BACKGROUND
seed protein and oil are crucial to the value of many crop species.
during seed development, carbon and nitrogen are partitioned among protein, oil, and carbohydrates  <cit> .
in legumes, particularly soybean  merrill), protein and oil are primary nutritional components of mature seed.
protein and oil comprise some 40% and 20%, respectively, of soybean seed.
protein meal is a major byproduct of soybean processing, and high seed protein content allows processors to derive meal with high nutritional value  <cit> .
a better understanding of the genetic basis of seed protein variation is important for developing strategies to improve seed quality traits not only in soybean but also in other legumes and cereal grains.
storage reserves account for the majority of the protein in the seed  <cit> .
the period of seed development where these reserves accumulate is commonly referred to as the seed filling stage, a 4- to 5-week period of cell expansion that occurs once cell division is complete  <cit> .
the most prevalent seed storage proteins in soybean are beta-conglycinin and glycinin  <cit> .
a number of diverse and interlinked processes, including photosynthesis, sucrose signaling, and transport, are associated with seed development and the regulation of complex traits  <cit> .
genetic control of seed constituents and size is inherited in a quantitative manner.
many quantitative trait loci  associated with seed protein and size have been identified in several species including wheat  <cit> , arabidopsis  <cit> , rice  <cit> , pea  <cit> , and barley  <cit> .
in soybean, numerous qtls associated with protein have been identified  <cit> .
the seed protein qtl mapped to soybean linkage group i  is of particular interest due to the large additive effect that accounts for its consistent detection in many soybean mapping populations  <cit>  and across multiple environments  <cit> .
inheritance of the high protein allele from g. soja at lg i resulted in a seed protein increase of  <dig> to  <dig> g/kg, and this increase was also associated with lower oil concentration  <cit> ; a negative phenotypic correlation between soybean seed protein and oil content is well documented  <cit> .
nichols et al.
<cit>  fine mapped the lg i protein qtl region to a  <dig> cm interval using bc5f5-derived near-isogenic lines  contrasting in seed protein and oil.
although linkage analysis is a valuable tool for localizing genetic regions of interest for a trait, the capabilities of mapping can be greatly enhanced by genomic approaches to identify genes that may control these traits.
analyses of transcript profiles by microarrays have provided insight into the genes and processes involved in developing seed of arabidopsis  <cit> , soybean  <cit> , medicago truncatula  <cit> , wheat  <cit> , barley  <cit> , and rice panicles  <cit> .
transcript changes, especially when used to contrast nils, have proven useful for the discovery of genes of interest in soybean and other species  <cit> .
in the present study, we leveraged a combination of resources - a nil pair that differed substantially in seed protein  <cit> , transcript profiling by affymetrix® soy genechip microarray, illumina® high-throughput transcriptome sequencing platforms, and the newly available soybean genome sequence--to assess genomic and genetic contributions to seed protein traits in soybean.
the objectives of our study were to: 1) define the borders of the genomic segment encompassing the lg i protein qtl region, 2) characterize transcript accumulation in the developing seed of a nil pair known to produce contrasting final seed protein content, and 3) identify candidate genes for this seed protein qtl.
the accomplishment of these objectives constitutes the first step toward understanding the genetic and molecular mechanisms underlying the regulation of seed protein.
in addition, the large dataset provided through this study is a valuable tool for further analysis of the soybean transcriptome.
RESULTS
demarcation of the qtl region
previous genetic studies  <cit>  localized the lg i protein qtl region to a  <dig> cm interval.
nil populations used to map the lg i protein qtl were created by backcrossing the high protein g. soja  allele into a g. max background   <cit> .
the nil population p-c609-45- <dig> was found to segregate for the smallest lg i qtl interval corresponding to high and low seed protein phenotypes in the field  <cit> .
in this study, these nils were used to link the genetic map  to the physical map  and to identify recombination break points in p-c609-45- <dig> to demarcate the protein qtl region .
to obtain a physical map of the protein qtl region, bac  libraries of soybean genomic dna were scanned for alignment to known markers, and a bac-based physical map was assembled to span markers satt <dig> and satt <dig> .
this bac-based map accounted for approximately  <dig>  mb of the qtl region.
newly derived ssr  markers from the bac sequence that were polymorphic between a81- <dig> and pi <dig> were screened to determine if they segregated in the p-c609-45- <dig> population.
because the introgressed qtl-containing segment was segregating in the p-c609-45- <dig> population, markers located in that region were expected to segregate in the population.
upon release of the soybean whole genome sequence, alignment of bac sequences to the soybean whole genome assembly  identified chromosome  <dig> as the best match to all the bacs in the lg i protein qtl physical map.
the order of bac sequence alignment to chromosome  <dig> was in agreement with the physical map .
forty-eight ssr markers , including  <dig> ssr markers  derived from the bac sequences and from the whole genome sequence spanning the qtl region plus six previously genetically mapped ssr markers  <cit> , were screened for segregation as described above.
thirty-four of the  <dig> ssr markers derived in this study segregated in the p-c609-45- <dig> population.
the high and low protein phenotypes of the segregating progeny corresponded to the expected parental marker alleles originating from the high and low protein parents  <cit> .
the protein qtl region was delineated to approximately  <dig>  mbp of genomic sequence between sat_ <dig> and ssrpqtl_ <dig>  the two closest non-segregating ssr markers flanking the left and right borders of the protein qtl region on chromosome  <dig> .
the coordinates of the borders stretch from  <dig>  mb to  <dig>  mb on chromosome  <dig>
phenotypic evaluation of seed protein and oil in nils
a nil pair derived from the p-c609-45- <dig> population was chosen for further study.
one line  retained the g. max  background at the lg i protein qtl region, and the other  inherited the high protein allele in that region from g. soja .
the protein and oil phenotypes in the nil pair were evaluated at four stages of seed fill .
these four stages during seed fill were defined by seed size and were harvested at the same time during the r <dig> stage of development from the same plants for direct comparison.
stage one corresponded to a seed size of  <dig> to  <dig> mg, stage two to greater than  <dig> to  <dig> mg seed, stage three to greater than  <dig> to  <dig> mg seed, and stage four to greater than  <dig> to  <dig> mg seed.
at stage one, seed organs and tissues are formed but have yet to increase in cell size .
it is noteworthy that seed protein differences between lopro  and hipro  genotypes were apparent at the earliest stage of evaluation .
moreover, that difference remained consistent through the subsequent stages.
seed oil values, however, did not show as marked a contrast in the early stages .
the protein and oil phenotypes for the nils at seed maturity were consistent with the previously reported values .
transcript accumulation changes during seed fill
to examine transcript accumulation changes during seed fill, transcript profiles were evaluated in seeds of each genotype  from the four stages above by soy genome affymetrix® genechip analyses.
out of  <dig>  soybean probesets on the genechip, 64-69% were defined as 'present' in three out of three replicates by mas <dig> analysis of the various seed stages in both genotypes.
these detection figures are comparable to those found in seed microarray studies of other species  <cit> .
differences in the transcriptomes of the nil pair may reflect or affect the high and low protein and oil phenotypes seen in the lines.
using student's t-test to evaluate significance, affymetrix® genechip probesets with at least  <dig> -fold change between stages were identified at an fdr  of less than 5%  <cit> .
transcript accumulation changes across stages were evaluated with reference to the stage one profiles .
in both genotypes, no probesets from the stage two versus stage one comparison qualified under the fdr <  <dig>  criterion, so this comparison was excluded from further analysis.
the number of probesets representing differentially accumulated transcripts with higher accumulation in stage three compared to stage one was greater in hipro than in lopro , and this difference was again apparent between stages four and one  .
analysis of all probeset expression changes revealed that  <dig> % of the genes that significantly increased in expression over time in either genotype were shared between lopro and hipro .
transcripts common to both genotypes that increase significantly in stage four seed as opposed to stage one seed include: beta-conglycinins and glycinins, sucrose binding proteins, heat shock chaperonins, late embryogenesis messages, seed maturation proteins, glutathione s-transferases and peroxidases, iron binding and flavonoid synthesis proteins, and numerous transporters.
interestingly,  <dig> transcripts with ubiquitin-related annotations were found to increase in accumulation over time in both genotypes while three were found to decrease in accumulation levels in both genotypes .
it is noteworthy that some  <dig> transcription factor messages showed enhanced abundance at stage three or four versus stage one seed.
of the genes that decreased in expression over time,  <dig> % of these genes were shared between lopro and hipro .
transcripts common to both genotypes that were reduced in abundance at stage four as opposed to stage one include genes involved in flavonoid metabolism, cell wall deposition, kinases , response to arachidonic acid, strictosidine synthesis, and disease resistance response.
twenty transcription factor annotations were common to both lines and displayed reduced abundance by stage three or four.
during seed development, the synthesis of seed storage products is coordinated with carbohydrate and nitrogen metabolic processes involving many transporters  <cit> .
some  <dig> transport-related transcripts increased in abundance in both genotypes, including gene transcripts annotated as ammonium, sugar, metal, and ion transporters .
meanwhile, some  <dig> transport-related transcripts decreased in accumulation levels in both genotypes, and these included transcripts annotated as ammonium, sugar, and abc transporters .
a high number of microtubule-related gene transcripts were also found to decrease in abundance, supporting a role for fundamental transport mechanisms  <cit>  and the slowing of cell expansion  <cit>  during these stages of seed development.
eleven microtubule-related transcripts, including those involved in activity and movement, were found to decrease in abundance versus four microtubule-related transcripts that increased in abundance in both genotypes .
cyclin-related transcripts were also found in both genotypes.
it is interesting to note that of the transcripts directly associated with cell division cycle annotations, those that increased in abundance included transcripts for cdc <dig> and five transcripts annotated as tyrosine kinase specific for activated  p21cdc42hs .
those that decreased in abundance included transcripts for cdc <dig> and cdc <dig> .
at least one transcript related to cdc <dig> was found to accumulate in both directions for both genotypes over time .
sucrose is well known for its many roles during seed development  <cit> .
sixteen transcripts with sucrose-related annotations were found to increase in accumulation in both genotypes, and these annotations included sucrose-binding protein and sucrose degradation and transport-related genes .
this number is in contrast to the five sucrose-related transcripts that were found to decrease in accumulation in both genotypes and that included sucrose degradation and sucrose response genes .
variation in transcriptome abundance profiles revealed differences between the two genotypes that may relate to their phenotypes.
tables  <dig> and  <dig> show the  <dig> transcripts that were most enhanced in abundance from each genotype in stage four seed versus stage one seed.
overall, hipro possessed  <dig> transcripts with greater than four-fold abundance in stage four versus stage one seed, compared to  <dig> transcripts in lopro.
in addition, the hipro line showed more than five times greater maximum fold change differences between stage four and stage one.
hipro revealed a striking abundance of transcripts related to protein accumulation, iron sequestration, sucrose binding, and seed maturation .
by comparison, the greatest abundance of transcripts in lopro related to chaperonin heat shock protein, peptide transporter kinases, and glutathionine s-transferase .
interestingly, transcripts related to chloroplast function were greatly reduced in lopro in both abundance and unique representation in comparison to hipro .
transcripts with accumulation changes were also assigned to gene ontology categories, and gene categories that were enriched under each condition within each genotype were identified .
transcripts for specific genes were also examined closely.
the effect of dof transcription factors on seed oil regulation have been previously documented  <cit> , where gmdof <dig> and gmdof <dig> were found to contribute to high seed oil phenotypes in arabidopsis.
in our study, dof <dig> and dof <dig> genes were upregulated in the hipro soy line, but no significant difference was seen in the transcript abundance for dof <dig> and dof <dig> in either genotype .
differentially accumulated transcripts between nils identified by microarray
direct comparisons of transcript accumulation between the two genotypes showed few significant differences by soy genome affymetrix® genechip analyses.
differentially expressed transcripts between the two genotypes were detected using student's t-test.
at a false discovery rate of 5% or less  <cit> , only  <dig> affymetrix® probesets displayed at least  <dig> -fold change between the two genotypes lopro and hipro .
strikingly, six probesets were detected at greater than four-fold change between the two genotypes .
examination of the six probesets above revealed that they likely represent three genes according to est and genbank data.
these three genes are labeled as pqi <dig>  pqi <dig>  and pqi <dig> .
all six of the probesets with the greatest fold change were detected as transcripts with greater abundance in lopro than in hipro at all four stages .
probesets representing transcripts with greater abundance in hipro than in lopro also existed .
each affymetrix® probeset identifier  is shown with corresponding normalized expression values for lopro and hipro and the ratio of mean lopro divided by mean hipro values.
criteria for the list: fdr <  <dig>  and fold-change >  <dig> .
the uniprot description for each affymetrix® id is accompanied by the e-value for the alignment; na = not applicable, no significant alignment.
an n-way anova test was also conducted to examine transcript accumulation differences simultaneously across multiple factors, genotype, and time  within the genotype.
at fdr <  <dig>   <cit> , a total of  <dig> soy affymetrix® probesets were detected with differential changes in transcript accumulation using this method .
interestingly, five transcription factor-related transcripts, annotated as bzip, ethylene-responsive, or heat shock, were detected with differential accumulation patterns .
again, the six probesets with the most highly differential accumulation values were represented .
because the affymetrix® genechip analysis was performed using transcripts from two different genotypes, the possibility of the presence of feature polymorphisms in the transcripts that could alter probe to transcript affinity was high.
therefore, single feature polymorphism  analysis  <cit>  was performed using the affymetrix® genechip data and an algorithm based on the li-wong model  <cit>  combined with a modified probe level statistical method  <cit> .
sfp analysis of the three genes above showed large affinity differences to multiple probes on the affymetrix® genechip .
these three genes were potentially polymorphic in one or more regions between the two genotypes or completely absent in one genotype.
to further validate the microarray data, quantitative reverse transcriptase-polymerase chain reaction  was performed.
specific primers were designed for the three genes and an actin control.
significant differences between lopro and hipro were observed for pqi <dig> and pqi <dig> .
however, no significant transcript level fold changes were observed for pqi <dig> .
thus, only two of the three genes identified as upregulated in lopro in prior analyses were determined to display differentially accumulating transcripts between the two genotypes by qrt-pcr.
genes with differentially accumulated transcripts between nils map to the lg i protein qtl
the three most highly differentially accumulating transcripts identified by affymetrix® genechip were aligned to the soybean genome sequence  and found to reside within the borders of the protein qtl region on chromosome  <dig>  .
even though only two of the three were confirmed to accumulate differential levels of transcripts, allelic differences at the segregating qtl region are a potential source for polymorphisms between the two genotypes that could also result in a candidate gene.
three additional differentially accumulating transcripts identified by affymetrix® genechip also mapped to the qtl region, one within  <dig> kb of pqi <dig> .
thus,  <dig> of the  <dig> differentially accumulating transcripts identified by affymetrix® genechip  resided within the defined boundaries of the protein qtl region at lg i.
transcripts identified by n-way anova  were aligned to the genome sequence to show the range and distribution along the soybean chromosomes .
the soybean genome sequence reveals a general bias toward gene-rich chromosome ends  <cit> , a phenomenon that has been observed in other plant genomes  <cit> .
however, a striking concentration of probes  mapped to chromosome  <dig> at the protein qtl region .
the presence of differentially accumulating transcripts in this region is consistent with the development of a near-isogenic line pair that displays variation in seed protein phenotype and segregation of markers within the protein qtl region.
recently, wei et al.
<cit>  also performed a transcriptome analysis using rice superhybrid lyp <dig> and mapped differentially expressed genes to yield-related qtls in the rice genome.
differentially accumulated transcripts between nils identified by high-throughput transcriptome sequencing
because the soy genome affymetrix® genechip does not represent the complete set of soybean genes, high-throughput transcriptome sequencing  was performed to confirm the microarray data and search for additional candidate genes.
using the same rna samples prepared for microarray analysis as templates for high-throughput deep sequencing, more than  <dig> million reads were sequenced, each  <dig> or  <dig> nucleotides in length, using the illumina® genome analyzer.
sequences were generated from random priming sites within transcript cdna from each of the four stages in lopro and in hipro, producing more than  <dig> million reads per stage.
of these reads, more than  <dig> million aligned uniquely to the genome sequence.
the soybean genome sequencing consortium predicted  <dig>  gene models and  <dig>  additional transposon-like gene models  <cit> .
from that initial set of gene models, the consortium identified  <dig>  "high-confidence" genes.
in the current seed nils sequencing effort,  <dig>  of the  <dig>   of the high-confidence genes show evidence of expression.
an additional  <dig>  predicted genes not in the high-confidence set show evidence of expression from the seed nils data.
twelve differentially accumulated transcripts between lopro and hipro were identified within the lg i protein qtl region with at least a two-fold change in expression at a p <  <dig>  using htts .
putative genes were annotated and compared with plant est and genbank data sets .
to further validate the htts data, quantitative reverse transcriptase-polymerase chain reaction  was performed.
specific primers were designed for four genes from table  <dig> with no available corresponding microarray data.
examination of three genes, glyma <dig> g <dig>  glyma <dig> g <dig>  and glyma <dig> g <dig>  by qrt-pcr confirmed higher transcript accumulation levels in lopro versus hipro , although the standard deviation among biological replicates in glyma <dig> g <dig> was high.
analysis by qrt-pcr also confirmed higher transcript accumulation levels of glyma <dig> g <dig> in hipro versus lopro.
the sequence identifier  is shown for each numbered gene candidate with differentially accumulated transcripts between genotypes at the lg i protein qtl region.
transcript sequencing read counts for lopro and hipro are reported along with the sequence location for the closest predicted gene.
the blastp description is reported for each gene at e-value < 10- <dig>  na = not applicable.
affymetrix® genechip vs. illumina® high-throughput transcriptome sequencing analysis
close comparison of the transcripts identified by htts  showed the presence of the two most highly differentially accumulated transcripts identified by affymetrix® genechip analysis .
examination of the coordinates of the most highly differentially accumulated transcripts revealed a distance of  <dig>  mb between pqi <dig> and pqi <dig> .
however, the positioning of the soybean target sequence from the affymetrix® genechip for these genes did not directly conform to the predicted gene models in the soybean genome .
interestingly, two pairs of transcripts identified from the illumina® deep sequencing analysis  appeared in the same region with overlapping chromosome coordinates but on opposite strands.
transcripts with sequence homology to known proteins included an ethylene receptor and a glutamyl-trna synthetase that presented differentially accumulated transcripts at only one stage, as well as a putative ammonium transporter .
examination of the available affymetrix® soy genechip target equivalents that overlapped the region, however, did not provide support for the ethylene receptor and ammonium transporter transcript accumulation differences .
in all, the union of affymetrix® soy genechip and illumina® deep sequencing transcriptome data yielded  <dig> genes with differentially accumulating transcripts that mapped to the protein qtl region at lg i on chromosome  <dig> .
genome-wide gene expression coverage
from htts of the near-isogenic line pair, a large amount of data was obtained.
uniquely mapped read counts for each genotype at each stage are provided for comparison of transcript accumulation levels at each gene within the defined boundaries of the lg i protein qtl region .
this list excludes genes annotated as transposon-related.
out of  <dig> genes on chromosome  <dig> at the lg i protein qtl region,  <dig> showed evidence of expression during the seed fill stages examined in this study.
the  <dig> genes in the lg i protein qtl region with the most transcript read counts are listed here .
additional file  <dig> lists all  <dig> genes in order of total read abundance.
listed in this table are the  <dig> genes with the most number of uniquely mapped reads contributing to transcript counts .
a <dig> to a <dig> correspond to lopro stages one to four, and b <dig> to b <dig> correspond to hipro stages one to four.
na = not applicable, no significant alignment
all htts transcript profiles from this study, for all predicted soybean genes, are available at http://soybase.org/gbrowse.
two gbrowse annotation tracks provide information on transcript read coverage and location .
a "seed development coverage depth" track  shows locations and counts of uniquely mapped htts reads, and a "seed development transcript count" track  shows a colored histogram of relative read accumulation counts in each of the eight libraries in this study: a <dig> to a <dig> correspond to lopro stages one to four, and b <dig> to b <dig> correspond to hipro stages one to four.
histograms for each gene are centered under their corresponding gene model.
an example of a screenshot depiction of transcript read accumulation coverage is shown for a gene at the lg i protein qtl region, glyma <dig> g <dig>  .
transcript coverage for glyma <dig> g <dig> is consistent with the predicted gene model.
discussion
seed protein and oil relationships
it has long been documented that seed protein and oil content are inversely correlated in the soybean seed  <cit> .
low oil alleles are consistently cotransmitted with high protein alleles in many instances  <cit> , and attempts to separate these two traits through chromosomal recombination in the nils used in this study have not been successful  <cit> .
it has been hypothesized that this relationship may be due to either very tight linkage or pleiotropic effects  <cit> .
whether one phenotype directly or indirectly results in the other is unknown, and the timing of events regarding differential accumulation of contrasting protein and oil levels in the seed is uncertain.
gmdof <dig> and gmdof <dig> transcription factors, however, have been reported to activate genes involved in lipid biosynthesis and simultaneously suppress the expression of storage protein genes  <cit> .
transcription factors have also been shown to influence seed traits in other studies.
for example, the putative ap2/erebp transcription factor wrinkled <dig> was found to be involved in the regulation of seed oil accumulation in arabidopsis  <cit> , and a qtl encoding a nac transcription factor was found to control grain protein and leaf senescence in wheat  <cit> .
in addition, seed mass in arabidopsis has been shown to be regulated by the apetala <dig>  class of transcription factors  <cit> .
verdier et al.
<cit>  evaluated the expression of transcription factors throughout seed development of medicago truncatula.
they found some  <dig> transcription factors were expressed equally throughout seed development while  <dig> had differential expression at one or more stages.
cluster analysis demonstrated six different clusters of transcription factor genes that corresponded to the developmental stages evaluated.
many of the  <dig> transcription factors that were found to be upregulated in this study during seed development of the soybean nils were similar to those described by verdier et al.
<cit> .
transcriptional suppression of some aspect of seed protein accumulation could be envisioned for the low protein/high oil nil homozygous for the g. max allele of the lg i qtl.
however, transcriptional suppression of seed oil accumulation in the nil homozygous for the g. soja allele  would be envisioned to occur in a time frame late in seed fill.
this assumption is due to the observation that the rate of seed oil accumulation in hipro did not differ from that of lopro until the last stage of seed fill .
although hipro matures slightly earlier and generally yields less seed than lopro  <cit> , these differences do not fully account for the striking differences in nil seed protein content observed at the early stages of seed fill.
whether additional differences in the morphology or composition of the seed exist between the near-isogenic lines remains to be seen.
further detailed investigation is in progress to study the temporal and spatial distribution and partitioning of candidate gene expression that may govern the relationship between protein and oil accumulation in the developing soybean seed.
processes and pathways influencing seed content
comprehensive evaluation of seed transcripts through microarray analyses have been reported for arabidopsis  <cit> , medicago truncatula  <cit> , barley  <cit> , and wheat  <cit> .
these studies, in common, report differential expression of hundreds of genes at one or more stages of seed development involved in processes related to carbon and nitrogen metabolism, protein processing, transport of nutrients, organ development , signal transduction, and phytohormone balance.
the transcript accumulation patterns we observed during nils seed fill by genechip® microarray data were consistent with these studies.
prior studies have demonstrated the transcription and accumulation of both mrna and protein for beta-conglycinin and glycinin genes during the seed fill stage of seed development  <cit> .
transcripts for these seed storage proteins were identified during seed fill with particular abundance in the hipro line .
additional classes of genes with roles in seed development and maturation, flavonoid metabolism, and sucrose binding were also identified.
proteome analyses of the seed filling stages in soybean have provided support for the presence of gene transcripts found in this study with the detection of proteins associated with protein destination and storage, metabolism, and disease/defense  <cit> .
expression of different protein isoforms have been shown to display different accumulation trends, and the activities of many genes may have multiple roles during seed filling.
this phenomenon may be reflected in the increase and decrease in accumulated transcripts of lipoxygenase-related genes in this study, consistent with proteomic data on various lipoxygenases in the developing soybean seed  <cit> .
carbon metabolism directed toward oil and protein deposition plays an important role in seed quality.
changes in seed protein or oil in many plant species have been linked to the activity of acetyl-ca carboxylase   <cit>  and phosphoenolpyruvate carboxylase   <cit> .
recent proteomic and microarray studies have shown the presence of peptides and transcripts for both enzymes during seed development  <cit> .
overexpression of arabidopsis acetyl-coa carboxylase led to increased oil content of brassica napus seeds  <cit>  and potato tubers  <cit> .
the acetyl-coa carboxylase gene has also been associated with a major groat oil content qtl  <cit> .
in addition, inhibition of plastid acetyl-coa carboxylase resulted in lower seed oil  <cit> .
in soybean, a significant correlation was found between phosphoenolpyruvate carboxylase activity and seed protein and oil concentrations  <cit> , although this correlation was found to be higher for seed protein.
furthermore, overexpression of phosphoenolypruvate carboxylase in vicia narbonensis seed was shown to increase seed storage capacity and protein content  <cit> .
although we found no significant differences in transcript expression of accase and pepc between nils, we observed that transcripts corresponding to several forms of accase and pepc were expressed at all stages of seed development in this study .
interestingly, some forms of accase were expressed at higher levels in the seed than others.
such data may reflect the importance of enhanced isoforms of accase and pepc in seed development compared to isoforms expressed elsewhere in the plant.
impaired storage metabolism has been linked with decreased sucrose levels  <cit> , and sucrose may affect carbon flux at the transcriptional or post-transcriptional levels  <cit> .
studies have shown the importance of photosynthesis in seed filling metabolism  <cit>  and for the biosynthesis of seed storage products  <cit>  consistent with the wide array of photosynthesis-related genes detected during seed fill in this study.
regulation of protein destination, storage, and proteolysis, as well as metabolic and photosynthetic pathways, may contribute to the contrasting seed phenotypes seen in the nil pair.
additional transcript accumulation changes have been documented during seed development.
a heat shock protein and peptide transporter were among the annotations of the transcripts with the greatest fold change increases from stage one to stage four in lopro .
both a peptide transporter and heat shock-related proteins were previously found to increase dramatically during seed development in a high oil soybean line  <cit> .
down-regulation of lipoxygenases and sucrose udp-glycosyltransferase during seed development in a high oil soybean line of a previous study  <cit>  is also consistent with the detection of down-regulated lipoxygenase and udp-glycosyltransferase transcripts in lopro .
the transcription accumulation patterns of these genes may be a feature common to soybean lines with high oil phenotypes.
candidates for regulation of seed protein and oil
we identified  <dig> genes mapping to the protein qtl region at lg i that may play a role in the regulation of seed protein and oil.
thirteen of these  <dig> genes displayed differentially accumulating transcripts.
of these  <dig>   <dig> were found at high levels in the low protein line with low or no detectable levels in the high protein line.
based on sequence homology searches to protein databases, these candidates include a potential regulatory protein in the mov34- <dig> family, a heat shock protein hsp <dig> , and an atp synthase .
although the mov34- <dig> candidate appeared to possess versatile domains for the potential regulation of multiple processes, transcripts isolated from this candidate region contained numerous stop codons, raising the possibility of non-coding genes.
the same was true for a number of the other candidates and may account for the high percentage of genes with no significant e-value returns to the uniprot protein database  <cit> .
there is increasing evidence for the role of riboregulators, either as long non-protein coding rnas or processed into small rnas in plant development  <cit> , and these molecules may play a role in seed protein and oil accumulation.
two pairs of genes among the candidates  were found to possess overlapping transcripts; one possibility is that these overlapping transcripts form double-stranded rnas that may be processed into small rnas  <cit> .
evidence for the expression of heat shock proteins during the stress-independent development of the seed has previously been observed  <cit> .
interestingly, heat shock protein genes were found to be expressed at higher levels in the low protein line of a near-isogenic line pair in barley  <cit> , a phenomenon also observed in the lopro line of this study.
previous studies have detailed an indirect relationship among the accumulation of storage proteins, lipid biosynthesis, and photosynthesis in the seed, correlating to the availability and distribution of atp  <cit> .
further investigation into the modulation of atp synthase levels on energy status and storage product accumulation in the soybean seed will shed light on the potential role for atp synthase as a candidate gene.
currently, the occurrence of additional candidate genes from even earlier stages of seed development is being evaluated through differential analysis of transcriptome profiles of the near-isogenic line pair.
potential modes of regulation for seed protein and oil
the lopro line was converted into the hipro line upon inheritance of a g. soja allele at the lg i protein qtl region.
however, the lopro line is also the high oil line, and a number of scenarios may explain how gene expression differences relate to variation in protein and oil phenotypes in the seed.
protein content may be positively regulated by the expression of a gene that increases protein production in hipro.
alternatively, protein content may be negatively regulated by expression of a gene in lopro that inhibits or reduces protein accumulation and thus allows for increased oil accumulation.
significant protein differences would then be observed at an earlier stage than oil differences, as in figure  <dig>  inhibition of protein accumulation could take place at many levels, including transcriptional and post-transcriptional control and regulation of protein synthesis, transport, and turnover.
the presence of candidate genes with non-coding segments raises the possibility of regulation at the transcriptional level that may affect the transcription of genes outside the list of candidates shown in this study.
differences in transcriptome profiles may correlate directly or indirectly with the differences in protein and oil accumulation between the nils.
previous studies have shown that seed storage proteins are largely controlled by transcriptional regulation during the seed fill stage .
extensive analysis of cis-regulatory elements of seed storage proteins has demonstrated interaction of these elements with bzip and myb factors  <cit> .
transcription of a candidate gene in lopro may result in negative regulation of transcriptional regulators or key factors involved in high protein accumulation.
the presence of sequence polymorphisms in gene sequences or promoter regions within the segregating region of the protein qtl may account for the low or absent levels of differentially accumulating gene transcripts in hipro versus lopro .
in an alternative scenario, oil content may be regulated.
gene expression or transcript accumulation leading to a higher oil phenotype may act in concert with other factors to directly or indirectly lead to reduced protein accumulation.
genes regulated by transcription factors could initiate this effect.
in support of this model, batch analysis of the promoter regions of the genes with the greatest differentially accumulated transcripts between the nils revealed a number of transcription factor binding sites and seed-specific motifs .
a regulatory factor expressed in the high oil lopro line may activate higher oil synthesis or accumulation pathways.
this is consistent with the greater abundance of candidate gene transcript accumulation seen in lopro .
inheritance of a g. soja allele that does not allow for expression or accumulation of the high oil gene could account for the low oil and high protein phenotype in hipro.
utility of the htts dataset for understanding the soybean genome
although we focused on the transcripts derived from the lg i region of the genome in this study, the high-throughput transcriptome sequencing data set we obtained compiles greater than  <dig> million reads and  <dig>  ×  <dig> nucleotides of transcript data and is an excellent resource for increasing our understanding of the soybean genome.
the use of htts in conjunction with microarrays allowed us to detect a more comprehensive set of soybean gene transcripts.
our observation that 86% of gene transcripts in soybean were present during seed development greatly extends previous microarray-dependent seed development studies.
recent reports demonstrate the value of high-throughput transcriptome sequencing in eukaryotes for identification of novel transcripts and transcript isoforms, untranslated regions, and gene structures, leading to improved genome annotation  <cit> .
for the soybean genome, current gene models using the 8× genome sequence assembly  were predicted based on protein coding sequences.
by comparison, our transcriptome dataset encompasses both protein coding and non-protein coding sequences and will be useful for identification of transcripts outside of gene models.
analyses of our dataset also show evidence for the existence of novel transcript isoforms, including alternative splicing, between genotypes and among seed stages .
moreover, beyond the detection of feature polymorphisms reported here, a comparative analysis of common transcripts between soybean lines will provide a multitude of single nucleotide polymorphisms useful in following agronomic traits in breeding populations.
currently we are analyzing high-throughput sequencing of transcripts from many soybean tissues.
that data, along with the seed transcriptome data, will compile an atlas of gene expression for soybean.
CONCLUSIONS
this study provided the rare opportunity to intersect structural mapping and molecular profiling studies.
here, we compared the transcript abundance profiles of the developing seed from a soybean nil pair with contrasting seed protein and identified gene candidates at the lg i protein qtl for potential involvement in the regulation of protein content in the soybean seed.
the entire transcriptome sequencing dataset generated from this study is also provided as a valuable resource.
control of protein and oil accumulation in the seed occurs at many different levels and is likely influenced by more than one gene.
of the candidates genes identified in this study, any combination could be responsible for the observed change in protein and oil and phenotypes conditioned by the alleles of the lg i qtl.
other protein/oil qtls have been identified in qtl mapping studies, but the lg i qtl is of great interest because its additive effect on seed protein and oil is the largest of any qtl identified to date.
the models presented here are compatible with the role of additional genes and pathways as well as mixed models for control of seed protein and oil.
resources that include the availability of additional recombinants and the use of markers derived from this study will allow for further demarcation of the qtl region.
further studies are being conducted on additional mapping populations to dissect the relationship between protein and oil levels, and functional studies are under way to identify and validate the role of candidate genes in the accumulation of protein and oil in the seed.

 seed protein and oil are crucial to the value of many crop species. during seed development, carbon and nitrogen are partitioned among protein, oil, and carbohydrates. in legumes, particularly soybean merrill), protein and oil are primary nutritional components of mature seed. protein and oil comprise some 40% and 20%, respectively, of soybean seed. protein meal is a major byproduct of soybean processing, and high seed protein content allows processors to derive meal with high nutritional value. a better understanding of the genetic basis of seed protein variation is important for developing strategies to improve seed quality traits not only in soybean but also in other legumes and cereal grains. storage reserves account for the majority of the protein in the seed. the period of seed development where these reserves accumulate is commonly referred to as the seed filling stage, a 4- to 5-week period of cell expansion that occurs once cell division is complete. the most prevalent seed storage proteins in soybean are beta-conglycinin and glycinin. a number of diverse and interlinked processes, including photosynthesis, sucrose signaling, and transport, are associated with seed development and the regulation of complex traits. genetic control of seed constituents and size is inherited in a quantitative manner. many quantitative trait loci associated with seed protein and size have been identified in several species including wheat, arabidopsis, rice, pea, and barley. in soybean, numerous qtls associated with protein have been identified. the seed protein qtl mapped to soybean linkage group i is of particular interest due to the large additive effect that accounts for its consistent detection in many soybean mapping populations and across multiple environments. inheritance of the high protein allele from g. soja at lg i resulted in a seed protein increase of to g/kg, and this increase was also associated with lower oil concentration; a negative phenotypic correlation between soybean seed protein and oil content is well documented. nichols et al. fine mapped the lg i protein qtl region to a cm interval using bc5f5-derived near-isogenic lines contrasting in seed protein and oil. although linkage analysis is a valuable tool for localizing genetic regions of interest for a trait, the capabilities of mapping can be greatly enhanced by genomic approaches to identify genes that may control these traits. analyses of transcript profiles by microarrays have provided insight into the genes and processes involved in developing seed of arabidopsis, soybean, medicago truncatula, wheat, barley, and rice panicles. transcript changes, especially when used to contrast nils, have proven useful for the discovery of genes of interest in soybean and other species. in the present study, we leveraged a combination of resources - a nil pair that differed substantially in seed protein, transcript profiling by affymetrix® soy genechip microarray, illumina® high-throughput transcriptome sequencing platforms, and the newly available soybean genome sequence--to assess genomic and genetic contributions to seed protein traits in soybean. the objectives of our study were to: 1) define the borders of the genomic segment encompassing the lg i protein qtl region, 2) characterize transcript accumulation in the developing seed of a nil pair known to produce contrasting final seed protein content, and 3) identify candidate genes for this seed protein qtl. the accomplishment of these objectives constitutes the first step toward understanding the genetic and molecular mechanisms underlying the regulation of seed protein. in addition, the large dataset provided through this study is a valuable tool for further analysis of the soybean transcriptome.  demarcation of the qtl region previous genetic studies localized the lg i protein qtl region to a cm interval. nil populations used to map the lg i protein qtl were created by backcrossing the high protein g. soja allele into a g. max background. the nil population p-c609-45- was found to segregate for the smallest lg i qtl interval corresponding to high and low seed protein phenotypes in the field. in this study, these nils were used to link the genetic map to the physical map and to identify recombination break points in p-c609-45- to demarcate the protein qtl region. to obtain a physical map of the protein qtl region, bac libraries of soybean genomic dna were scanned for alignment to known markers, and a bac-based physical map was assembled to span markers satt and satt. this bac-based map accounted for approximately mb of the qtl region. newly derived ssr markers from the bac sequence that were polymorphic between a81- and pi were screened to determine if they segregated in the p-c609-45- population. because the introgressed qtl-containing segment was segregating in the p-c609-45- population, markers located in that region were expected to segregate in the population. upon release of the soybean whole genome sequence, alignment of bac sequences to the soybean whole genome assembly identified chromosome as the best match to all the bacs in the lg i protein qtl physical map. the order of bac sequence alignment to chromosome was in agreement with the physical map. forty-eight ssr markers, including ssr markers derived from the bac sequences and from the whole genome sequence spanning the qtl region plus six previously genetically mapped ssr markers, were screened for segregation as described above. thirty-four of the ssr markers derived in this study segregated in the p-c609-45- population. the high and low protein phenotypes of the segregating progeny corresponded to the expected parental marker alleles originating from the high and low protein parents. the protein qtl region was delineated to approximately mbp of genomic sequence between sat_ and ssrpqtl_ the two closest non-segregating ssr markers flanking the left and right borders of the protein qtl region on chromosome. the coordinates of the borders stretch from mb to mb on chromosome phenotypic evaluation of seed protein and oil in nils a nil pair derived from the p-c609-45- population was chosen for further study. one line retained the g. max background at the lg i protein qtl region, and the other inherited the high protein allele in that region from g. soja. the protein and oil phenotypes in the nil pair were evaluated at four stages of seed fill. these four stages during seed fill were defined by seed size and were harvested at the same time during the r stage of development from the same plants for direct comparison. stage one corresponded to a seed size of to mg, stage two to greater than to mg seed, stage three to greater than to mg seed, and stage four to greater than to mg seed. at stage one, seed organs and tissues are formed but have yet to increase in cell size. it is noteworthy that seed protein differences between lopro and hipro genotypes were apparent at the earliest stage of evaluation. moreover, that difference remained consistent through the subsequent stages. seed oil values, however, did not show as marked a contrast in the early stages. the protein and oil phenotypes for the nils at seed maturity were consistent with the previously reported values. transcript accumulation changes during seed fill to examine transcript accumulation changes during seed fill, transcript profiles were evaluated in seeds of each genotype from the four stages above by soy genome affymetrix® genechip analyses. out of soybean probesets on the genechip, 64-69% were defined as'present' in three out of three replicates by mas analysis of the various seed stages in both genotypes. these detection figures are comparable to those found in seed microarray studies of other species. differences in the transcriptomes of the nil pair may reflect or affect the high and low protein and oil phenotypes seen in the lines. using student's t-test to evaluate significance, affymetrix® genechip probesets with at least -fold change between stages were identified at an fdr of less than 5%. transcript accumulation changes across stages were evaluated with reference to the stage one profiles. in both genotypes, no probesets from the stage two versus stage one comparison qualified under the fdr < criterion, so this comparison was excluded from further analysis. the number of probesets representing differentially accumulated transcripts with higher accumulation in stage three compared to stage one was greater in hipro than in lopro, and this difference was again apparent between stages four and one. analysis of all probeset expression changes revealed that % of the genes that significantly increased in expression over time in either genotype were shared between lopro and hipro. transcripts common to both genotypes that increase significantly in stage four seed as opposed to stage one seed include: beta-conglycinins and glycinins, sucrose binding proteins, heat shock chaperonins, late embryogenesis messages, seed maturation proteins, glutathione s-transferases and peroxidases, iron binding and flavonoid synthesis proteins, and numerous transporters. interestingly, transcripts with ubiquitin-related annotations were found to increase in accumulation over time in both genotypes while three were found to decrease in accumulation levels in both genotypes. it is noteworthy that some transcription factor messages showed enhanced abundance at stage three or four versus stage one seed. of the genes that decreased in expression over time, % of these genes were shared between lopro and hipro. transcripts common to both genotypes that were reduced in abundance at stage four as opposed to stage one include genes involved in flavonoid metabolism, cell wall deposition, kinases, response to arachidonic acid, strictosidine synthesis, and disease resistance response. twenty transcription factor annotations were common to both lines and displayed reduced abundance by stage three or four. during seed development, the synthesis of seed storage products is coordinated with carbohydrate and nitrogen metabolic processes involving many transporters. some transport-related transcripts increased in abundance in both genotypes, including gene transcripts annotated as ammonium, sugar, metal, and ion transporters. meanwhile, some transport-related transcripts decreased in accumulation levels in both genotypes, and these included transcripts annotated as ammonium, sugar, and abc transporters. a high number of microtubule-related gene transcripts were also found to decrease in abundance, supporting a role for fundamental transport mechanisms and the slowing of cell expansion during these stages of seed development. eleven microtubule-related transcripts, including those involved in activity and movement, were found to decrease in abundance versus four microtubule-related transcripts that increased in abundance in both genotypes. cyclin-related transcripts were also found in both genotypes. it is interesting to note that of the transcripts directly associated with cell division cycle annotations, those that increased in abundance included transcripts for cdc and five transcripts annotated as tyrosine kinase specific for activated p21cdc42hs. those that decreased in abundance included transcripts for cdc and cdc. at least one transcript related to cdc was found to accumulate in both directions for both genotypes over time. sucrose is well known for its many roles during seed development. sixteen transcripts with sucrose-related annotations were found to increase in accumulation in both genotypes, and these annotations included sucrose-binding protein and sucrose degradation and transport-related genes. this number is in contrast to the five sucrose-related transcripts that were found to decrease in accumulation in both genotypes and that included sucrose degradation and sucrose response genes. variation in transcriptome abundance profiles revealed differences between the two genotypes that may relate to their phenotypes. tables and show the transcripts that were most enhanced in abundance from each genotype in stage four seed versus stage one seed. overall, hipro possessed transcripts with greater than four-fold abundance in stage four versus stage one seed, compared to transcripts in lopro. in addition, the hipro line showed more than five times greater maximum fold change differences between stage four and stage one. hipro revealed a striking abundance of transcripts related to protein accumulation, iron sequestration, sucrose binding, and seed maturation. by comparison, the greatest abundance of transcripts in lopro related to chaperonin heat shock protein, peptide transporter kinases, and glutathionine s-transferase. interestingly, transcripts related to chloroplast function were greatly reduced in lopro in both abundance and unique representation in comparison to hipro. transcripts with accumulation changes were also assigned to gene ontology categories, and gene categories that were enriched under each condition within each genotype were identified. transcripts for specific genes were also examined closely. the effect of dof transcription factors on seed oil regulation have been previously documented, where gmdof and gmdof were found to contribute to high seed oil phenotypes in arabidopsis. in our study, dof and dof genes were upregulated in the hipro soy line, but no significant difference was seen in the transcript abundance for dof and dof in either genotype. differentially accumulated transcripts between nils identified by microarray direct comparisons of transcript accumulation between the two genotypes showed few significant differences by soy genome affymetrix® genechip analyses. differentially expressed transcripts between the two genotypes were detected using student's t-test. at a false discovery rate of 5% or less, only affymetrix® probesets displayed at least -fold change between the two genotypes lopro and hipro. strikingly, six probesets were detected at greater than four-fold change between the two genotypes. examination of the six probesets above revealed that they likely represent three genes according to est and genbank data. these three genes are labeled as pqi pqi and pqi. all six of the probesets with the greatest fold change were detected as transcripts with greater abundance in lopro than in hipro at all four stages. probesets representing transcripts with greater abundance in hipro than in lopro also existed. each affymetrix® probeset identifier is shown with corresponding normalized expression values for lopro and hipro and the ratio of mean lopro divided by mean hipro values. criteria for the list: fdr < and fold-change >. the uniprot description for each affymetrix® id is accompanied by the e-value for the alignment; na = not applicable, no significant alignment. an n-way anova test was also conducted to examine transcript accumulation differences simultaneously across multiple factors, genotype, and time within the genotype. at fdr <, a total of soy affymetrix® probesets were detected with differential changes in transcript accumulation using this method. interestingly, five transcription factor-related transcripts, annotated as bzip, ethylene-responsive, or heat shock, were detected with differential accumulation patterns. again, the six probesets with the most highly differential accumulation values were represented. because the affymetrix® genechip analysis was performed using transcripts from two different genotypes, the possibility of the presence of feature polymorphisms in the transcripts that could alter probe to transcript affinity was high. therefore, single feature polymorphism analysis was performed using the affymetrix® genechip data and an algorithm based on the li-wong model combined with a modified probe level statistical method. sfp analysis of the three genes above showed large affinity differences to multiple probes on the affymetrix® genechip. these three genes were potentially polymorphic in one or more regions between the two genotypes or completely absent in one genotype. to further validate the microarray data, quantitative reverse transcriptase-polymerase chain reaction was performed. specific primers were designed for the three genes and an actin control. significant differences between lopro and hipro were observed for pqi and pqi. however, no significant transcript level fold changes were observed for pqi. thus, only two of the three genes identified as upregulated in lopro in prior analyses were determined to display differentially accumulating transcripts between the two genotypes by qrt-pcr. genes with differentially accumulated transcripts between nils map to the lg i protein qtl the three most highly differentially accumulating transcripts identified by affymetrix® genechip were aligned to the soybean genome sequence and found to reside within the borders of the protein qtl region on chromosome. even though only two of the three were confirmed to accumulate differential levels of transcripts, allelic differences at the segregating qtl region are a potential source for polymorphisms between the two genotypes that could also result in a candidate gene. three additional differentially accumulating transcripts identified by affymetrix® genechip also mapped to the qtl region, one within kb of pqi. thus, of the differentially accumulating transcripts identified by affymetrix® genechip resided within the defined boundaries of the protein qtl region at lg i. transcripts identified by n-way anova were aligned to the genome sequence to show the range and distribution along the soybean chromosomes. the soybean genome sequence reveals a general bias toward gene-rich chromosome ends, a phenomenon that has been observed in other plant genomes. however, a striking concentration of probes mapped to chromosome at the protein qtl region. the presence of differentially accumulating transcripts in this region is consistent with the development of a near-isogenic line pair that displays variation in seed protein phenotype and segregation of markers within the protein qtl region. recently, wei et al. also performed a transcriptome analysis using rice superhybrid lyp and mapped differentially expressed genes to yield-related qtls in the rice genome. differentially accumulated transcripts between nils identified by high-throughput transcriptome sequencing because the soy genome affymetrix® genechip does not represent the complete set of soybean genes, high-throughput transcriptome sequencing was performed to confirm the microarray data and search for additional candidate genes. using the same rna samples prepared for microarray analysis as templates for high-throughput deep sequencing, more than million reads were sequenced, each or nucleotides in length, using the illumina® genome analyzer. sequences were generated from random priming sites within transcript cdna from each of the four stages in lopro and in hipro, producing more than million reads per stage. of these reads, more than million aligned uniquely to the genome sequence. the soybean genome sequencing consortium predicted gene models and additional transposon-like gene models. from that initial set of gene models, the consortium identified "high-confidence" genes. in the current seed nils sequencing effort, of the of the high-confidence genes show evidence of expression. an additional predicted genes not in the high-confidence set show evidence of expression from the seed nils data. twelve differentially accumulated transcripts between lopro and hipro were identified within the lg i protein qtl region with at least a two-fold change in expression at a p < using htts. putative genes were annotated and compared with plant est and genbank data sets. to further validate the htts data, quantitative reverse transcriptase-polymerase chain reaction was performed. specific primers were designed for four genes from table with no available corresponding microarray data. examination of three genes, glyma g glyma g and glyma g by qrt-pcr confirmed higher transcript accumulation levels in lopro versus hipro, although the standard deviation among biological replicates in glyma g was high. analysis by qrt-pcr also confirmed higher transcript accumulation levels of glyma g in hipro versus lopro. the sequence identifier is shown for each numbered gene candidate with differentially accumulated transcripts between genotypes at the lg i protein qtl region. transcript sequencing read counts for lopro and hipro are reported along with the sequence location for the closest predicted gene. the blastp description is reported for each gene at e-value < 10- na = not applicable. affymetrix® genechip vs. illumina® high-throughput transcriptome sequencing analysis close comparison of the transcripts identified by htts showed the presence of the two most highly differentially accumulated transcripts identified by affymetrix® genechip analysis. examination of the coordinates of the most highly differentially accumulated transcripts revealed a distance of mb between pqi and pqi. however, the positioning of the soybean target sequence from the affymetrix® genechip for these genes did not directly conform to the predicted gene models in the soybean genome. interestingly, two pairs of transcripts identified from the illumina® deep sequencing analysis appeared in the same region with overlapping chromosome coordinates but on opposite strands. transcripts with sequence homology to known proteins included an ethylene receptor and a glutamyl-trna synthetase that presented differentially accumulated transcripts at only one stage, as well as a putative ammonium transporter. examination of the available affymetrix® soy genechip target equivalents that overlapped the region, however, did not provide support for the ethylene receptor and ammonium transporter transcript accumulation differences. in all, the union of affymetrix® soy genechip and illumina® deep sequencing transcriptome data yielded genes with differentially accumulating transcripts that mapped to the protein qtl region at lg i on chromosome. genome-wide gene expression coverage from htts of the near-isogenic line pair, a large amount of data was obtained. uniquely mapped read counts for each genotype at each stage are provided for comparison of transcript accumulation levels at each gene within the defined boundaries of the lg i protein qtl region. this list excludes genes annotated as transposon-related. out of genes on chromosome at the lg i protein qtl region, showed evidence of expression during the seed fill stages examined in this study. the genes in the lg i protein qtl region with the most transcript read counts are listed here. additional file lists all genes in order of total read abundance. listed in this table are the genes with the most number of uniquely mapped reads contributing to transcript counts. a to a correspond to lopro stages one to four, and b to b correspond to hipro stages one to four. na = not applicable, no significant alignment all htts transcript profiles from this study, for all predicted soybean genes, are available at http://soybase.org/gbrowse. two gbrowse annotation tracks provide information on transcript read coverage and location. a "seed development coverage depth" track shows locations and counts of uniquely mapped htts reads, and a "seed development transcript count" track shows a colored histogram of relative read accumulation counts in each of the eight libraries in this study: a to a correspond to lopro stages one to four, and b to b correspond to hipro stages one to four. histograms for each gene are centered under their corresponding gene model. an example of a screenshot depiction of transcript read accumulation coverage is shown for a gene at the lg i protein qtl region, glyma g. transcript coverage for glyma g is consistent with the predicted gene model. discussion seed protein and oil relationships it has long been documented that seed protein and oil content are inversely correlated in the soybean seed. low oil alleles are consistently cotransmitted with high protein alleles in many instances, and attempts to separate these two traits through chromosomal recombination in the nils used in this study have not been successful. it has been hypothesized that this relationship may be due to either very tight linkage or pleiotropic effects. whether one phenotype directly or indirectly results in the other is unknown, and the timing of events regarding differential accumulation of contrasting protein and oil levels in the seed is uncertain. gmdof and gmdof transcription factors, however, have been reported to activate genes involved in lipid biosynthesis and simultaneously suppress the expression of storage protein genes. transcription factors have also been shown to influence seed traits in other studies. for example, the putative ap2/erebp transcription factor wrinkled was found to be involved in the regulation of seed oil accumulation in arabidopsis, and a qtl encoding a nac transcription factor was found to control grain protein and leaf senescence in wheat. in addition, seed mass in arabidopsis has been shown to be regulated by the apetala class of transcription factors. verdier et al. evaluated the expression of transcription factors throughout seed development of medicago truncatula. they found some transcription factors were expressed equally throughout seed development while had differential expression at one or more stages. cluster analysis demonstrated six different clusters of transcription factor genes that corresponded to the developmental stages evaluated. many of the transcription factors that were found to be upregulated in this study during seed development of the soybean nils were similar to those described by verdier et al.. transcriptional suppression of some aspect of seed protein accumulation could be envisioned for the low protein/high oil nil homozygous for the g. max allele of the lg i qtl. however, transcriptional suppression of seed oil accumulation in the nil homozygous for the g. soja allele would be envisioned to occur in a time frame late in seed fill. this assumption is due to the observation that the rate of seed oil accumulation in hipro did not differ from that of lopro until the last stage of seed fill. although hipro matures slightly earlier and generally yields less seed than lopro, these differences do not fully account for the striking differences in nil seed protein content observed at the early stages of seed fill. whether additional differences in the morphology or composition of the seed exist between the near-isogenic lines remains to be seen. further detailed investigation is in progress to study the temporal and spatial distribution and partitioning of candidate gene expression that may govern the relationship between protein and oil accumulation in the developing soybean seed. processes and pathways influencing seed content comprehensive evaluation of seed transcripts through microarray analyses have been reported for arabidopsis, medicago truncatula, barley, and wheat. these studies, in common, report differential expression of hundreds of genes at one or more stages of seed development involved in processes related to carbon and nitrogen metabolism, protein processing, transport of nutrients, organ development, signal transduction, and phytohormone balance. the transcript accumulation patterns we observed during nils seed fill by genechip® microarray data were consistent with these studies. prior studies have demonstrated the transcription and accumulation of both mrna and protein for beta-conglycinin and glycinin genes during the seed fill stage of seed development. transcripts for these seed storage proteins were identified during seed fill with particular abundance in the hipro line. additional classes of genes with roles in seed development and maturation, flavonoid metabolism, and sucrose binding were also identified. proteome analyses of the seed filling stages in soybean have provided support for the presence of gene transcripts found in this study with the detection of proteins associated with protein destination and storage, metabolism, and disease/defense. expression of different protein isoforms have been shown to display different accumulation trends, and the activities of many genes may have multiple roles during seed filling. this phenomenon may be reflected in the increase and decrease in accumulated transcripts of lipoxygenase-related genes in this study, consistent with proteomic data on various lipoxygenases in the developing soybean seed. carbon metabolism directed toward oil and protein deposition plays an important role in seed quality. changes in seed protein or oil in many plant species have been linked to the activity of acetyl-ca carboxylase and phosphoenolpyruvate carboxylase. recent proteomic and microarray studies have shown the presence of peptides and transcripts for both enzymes during seed development. overexpression of arabidopsis acetyl-coa carboxylase led to increased oil content of brassica napus seeds and potato tubers. the acetyl-coa carboxylase gene has also been associated with a major groat oil content qtl. in addition, inhibition of plastid acetyl-coa carboxylase resulted in lower seed oil. in soybean, a significant correlation was found between phosphoenolpyruvate carboxylase activity and seed protein and oil concentrations, although this correlation was found to be higher for seed protein. furthermore, overexpression of phosphoenolypruvate carboxylase in vicia narbonensis seed was shown to increase seed storage capacity and protein content. although we found no significant differences in transcript expression of accase and pepc between nils, we observed that transcripts corresponding to several forms of accase and pepc were expressed at all stages of seed development in this study. interestingly, some forms of accase were expressed at higher levels in the seed than others. such data may reflect the importance of enhanced isoforms of accase and pepc in seed development compared to isoforms expressed elsewhere in the plant. impaired storage metabolism has been linked with decreased sucrose levels, and sucrose may affect carbon flux at the transcriptional or post-transcriptional levels. studies have shown the importance of photosynthesis in seed filling metabolism and for the biosynthesis of seed storage products consistent with the wide array of photosynthesis-related genes detected during seed fill in this study. regulation of protein destination, storage, and proteolysis, as well as metabolic and photosynthetic pathways, may contribute to the contrasting seed phenotypes seen in the nil pair. additional transcript accumulation changes have been documented during seed development. a heat shock protein and peptide transporter were among the annotations of the transcripts with the greatest fold change increases from stage one to stage four in lopro. both a peptide transporter and heat shock-related proteins were previously found to increase dramatically during seed development in a high oil soybean line. down-regulation of lipoxygenases and sucrose udp-glycosyltransferase during seed development in a high oil soybean line of a previous study is also consistent with the detection of down-regulated lipoxygenase and udp-glycosyltransferase transcripts in lopro. the transcription accumulation patterns of these genes may be a feature common to soybean lines with high oil phenotypes. candidates for regulation of seed protein and oil we identified genes mapping to the protein qtl region at lg i that may play a role in the regulation of seed protein and oil. thirteen of these genes displayed differentially accumulating transcripts. of these were found at high levels in the low protein line with low or no detectable levels in the high protein line. based on sequence homology searches to protein databases, these candidates include a potential regulatory protein in the mov34- family, a heat shock protein hsp, and an atp synthase. although the mov34- candidate appeared to possess versatile domains for the potential regulation of multiple processes, transcripts isolated from this candidate region contained numerous stop codons, raising the possibility of non-coding genes. the same was true for a number of the other candidates and may account for the high percentage of genes with no significant e-value returns to the uniprot protein database. there is increasing evidence for the role of riboregulators, either as long non-protein coding rnas or processed into small rnas in plant development, and these molecules may play a role in seed protein and oil accumulation. two pairs of genes among the candidates were found to possess overlapping transcripts; one possibility is that these overlapping transcripts form double-stranded rnas that may be processed into small rnas. evidence for the expression of heat shock proteins during the stress-independent development of the seed has previously been observed. interestingly, heat shock protein genes were found to be expressed at higher levels in the low protein line of a near-isogenic line pair in barley, a phenomenon also observed in the lopro line of this study. previous studies have detailed an indirect relationship among the accumulation of storage proteins, lipid biosynthesis, and photosynthesis in the seed, correlating to the availability and distribution of atp. further investigation into the modulation of atp synthase levels on energy status and storage product accumulation in the soybean seed will shed light on the potential role for atp synthase as a candidate gene. currently, the occurrence of additional candidate genes from even earlier stages of seed development is being evaluated through differential analysis of transcriptome profiles of the near-isogenic line pair. potential modes of regulation for seed protein and oil the lopro line was converted into the hipro line upon inheritance of a g. soja allele at the lg i protein qtl region. however, the lopro line is also the high oil line, and a number of scenarios may explain how gene expression differences relate to variation in protein and oil phenotypes in the seed. protein content may be positively regulated by the expression of a gene that increases protein production in hipro. alternatively, protein content may be negatively regulated by expression of a gene in lopro that inhibits or reduces protein accumulation and thus allows for increased oil accumulation. significant protein differences would then be observed at an earlier stage than oil differences, as in figure inhibition of protein accumulation could take place at many levels, including transcriptional and post-transcriptional control and regulation of protein synthesis, transport, and turnover. the presence of candidate genes with non-coding segments raises the possibility of regulation at the transcriptional level that may affect the transcription of genes outside the list of candidates shown in this study. differences in transcriptome profiles may correlate directly or indirectly with the differences in protein and oil accumulation between the nils. previous studies have shown that seed storage proteins are largely controlled by transcriptional regulation during the seed fill stage. extensive analysis of cis-regulatory elements of seed storage proteins has demonstrated interaction of these elements with bzip and myb factors. transcription of a candidate gene in lopro may result in negative regulation of transcriptional regulators or key factors involved in high protein accumulation. the presence of sequence polymorphisms in gene sequences or promoter regions within the segregating region of the protein qtl may account for the low or absent levels of differentially accumulating gene transcripts in hipro versus lopro. in an alternative scenario, oil content may be regulated. gene expression or transcript accumulation leading to a higher oil phenotype may act in concert with other factors to directly or indirectly lead to reduced protein accumulation. genes regulated by transcription factors could initiate this effect. in support of this model, batch analysis of the promoter regions of the genes with the greatest differentially accumulated transcripts between the nils revealed a number of transcription factor binding sites and seed-specific motifs. a regulatory factor expressed in the high oil lopro line may activate higher oil synthesis or accumulation pathways. this is consistent with the greater abundance of candidate gene transcript accumulation seen in lopro. inheritance of a g. soja allele that does not allow for expression or accumulation of the high oil gene could account for the low oil and high protein phenotype in hipro. utility of the htts dataset for understanding the soybean genome although we focused on the transcripts derived from the lg i region of the genome in this study, the high-throughput transcriptome sequencing data set we obtained compiles greater than million reads and × nucleotides of transcript data and is an excellent resource for increasing our understanding of the soybean genome. the use of htts in conjunction with microarrays allowed us to detect a more comprehensive set of soybean gene transcripts. our observation that 86% of gene transcripts in soybean were present during seed development greatly extends previous microarray-dependent seed development studies. recent reports demonstrate the value of high-throughput transcriptome sequencing in eukaryotes for identification of novel transcripts and transcript isoforms, untranslated regions, and gene structures, leading to improved genome annotation. for the soybean genome, current gene models using the 8× genome sequence assembly were predicted based on protein coding sequences. by comparison, our transcriptome dataset encompasses both protein coding and non-protein coding sequences and will be useful for identification of transcripts outside of gene models. analyses of our dataset also show evidence for the existence of novel transcript isoforms, including alternative splicing, between genotypes and among seed stages. moreover, beyond the detection of feature polymorphisms reported here, a comparative analysis of common transcripts between soybean lines will provide a multitude of single nucleotide polymorphisms useful in following agronomic traits in breeding populations. currently we are analyzing high-throughput sequencing of transcripts from many soybean tissues. that data, along with the seed transcriptome data, will compile an atlas of gene expression for soybean.  this study provided the rare opportunity to intersect structural mapping and molecular profiling studies. here, we compared the transcript abundance profiles of the developing seed from a soybean nil pair with contrasting seed protein and identified gene candidates at the lg i protein qtl for potential involvement in the regulation of protein content in the soybean seed. the entire transcriptome sequencing dataset generated from this study is also provided as a valuable resource. control of protein and oil accumulation in the seed occurs at many different levels and is likely influenced by more than one gene. of the candidates genes identified in this study, any combination could be responsible for the observed change in protein and oil and phenotypes conditioned by the alleles of the lg i qtl. other protein/oil qtls have been identified in qtl mapping studies, but the lg i qtl is of great interest because its additive effect on seed protein and oil is the largest of any qtl identified to date. the models presented here are compatible with the role of additional genes and pathways as well as mixed models for control of seed protein and oil. resources that include the availability of additional recombinants and the use of markers derived from this study will allow for further demarcation of the qtl region. further studies are being conducted on additional mapping populations to dissect the relationship between protein and oil levels, and functional studies are under way to identify and validate the role of candidate genes in the accumulation of protein and oil in the seed. 
a soybean near-isogenic line pair contrasting in seed protein and differing in an introgressed genomic segment containing the lg i protein qtl was used as a resource to demarcate the qtl region and to study variation in transcript abundance in developing seed. the lg i qtl region was delineated to less than mbp of genomic sequence on chromosome using affymetrix® soy genechip and high-throughput illumina® whole transcriptome sequencing platforms, genes displaying significant seed transcript accumulation differences between nils were identified that mapped to the mbp lg i protein qtl region. this study identifies gene candidates at the lg i protein qtl for potential involvement in the regulation of protein content in the soybean seed. the results demonstrate the power of complementary approaches to characterize contrasting nils and provide genome-wide transcriptome insight towards understanding seed biology and the soybean genome. a quantitative trait locus on linkage group i of soybean merrill) has a striking effect on seed protein content. the nutritional and economic value of many crops is effectively a function of seed protein and oil content.
 the nutritional and economic value of many crops is effectively a function of seed protein and oil content. insight into the genetic and molecular control mechanisms involved in the deposition of these constituents in the developing seed is needed to guide crop improvement. a quantitative trait locus on linkage group i of soybean merrill) has a striking effect on seed protein content.  a soybean near-isogenic line pair contrasting in seed protein and differing in an introgressed genomic segment containing the lg i protein qtl was used as a resource to demarcate the qtl region and to study variation in transcript abundance in developing seed. the lg i qtl region was delineated to less than mbp of genomic sequence on chromosome using affymetrix® soy genechip and high-throughput illumina® whole transcriptome sequencing platforms, genes displaying significant seed transcript accumulation differences between nils were identified that mapped to the mbp lg i protein qtl region.  this study identifies gene candidates at the lg i protein qtl for potential involvement in the regulation of protein content in the soybean seed. the results demonstrate the power of complementary approaches to characterize contrasting nils and provide genome-wide transcriptome insight towards understanding seed biology and the soybean genome. 