gene expression in maize crown roots, and describe the pitfalls of detecting false cis- or trans-acting eqtl in the absence of perfect genomic sequences from both parents.
in addition to this genome-wide analysis of regulating factors, we have positionally cloned a trans-acting factor.
RESULTS
global analysis of cis-acting and trans-acting eqtl in maize root
the expression profile of the maize ibm <dig> syn <dig> double haploid population  <cit>  was determined by microarray hybridization to 60-mer probes.
the mapping population was a subset of a population created through ten generations of intermating between the b <dig> and mo <dig> maize lines followed by the generation of double haploid lines, which creates highly recombinant but fixed alleles.
the more than  <dig>  probes were designed to measure expression of the full complement of approximately  <dig>  maize genes  <cit> .
these expression level values were used as phenotypes for initial mapping analysis.
while the array theoretically measures all genes, several factors must be considered: 1) the genomic origin remains unknown for a moderate number of genes; 2) probes may not be unique to individual genes but rather simultaneously measure several family members; 3) probes may not measure known genes, but rather expression originating from "non-genic" regions such as regulatory rnas.
therefore, to ensure that appropriate mode of action can be assigned to eqtl, only those unique  genomic and/or genic probes were considered for eqtl analysis.
only the most significant eqtl was assigned for each probe, as described in methods.
a genetic distance window of  <dig> cm was used to define the mechanism of action for the eqtl.
when the distance was less than  <dig> cm between the target gene and eqtl, the mechanism of action was considered cis-acting.
any eqtl greater than  <dig> cm from the target was deemed trans-acting.
the filtering of eqtl by p-value significance, reproducibility between replicates, and confidence in unique probe hybridization eliminated nearly 90% of the probes from further analysis, resulting in  <dig>  high quality eqtl.
of the most significant eqtl for each probe, the majority of eqtl analyzed functioned through a cis mechanism  and of those, 54%  functioned with a kolmogorov-smirnov  p-value of ≤ 1e- <dig>  trans-acting eqtl were the minority of the eqtl identified , and of those 44%  functioned with a ks p-value of ≤ 1e- <dig>  altogether, these data suggest that cis-acting eqtl in general regulate gene expression with more significance than trans-acting eqtl .
significant eqtl  classified based on strength and trans- or cis-acting function for the  <dig>  confirmed eqtl identified
high reproducibility of biological replicates
the experimental design, utilizing a supported hydroponics system in growth chamber conditions, was intended to minimize false eqtl that resulted from environmental effects.
while biological replicates were grown and harvested several months apart, the time and duration of harvest were comparable between replicates to minimize diurnal effects on transcription.
reproducibility between replicates was determined by the eqtl map position identified, as well as the confidence in the eqtl .
initially, eqtl were identified in the first replicate with a p-value cut-off of 1e- <dig>  but were validated in replicate  <dig> with a less stringent cut-off of 1e- <dig>  <cit> .
based on high quality eqtl identified in replicate  <dig> alone, ,  <dig> %  were validated in replicate  <dig> and used for all subsequent global analysis.
those eqtl that failed to validate between replicates fell into two categories: 1) non-significant p-value in replicate  <dig>  or 2) inconsistent map position  such that the position of the strongest eqtl failed to reproduce between replicates .
further investigation of the  <dig> inconsistent eqtl suggests that the reasons for the inconsistency are complex and detailed analysis is required for appropriate determination of reproducibility.
if several trans-eqtl in combination with a cis-eqtl contribute to expression control, it is possible that the eqtl deemed strongest in one replicate may be second strongest in another replicate, and vice versa.
additionally, if the eqtl peak broadly extends across a greater than the  <dig> cm interval, then significance variations between replicates may falsely suggest a failure of reproducibility for that eqtl.
therefore, the actual reproducibility might be slightly higher than the number indicated.
for the eqtl that mapped reproducibly it is also important to note that the significance of each eqtl was consistent as well .
altogether, this suggests that the hydroponics system and analyses enriched for the identification of genetic components of gene expression regulation.
cooperative regulation of gene expression
previously published work  <cit>  suggests that the vast majority of eqtl detected will function through a trans-acting mechanism , while the strongest  eqtl will function through a cis-acting mechanism.
however, a breakdown of strengths and quantities of each mode of action identified using the ibm <dig> syn <dig> population showed nearly  <dig> fold more cis-acting than trans-acting eqtl, yet the cis- did function with greater significance .
there are several possible explanation for the discrepancy: 1) a conservative p-value cut-off of 1e- <dig> is used to minimize the number of false positive eqtl identified, however, it is expected that many trans-acting eqtl would weakly regulate gene expression, perhaps with significance less than the cut-off value, and may be lost in the analysis; 2) only the most significant eqtl was selected for each probe, which is more likely to be cis-acting ; 3) the microarray probes were strongly b73-biased  and any imperfect hybridization of the mo <dig> derived alleles to the probe set will map as cis-acting eqtl regardless of the genome-mediated functional expression regulation in situ; 4) the maize genome sequence is not completed.
many of the genomic origins of array probes remain unidentified or are not unique within the genome, therefore the transcripts being measured by the microarray cannot be accurately assigned to the current public genome assemblies, therefore the modes of action cannot be distinguished, potentially ignoring significant trans-eqtl; 5) a relatively large window  was used to assign cis-eqtl potentially incorrectly characterizing eqtl.
a more comprehensive regulatory analysis can be performed in cases where multiple eqtl were identified for any particular target .
the general global gene regulation analysis identifies the most significant eqtl regulating any particular gene.
therefore, any secondary regulation of that gene is masked by the initial analysis.
this second-level analysis enables the identification of regulatory elements that function with less significance than the primary eqtl that was initially identified.
of the eqtl analyzed, the vast majority  functioned independently to regulate target gene expression with the current statistic cut-off.
mapped genes that were regulated by  <dig> or more eqtl were further investigated.
in total,  <dig>  eqtl were detected for  <dig> target genes .
the majority of the initial primary  eqtl  mapped as cis.
an additional  <dig> weaker secondary and  <dig> tertiary trans-acting peaks were found to regulate those same target genes.
a minority  of initial primary eqtl mapped as trans-.
an additional  <dig> secondary and  <dig> tertiary trans-acting eqtl were detected regulating those same target genes.
interestingly,  <dig> cis-acting eqtl were identified that function more weakly than the primary trans-acting eqtl.
using the stringent ks p-value cutoff of 10- <dig>  the maximum number of cooperative eqtl identified for any particular gene was  <dig>  this analysis demonstrates that although the strongest eqtl identified will likely function in cis, more trans-acting eqtl regulate gene expression, although with less significance.
the strongest eqtl identified regulating the expression of any particular gene is considered the primary identified eqtl.
up to two additional, yet weaker, functional expression regulators were identified regulating the same gene and are considered secondary or tertiary eqtl.
total verifiable cis-acting eqtl identified =  <dig> ; total verifiable trans-acting eqtl identified =  <dig> .
due to the resolution of the genetic map, only one cis-acting eqtl can be identified per gene being measured, n = not possible.
lack of perfect genome sequences from both parental lines caused the identification of false cis- and trans-acting eqtl
while the b <dig> genome draft sequences are available, the mo <dig> genome sequence is currently in a primitive state.
failure of mo <dig> sequences to hybridize to their analogous probe due to sequence polymorphisms would map as cis-acting eqtl regardless of the actual expression levels or modes of action in situ.
of all the cis-eqtl, only  <dig> % have higher expression levels for the mo <dig> allele, which deviates significantly from the expected 50%.
this implies that about 35% of the cis-eqtl are false positives.
of the most significant cis-eqtl , similar analysis indicates that 46% of them are false positives due to sequence polymorphism, to identify cases of such false positive eqtl, twenty of the strongest eqtl with unique genic/genomic probe positions in b <dig> were analyzed for sequence similarity in the mo <dig> genome.
blast of those probes to the publicly available paired-end read sequences of mo <dig> generated by the joint genome institute  found only two probes  that match perfectly between b <dig> and mo <dig> .
for the remaining eighteen b <dig> probes for which no identical mo <dig> sequences were detected, it is not clear how many are caused by the incomplete mo <dig> genome sequence and how many by sequence polymorphism.
using the b <dig> reference genome, primers were designed to amplify the intervals surrounding the probe hybridization sites for four of the potentially false-positive cis-eqtl.
two of the four intervals failed to amplify from mo <dig> derived genomic and cdna templates whereas they successfully amplified from b <dig> templates .
without the mo <dig> reference sequence, it remains unclear if polymorphisms prevented adequate primer annealing in mo <dig> for pcr amplification.
two intervals amplified successfully from mo <dig> template allowing for sequence analysis of the probe hybridization loci.
comparison of the amplified sequences from mo <dig> to the b <dig> probe sequences showed that polymorphisms likely caused differential hybridization to the microarray probes .
while trans- or cis-acting factors may function in the expression regulation of these genes in situ, the probe homology issues inappropriately suggest strong cis-eqtl.
therefore these strong cis-eqtl are most likely false positives.
despite the availability of the draft b <dig> genome sequences, incompleteness and inaccuracy in genome/bac sequencing or gene annotations remain problematic for eqtl analysis.
additionally, the ancestral genome duplication in maize complicates mapping analysis when markers, genes, and genomic regions are indistinguishable.
we identified a strong eqtl  that functioned apparently in trans- to regulate two iron superoxide dismutase  genes.
initially the eqtl was regarded as a master regulator considering its ability to regulate the expression of multiple biochemically related genes.
importantly, blast analysis of the microarray probes suggested that they measured their respective fesod transcripts uniquely.
however further analysis of the eqtl interval and sequencing of the single bac that spanned the interval revealed a sequencing gap in the available b <dig> genomic sequences.
within that gap resided a third fesod gene which the probes would recognize during microarray hybridization .
rather than identifying a strong trans-acting master regulator, most likely we have inadvertently revealed an unknown gene that was differentially expressed between b <dig> and mo <dig> due to cis-acting factors, yet had high sequence similarity to other known fesods.
it is obvious that some of the cis- and trans-acting eqtl identified are false due to the unavailability of complete genome sequences for both parental lines.
it is not clear how extensive the problem is, although it seems the strongest eqtl are more prone to be false positive than the weaker ones.
differential gene expression confirms presence/absence variations
in addition to nucleotide polymorphisms or minor insertion/deletion differences between the b <dig> and mo <dig> parent genomes, several studies have identified large deletions in the mo <dig> genome as compared to b <dig>  <cit> .
of particular interest is a deletion spanning many bacs on the short arm of chromosome  <dig> which contains at least  <dig> genes and pseudogenes  <cit> .
eqtl mapping using the ibm <dig> syn <dig> population found  <dig> strong  cis-acting eqtl, measuring  <dig> probes  located on the bacs in question.
for the genes in the region, b <dig> in general shows robust expression while mo <dig> expression is essentially off .
the eqtl results are consistent with the presence/absence variations  detected through array genome hybridization.
identification of a trans-acting regulator by map-based cloning
a strong trans-acting eqtl was selected for fine mapping and positional cloning to identify the gene responsible for expression regulation.
we identified a strong eqtl  located on the long arm of chromosome  <dig>  on ibm <dig> neighbors map  <cit> )) that regulated the expression of a gene annotated as aba 8'-hydroxylase, located  <dig> cm away from the eqtl near the centrosome of chromosome  <dig> .
within the mapping population, a b <dig> derived allele at the eqtl resulted in 6-fold greater expression  of the target gene as compared to a mo <dig> derived allele at the eqtl .
the recombinant individuals from the ibm <dig> syn <dig> population grown for the global eqtl analysis were used to define the genetic interval to within  <dig> base pairs in a single gene .
the candidate gene underlying the eqtl was determined to be a class i glutamine amidotransferase domain containing gene .
sequence analysis of the b <dig> and mo <dig> alleles of the full length gene revealed multiple snps coding for five amino acid residue differences between the parents plus a  <dig> residue truncation in mo <dig> as compared to b <dig> .
only the  <dig> residue truncation is located within the  <dig> base pair eqtl interval, thus presumably the causative variation for the differentially gene expression.
re-phenotyping the ibm <dig> syn <dig> population by rt-pcr specific for aba 8'-hydroxylase confirmed the gene expression results from the microarray analysis.
glutamine amidotransferase genes are well characterized players in several biosynthesis pathways including the purine, pyrimidine, histidine, tryptophan, and arginine pathways  <cit> , however, a transcript-abundance mediating function has never been published.
while steady state transcript abundance is determined by many factors including transcription factors, enhancers, mrna degradation regulators, and rate of translation  <cit> , the role of glutamine amidotransferase in aba 8'-hydroxylase transcript abundance remains elusive.
despite the role that glutamine amidotransferase plays on aba 8'-hydroxylase transcription, no other eqtl mapped to the same genetic region in this population suggesting that any broad transcriptional regulatory function is unlikely.
eqtl are determined by expression phenotypes, and not based on physiological or morphological phenotypes as in classic qtl identification.
therefore, further analysis of aba biosynthesis in the ibm <dig> syn <dig> population could prove a physiological function downstream of transcript abundance in regards to its trans-regulation.
in anticipation of aba biosynthesis analysis, the aba 8'-hydroxylase gene being regulated by glutamine amidotransferase was sequenced from b <dig> and mo <dig> cdna.
interestingly, the results showed that the annotated aba 8'-hydroxylase gene was actually a product of genomic shuffling that occurred sometime prior to the genetic diversification of b <dig> and mo <dig>  in both parentages, the aba 8'-hydroxylase pseudogene  is a chimera of the functional maize aba 8'-hydroxylase gene fragment, non-genic genomic sequence originating from chromosome  <dig>  and repetitive genomic sequence .
thus it seems that a pseudogene expression level is regulated.
the aba 8'p is not present in all maize inbred lines.
actually, validation of glutamine amidotransferase as the candidate gene for the eqtl was hampered by the absence of this pseudogene in certain lines .
whether this pseudogene or its expression regulation has any physiological function remains to be determined.
discussion
while the field of genomics has afforded scientists with access to genomic sequences of countless numbers of model species, strains, and lines, our understanding of the function of those sequences remains rather limited.
forward and reverse genetics have given meaning to sequence polymorphisms in a fair number of genes, but in addition to gene function analysis, it is equally important to understand how and when those genes are activated and the roles the translated proteins play within biochemical pathways.
only through the continued efforts in the fields of transcriptomics and proteomics can the full power of genomics be realized.
expression qtl studies are generating vast amounts of data from the perspective of gene regulation, both from cis-acting elements and trans-acting factors, that begin to fill in gaps in the understanding of transcriptional regulation and gene interactions.
results from our study help to elucidate the genome-wide expression regulation in play during the development of crown root tips in maize.
despite stringent statistical analysis, we identified more than  <dig>  eqtl that function through both cis- and trans-acting mechanisms.
in addition to the identification of cis- and trans-factors, we described the relative regulatory contribution each of those factors plays by means of a ks p-value.
despite our expectations, as well as those set forth in previously published maize eqtl studies  <cit> , we identified vastly more cis-acting eqtl than trans-suggesting that the most significant eqtl will act in cis for most genes.
the statistical methodology and stringency we employed was designed to minimize false positive eqtl from the analysis, however at the probable expense of the lesser significant eqtl which will most likely function in trans  <cit> .
additionally, the definition of trans can be "arbitrarily" set for each study.
while we defined trans-acting to mean any regulatory element greater than  <dig> cm from the target, others have set the boundary at  <dig> cm  <cit> .
either limit is appropriate for maize eqtl studies but will affect the classification and quantification of trans-eqtl.
among the tens of thousands of cis- and trans-acting eqtl identified in the current study, we have demonstrated that some of them are false-positives due to the lack of complete genome sequences from both parents of the mapping population.
polymorphism in the microarray probe regions, which could affect hybridization intensities for mrna quantification, will lead to the occurrence of false cis-acting eqtl.
likewise, sequencing gaps at the trans-acting eqtl regions could result in the detection of false trans-acting eqtl.
with these mechanisms, the stronger, large-effect eqtl  are more prone to be false positives than the weaker eqtl.
we estimated that 35% of all the cis-eqtl is false positive, but the false trans-eqtl discovery rate is unknown in the absence of the complete genome sequences.
it is likely that the whole-genome eqtl analysis reported in other systems could suffer similar false positive issues, and caution should be taken when interpreting the results and selecting eqtl for further analysis.
although tens of thousands of eqtl have been reported, very few have been cloned and characterized, especially in higher organisms where positional cloning could still be challenging.
to test the feasibility of eqtl cloning in maize and to understand the regulatory mechanism of transcript abundance, a strong trans-eqtl for a putative aba hydroxylase was selected for further mapping and cloning.
the eqtl was fine mapped to a very small physical interval  and a putative glutamine amidotransferase gene was identified as the candidate gene for this eqtl.
unfortunately, the target gene identified turned out to be a pseudogene.
it is not clear if this aba hydroxylase pseudogene has any physiological function, why the expression of a pseudogene is regulated, or how the two genes interact with each other.
nevertheless, we have clearly demonstrated the feasibility of cloning trans-eqtl with large effect in maize.
the cloning of trans-eqtl would help to understand the mechanism of transcript abundance regulation and identify regulatory genes for biochemical pathways.
the ibm <dig> syn <dig> population is a set of  <dig> doubled haploid lines from a randomly mated population derived from b <dig> and mo <dig>  <cit> .
having undergone  <dig> generations of random intermating/recombination, these dh lines exhibited a high degree of phenotypic variability and high frequency of recombination.
we were able to fine map an eqtl into a  <dig> bp interval with only  <dig> ibm <dig> syn <dig> dh lines.
although recombination frequency varies widely across the genome and more genomic loci need to be analyzed before this population can be better assessed for recombination frequency, the results suggest that the ibm syn <dig> population is suitable for high resolution eqtl mapping and recombination studies.
as in previous reports, we have shown that most of the strong eqtl act in cis.
moreover, for the relatively small number of strong trans-eqtl detected, some of them could be false positive.
therefore, it may be a challenge to identify a large number of strong trans-eqtl which are more amenable for positional cloning.
a recently study in human b cells which maps the regulatory elements that influence radiation-induced changes in gene expression indicated that nearly all the strong regulators act in trans to influence the expression of their target genes  <cit> .
therefore, instead of mapping the steady-state level of mrna under one constant condition, mapping eqtl which regulate the differential responsiveness in gene expression to biotic and abiotic factors could be a promising approach to enrich and identify strong trans-eqtl.
these trans-eqtl should be ideal targets for cloning.
they are important for understanding plant responses to biotic and abiotic stresses, and technically feasible to isolate.
CONCLUSIONS
we have shown the feasibility of eqtl analysis as a means to identify, clone and analyze trans-acting regulatory factors through large-scale screening analysis.
a glutamine amidotransferase regulator was identified as a trans-acting factor.
harnessing the regulatory function of trans-acting factors could allow for better control of important agronomical genes.
we also described the pitfall of identifying false-positive eqtl in the absence of complete genome sequences, which has broad implications in similar eqtl studies.

 genomic sequencing of crop species has shed light on causative relationships between sequence polymorphisms and traits of agronomic interest. ongoing efforts in maize qtl mapping have identified genetic intervals whose underlying genes variably contribute to interesting phenotypes such as oil content, root architecture, and pest resistance. while many trait variations result from amino acid differences, gene expression differences can also result in observable phenotypes. considering the burgeoning fields of epigenetics and transcriptomics, analysis of gene expression regulation is playing an important role in understanding gene interactions that lead to traits of interest. the concept of "genetical genomics" was proposed with the advance of high throughput gene expression profiling technologies. in traditional qtl analyses, linkage mapping leads to the detection of genomic regions which are associated with phenotypic variations within a population. genetical genomics employs this same approach, except that the phenotypes are levels in gene expression resulting in the detection of expression qtl. eqtl do not necessarily result from sequence polymorphisms proximal to the gene being measured but could result from differences in genes unlinked to the target. in these cases, the eqtl function in a trans-acting manner. the field of genetical genomics has allowed eqtl analysis within mapping populations in a multitude of species of plants, yeast, and mammals. an arabidopsis study describes that while there are more trans-acting factors within the genome, cis-acting elements are more significant and therefore stronger in regulatory ability than those acting in trans. the results suggest a generalization that while multiple trans-acting factors can each weakly contribute to the total expression regulation of a given gene, a single cis-acting variation plays a far greater role. the mapping and positional cloning of a trans-acting eqtl may reveal an expression regulator such as a transcription factor or small regulatory rna. while many eqtl have been identified, few trans-acting factors have been cloned. yvert et al. mapped and cloned two yeast trans-eqtl that regulate genes known to be involved in pathways regulating pheromone response and daughter cell separation following budding. interestingly, neither of the causative genes functions as expression regulators such as transcription factors nor through other expected mechanisms. however, their work suggests that the continued high-throughput eqtl studies will identify novel genes to better understand the regulation of known biochemical pathways. master regulators such as leafy, an arabidopsis regulator of at least seven components involved in reproductive development, were previously only identifiable through mutagenesis. genome-wide trans-eqtl analyses may identify more master regulators that function to control several components of a single pathway. we report a genome wide eqtl analysis in a highly utilized maize mapping population. we successfully identify both cis-acting and trans-acting genetic elements that cooperate to regulate gene expression in maize crown roots, and describe the pitfalls of detecting false cis- or trans-acting eqtl in the absence of perfect genomic sequences from both parents. in addition to this genome-wide analysis of regulating factors, we have positionally cloned a trans-acting factor.  global analysis of cis-acting and trans-acting eqtl in maize root the expression profile of the maize ibm syn double haploid population was determined by microarray hybridization to 60-mer probes. the mapping population was a subset of a population created through ten generations of intermating between the b and mo maize lines followed by the generation of double haploid lines, which creates highly recombinant but fixed alleles. the more than probes were designed to measure expression of the full complement of approximately maize genes. these expression level values were used as phenotypes for initial mapping analysis. while the array theoretically measures all genes, several factors must be considered: 1) the genomic origin remains unknown for a moderate number of genes; 2) probes may not be unique to individual genes but rather simultaneously measure several family members; 3) probes may not measure known genes, but rather expression originating from "non-genic" regions such as regulatory rnas. therefore, to ensure that appropriate mode of action can be assigned to eqtl, only those unique genomic and/or genic probes were considered for eqtl analysis. only the most significant eqtl was assigned for each probe, as described in methods. a genetic distance window of cm was used to define the mechanism of action for the eqtl. when the distance was less than cm between the target gene and eqtl, the mechanism of action was considered cis-acting. any eqtl greater than cm from the target was deemed trans-acting. the filtering of eqtl by p-value significance, reproducibility between replicates, and confidence in unique probe hybridization eliminated nearly 90% of the probes from further analysis, resulting in high quality eqtl. of the most significant eqtl for each probe, the majority of eqtl analyzed functioned through a cis mechanism and of those, 54% functioned with a kolmogorov-smirnov p-value of ≤ 1e- trans-acting eqtl were the minority of the eqtl identified, and of those 44% functioned with a ks p-value of ≤ 1e- altogether, these data suggest that cis-acting eqtl in general regulate gene expression with more significance than trans-acting eqtl. significant eqtl classified based on strength and trans- or cis-acting function for the confirmed eqtl identified high reproducibility of biological replicates the experimental design, utilizing a supported hydroponics system in growth chamber conditions, was intended to minimize false eqtl that resulted from environmental effects. while biological replicates were grown and harvested several months apart, the time and duration of harvest were comparable between replicates to minimize diurnal effects on transcription. reproducibility between replicates was determined by the eqtl map position identified, as well as the confidence in the eqtl. initially, eqtl were identified in the first replicate with a p-value cut-off of 1e- but were validated in replicate with a less stringent cut-off of 1e-. based on high quality eqtl identified in replicate alone,, % were validated in replicate and used for all subsequent global analysis. those eqtl that failed to validate between replicates fell into two categories: 1) non-significant p-value in replicate or 2) inconsistent map position such that the position of the strongest eqtl failed to reproduce between replicates. further investigation of the inconsistent eqtl suggests that the reasons for the inconsistency are complex and detailed analysis is required for appropriate determination of reproducibility. if several trans-eqtl in combination with a cis-eqtl contribute to expression control, it is possible that the eqtl deemed strongest in one replicate may be second strongest in another replicate, and vice versa. additionally, if the eqtl peak broadly extends across a greater than the cm interval, then significance variations between replicates may falsely suggest a failure of reproducibility for that eqtl. therefore, the actual reproducibility might be slightly higher than the number indicated. for the eqtl that mapped reproducibly it is also important to note that the significance of each eqtl was consistent as well. altogether, this suggests that the hydroponics system and analyses enriched for the identification of genetic components of gene expression regulation. cooperative regulation of gene expression previously published work suggests that the vast majority of eqtl detected will function through a trans-acting mechanism, while the strongest eqtl will function through a cis-acting mechanism. however, a breakdown of strengths and quantities of each mode of action identified using the ibm syn population showed nearly fold more cis-acting than trans-acting eqtl, yet the cis- did function with greater significance. there are several possible explanation for the discrepancy: 1) a conservative p-value cut-off of 1e- is used to minimize the number of false positive eqtl identified, however, it is expected that many trans-acting eqtl would weakly regulate gene expression, perhaps with significance less than the cut-off value, and may be lost in the analysis; 2) only the most significant eqtl was selected for each probe, which is more likely to be cis-acting; 3) the microarray probes were strongly b73-biased and any imperfect hybridization of the mo derived alleles to the probe set will map as cis-acting eqtl regardless of the genome-mediated functional expression regulation in situ; 4) the maize genome sequence is not completed. many of the genomic origins of array probes remain unidentified or are not unique within the genome, therefore the transcripts being measured by the microarray cannot be accurately assigned to the current public genome assemblies, therefore the modes of action cannot be distinguished, potentially ignoring significant trans-eqtl; 5) a relatively large window was used to assign cis-eqtl potentially incorrectly characterizing eqtl. a more comprehensive regulatory analysis can be performed in cases where multiple eqtl were identified for any particular target. the general global gene regulation analysis identifies the most significant eqtl regulating any particular gene. therefore, any secondary regulation of that gene is masked by the initial analysis. this second-level analysis enables the identification of regulatory elements that function with less significance than the primary eqtl that was initially identified. of the eqtl analyzed, the vast majority functioned independently to regulate target gene expression with the current statistic cut-off. mapped genes that were regulated by or more eqtl were further investigated. in total, eqtl were detected for target genes. the majority of the initial primary eqtl mapped as cis. an additional weaker secondary and tertiary trans-acting peaks were found to regulate those same target genes. a minority of initial primary eqtl mapped as trans-. an additional secondary and tertiary trans-acting eqtl were detected regulating those same target genes. interestingly, cis-acting eqtl were identified that function more weakly than the primary trans-acting eqtl. using the stringent ks p-value cutoff of 10- the maximum number of cooperative eqtl identified for any particular gene was this analysis demonstrates that although the strongest eqtl identified will likely function in cis, more trans-acting eqtl regulate gene expression, although with less significance. the strongest eqtl identified regulating the expression of any particular gene is considered the primary identified eqtl. up to two additional, yet weaker, functional expression regulators were identified regulating the same gene and are considered secondary or tertiary eqtl. total verifiable cis-acting eqtl identified =; total verifiable trans-acting eqtl identified =. due to the resolution of the genetic map, only one cis-acting eqtl can be identified per gene being measured, n = not possible. lack of perfect genome sequences from both parental lines caused the identification of false cis- and trans-acting eqtl while the b genome draft sequences are available, the mo genome sequence is currently in a primitive state. failure of mo sequences to hybridize to their analogous probe due to sequence polymorphisms would map as cis-acting eqtl regardless of the actual expression levels or modes of action in situ. of all the cis-eqtl, only % have higher expression levels for the mo allele, which deviates significantly from the expected 50%. this implies that about 35% of the cis-eqtl are false positives. of the most significant cis-eqtl, similar analysis indicates that 46% of them are false positives due to sequence polymorphism, to identify cases of such false positive eqtl, twenty of the strongest eqtl with unique genic/genomic probe positions in b were analyzed for sequence similarity in the mo genome. blast of those probes to the publicly available paired-end read sequences of mo generated by the joint genome institute found only two probes that match perfectly between b and mo. for the remaining eighteen b probes for which no identical mo sequences were detected, it is not clear how many are caused by the incomplete mo genome sequence and how many by sequence polymorphism. using the b reference genome, primers were designed to amplify the intervals surrounding the probe hybridization sites for four of the potentially false-positive cis-eqtl. two of the four intervals failed to amplify from mo derived genomic and cdna templates whereas they successfully amplified from b templates. without the mo reference sequence, it remains unclear if polymorphisms prevented adequate primer annealing in mo for pcr amplification. two intervals amplified successfully from mo template allowing for sequence analysis of the probe hybridization loci. comparison of the amplified sequences from mo to the b probe sequences showed that polymorphisms likely caused differential hybridization to the microarray probes. while trans- or cis-acting factors may function in the expression regulation of these genes in situ, the probe homology issues inappropriately suggest strong cis-eqtl. therefore these strong cis-eqtl are most likely false positives. despite the availability of the draft b genome sequences, incompleteness and inaccuracy in genome/bac sequencing or gene annotations remain problematic for eqtl analysis. additionally, the ancestral genome duplication in maize complicates mapping analysis when markers, genes, and genomic regions are indistinguishable. we identified a strong eqtl that functioned apparently in trans- to regulate two iron superoxide dismutase genes. initially the eqtl was regarded as a master regulator considering its ability to regulate the expression of multiple biochemically related genes. importantly, blast analysis of the microarray probes suggested that they measured their respective fesod transcripts uniquely. however further analysis of the eqtl interval and sequencing of the single bac that spanned the interval revealed a sequencing gap in the available b genomic sequences. within that gap resided a third fesod gene which the probes would recognize during microarray hybridization. rather than identifying a strong trans-acting master regulator, most likely we have inadvertently revealed an unknown gene that was differentially expressed between b and mo due to cis-acting factors, yet had high sequence similarity to other known fesods. it is obvious that some of the cis- and trans-acting eqtl identified are false due to the unavailability of complete genome sequences for both parental lines. it is not clear how extensive the problem is, although it seems the strongest eqtl are more prone to be false positive than the weaker ones. differential gene expression confirms presence/absence variations in addition to nucleotide polymorphisms or minor insertion/deletion differences between the b and mo parent genomes, several studies have identified large deletions in the mo genome as compared to b. of particular interest is a deletion spanning many bacs on the short arm of chromosome which contains at least genes and pseudogenes. eqtl mapping using the ibm syn population found strong cis-acting eqtl, measuring probes located on the bacs in question. for the genes in the region, b in general shows robust expression while mo expression is essentially off. the eqtl results are consistent with the presence/absence variations detected through array genome hybridization. identification of a trans-acting regulator by map-based cloning a strong trans-acting eqtl was selected for fine mapping and positional cloning to identify the gene responsible for expression regulation. we identified a strong eqtl located on the long arm of chromosome on ibm neighbors map )) that regulated the expression of a gene annotated as aba 8'-hydroxylase, located cm away from the eqtl near the centrosome of chromosome. within the mapping population, a b derived allele at the eqtl resulted in 6-fold greater expression of the target gene as compared to a mo derived allele at the eqtl. the recombinant individuals from the ibm syn population grown for the global eqtl analysis were used to define the genetic interval to within base pairs in a single gene. the candidate gene underlying the eqtl was determined to be a class i glutamine amidotransferase domain containing gene. sequence analysis of the b and mo alleles of the full length gene revealed multiple snps coding for five amino acid residue differences between the parents plus a residue truncation in mo as compared to b. only the residue truncation is located within the base pair eqtl interval, thus presumably the causative variation for the differentially gene expression. re-phenotyping the ibm syn population by rt-pcr specific for aba 8'-hydroxylase confirmed the gene expression results from the microarray analysis. glutamine amidotransferase genes are well characterized players in several biosynthesis pathways including the purine, pyrimidine, histidine, tryptophan, and arginine pathways, however, a transcript-abundance mediating function has never been published. while steady state transcript abundance is determined by many factors including transcription factors, enhancers, mrna degradation regulators, and rate of translation, the role of glutamine amidotransferase in aba 8'-hydroxylase transcript abundance remains elusive. despite the role that glutamine amidotransferase plays on aba 8'-hydroxylase transcription, no other eqtl mapped to the same genetic region in this population suggesting that any broad transcriptional regulatory function is unlikely. eqtl are determined by expression phenotypes, and not based on physiological or morphological phenotypes as in classic qtl identification. therefore, further analysis of aba biosynthesis in the ibm syn population could prove a physiological function downstream of transcript abundance in regards to its trans-regulation. in anticipation of aba biosynthesis analysis, the aba 8'-hydroxylase gene being regulated by glutamine amidotransferase was sequenced from b and mo cdna. interestingly, the results showed that the annotated aba 8'-hydroxylase gene was actually a product of genomic shuffling that occurred sometime prior to the genetic diversification of b and mo in both parentages, the aba 8'-hydroxylase pseudogene is a chimera of the functional maize aba 8'-hydroxylase gene fragment, non-genic genomic sequence originating from chromosome and repetitive genomic sequence. thus it seems that a pseudogene expression level is regulated. the aba 8'p is not present in all maize inbred lines. actually, validation of glutamine amidotransferase as the candidate gene for the eqtl was hampered by the absence of this pseudogene in certain lines. whether this pseudogene or its expression regulation has any physiological function remains to be determined. discussion while the field of genomics has afforded scientists with access to genomic sequences of countless numbers of model species, strains, and lines, our understanding of the function of those sequences remains rather limited. forward and reverse genetics have given meaning to sequence polymorphisms in a fair number of genes, but in addition to gene function analysis, it is equally important to understand how and when those genes are activated and the roles the translated proteins play within biochemical pathways. only through the continued efforts in the fields of transcriptomics and proteomics can the full power of genomics be realized. expression qtl studies are generating vast amounts of data from the perspective of gene regulation, both from cis-acting elements and trans-acting factors, that begin to fill in gaps in the understanding of transcriptional regulation and gene interactions. results from our study help to elucidate the genome-wide expression regulation in play during the development of crown root tips in maize. despite stringent statistical analysis, we identified more than eqtl that function through both cis- and trans-acting mechanisms. in addition to the identification of cis- and trans-factors, we described the relative regulatory contribution each of those factors plays by means of a ks p-value. despite our expectations, as well as those set forth in previously published maize eqtl studies, we identified vastly more cis-acting eqtl than trans-suggesting that the most significant eqtl will act in cis for most genes. the statistical methodology and stringency we employed was designed to minimize false positive eqtl from the analysis, however at the probable expense of the lesser significant eqtl which will most likely function in trans. additionally, the definition of trans can be "arbitrarily" set for each study. while we defined trans-acting to mean any regulatory element greater than cm from the target, others have set the boundary at cm. either limit is appropriate for maize eqtl studies but will affect the classification and quantification of trans-eqtl. among the tens of thousands of cis- and trans-acting eqtl identified in the current study, we have demonstrated that some of them are false-positives due to the lack of complete genome sequences from both parents of the mapping population. polymorphism in the microarray probe regions, which could affect hybridization intensities for mrna quantification, will lead to the occurrence of false cis-acting eqtl. likewise, sequencing gaps at the trans-acting eqtl regions could result in the detection of false trans-acting eqtl. with these mechanisms, the stronger, large-effect eqtl are more prone to be false positives than the weaker eqtl. we estimated that 35% of all the cis-eqtl is false positive, but the false trans-eqtl discovery rate is unknown in the absence of the complete genome sequences. it is likely that the whole-genome eqtl analysis reported in other systems could suffer similar false positive issues, and caution should be taken when interpreting the results and selecting eqtl for further analysis. although tens of thousands of eqtl have been reported, very few have been cloned and characterized, especially in higher organisms where positional cloning could still be challenging. to test the feasibility of eqtl cloning in maize and to understand the regulatory mechanism of transcript abundance, a strong trans-eqtl for a putative aba hydroxylase was selected for further mapping and cloning. the eqtl was fine mapped to a very small physical interval and a putative glutamine amidotransferase gene was identified as the candidate gene for this eqtl. unfortunately, the target gene identified turned out to be a pseudogene. it is not clear if this aba hydroxylase pseudogene has any physiological function, why the expression of a pseudogene is regulated, or how the two genes interact with each other. nevertheless, we have clearly demonstrated the feasibility of cloning trans-eqtl with large effect in maize. the cloning of trans-eqtl would help to understand the mechanism of transcript abundance regulation and identify regulatory genes for biochemical pathways. the ibm syn population is a set of doubled haploid lines from a randomly mated population derived from b and mo. having undergone generations of random intermating/recombination, these dh lines exhibited a high degree of phenotypic variability and high frequency of recombination. we were able to fine map an eqtl into a bp interval with only ibm syn dh lines. although recombination frequency varies widely across the genome and more genomic loci need to be analyzed before this population can be better assessed for recombination frequency, the results suggest that the ibm syn population is suitable for high resolution eqtl mapping and recombination studies. as in previous reports, we have shown that most of the strong eqtl act in cis. moreover, for the relatively small number of strong trans-eqtl detected, some of them could be false positive. therefore, it may be a challenge to identify a large number of strong trans-eqtl which are more amenable for positional cloning. a recently study in human b cells which maps the regulatory elements that influence radiation-induced changes in gene expression indicated that nearly all the strong regulators act in trans to influence the expression of their target genes. therefore, instead of mapping the steady-state level of mrna under one constant condition, mapping eqtl which regulate the differential responsiveness in gene expression to biotic and abiotic factors could be a promising approach to enrich and identify strong trans-eqtl. these trans-eqtl should be ideal targets for cloning. they are important for understanding plant responses to biotic and abiotic stresses, and technically feasible to isolate.  we have shown the feasibility of eqtl analysis as a means to identify, clone and analyze trans-acting regulatory factors through large-scale screening analysis. a glutamine amidotransferase regulator was identified as a trans-acting factor. harnessing the regulatory function of trans-acting factors could allow for better control of important agronomical genes. we also described the pitfall of identifying false-positive eqtl in the absence of complete genome sequences, which has broad implications in similar eqtl studies. 
the unexpected regulatory function of a class i glutamine amidotransferase controls the expression of an aba 8'-hydroxylase pseudogene. these microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes. a candidate gene for a trans-acting regulatory factor was identified through positional cloning. identification of a candidate gene underlying a trans-eqtl demonstrated the feasibility of eqtl cloning in maize and could help to understand the mechanism of gene expression regulation. expression qtl analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts. cases of false-positive eqtl, which results from the lack of complete genomic sequences from both parental genomes, were described. lack of complete genome sequences from both parents could cause the identification of false-positive cis- and trans-acting eqtl.
 expression qtl analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts. these microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes.  a hydroponics-based genetical genomics study in roots of a zea mays ibm syn double haploid population identified tens of thousands of cis-acting and trans-acting eqtl. cases of false-positive eqtl, which results from the lack of complete genomic sequences from both parental genomes, were described. a candidate gene for a trans-acting regulatory factor was identified through positional cloning. the unexpected regulatory function of a class i glutamine amidotransferase controls the expression of an aba 8'-hydroxylase pseudogene.  identification of a candidate gene underlying a trans-eqtl demonstrated the feasibility of eqtl cloning in maize and could help to understand the mechanism of gene expression regulation. lack of complete genome sequences from both parents could cause the identification of false-positive cis- and trans-acting eqtl. 