BACKGROUND
interest in biofuels has prompted researchers to critically evaluate alternative feedstocks for biofuel production.
one important, emerging biofuel crop is camelina sativa l. cranz , commonly referred to as "false flax" or "gold-of-pleasure".
renewed interest in c. sativa as a biofuel feedstock is due in part to its drought tolerance and minimal requirements for supplemental nitrogen and other agricultural inputs  <cit> .
similar to other non-traditional, renewable oilseed feedstocks such as jatropha curcas l. , c. sativa grows on marginal land.
unlike jatropha, which is a tropical and subtropical shrub, c. sativa is native to europe and is naturalized in north america, where it grows well in the northern united states and southern canada.
in addition to its drought tolerance and broad distribution, several other aspects of c. sativa biology make it well suited for development as an oilseed crop.
first, c. sativa is a member of the family brassicaceae, and thus is a relative of both the genetic model organism arabidopsis thaliana and the oilseed crop brassica napus.
the close relationship between c. sativa and arabidopsis  <cit>  makes the arabidopsis genome an ideal reference point for the development of genetic and genomic tools in c. sativa.
second, the oil content of c. sativa seeds is comparable to that of b. napus, ranging from  <dig> to 40%  <cit> , suggesting that agronomic lessons from the cultivation of b. napus are applicable to c. sativa cultivation.
finally, the properties of c. sativa biodiesel are already well described  <cit> , and both seed oil and biodiesel from c. sativa were used as fuel in engine trials with promising results  <cit> .
notwithstanding its potential for oil production, there is limited molecular and genomic information on this crop.
published studies detailing the biology of c. sativa and its closest relatives in the genus camelina are few.
however, several important findings can be drawn from the literature.
taxonomic treatments describe  <dig> species in the genus with a center of diversity in eurasia  <cit> , although c. sativa, c. rumelica, c. microcarpa, and c. alyssum are naturalized weeds with broad distributions.
camelina species can be annual or biennial, with some species requiring vernalization to induce flowering  <cit> .
chromosome counts range from n =  <dig> in c. rumelica  <cit> , or n =  <dig> in c. hispida  <cit> , upwards to n =  <dig> in c. sativa, c. microcarpa, and c. alyssum  <cit> .
some camelina species are interfertile; crosses of c. sativa with c. alyssum, and c. sativa with c. microcarpa, produce viable seed  <cit> .
in addition to these studies, a limited amount of molecular and sequence information is available for c. sativa  <cit> .
understanding the camelina sativa genome is essential if agronomic properties are to be improved through molecular assisted breeding, mutation breeding, and/or genetic manipulation.
for example, modification of the oil composition for superior biodiesel is a natural goal for this oilseed crop.
c. sativa is high in polyunsaturated fatty acids such as linoleic acid  and alpha-linolenic acid  as well as very long chain fatty acids  such as 11-eicosenoic acid   <cit> , while an ideal biodiesel blend is high in oleic acid   <cit> .
target genes for modification could therefore include fatty acid desaturase  <dig> , a membrane bound delta-12-desaturase which converts oleic acid to linoleic acid  <cit> , and fatty acid elongase  <dig>  which sequentially adds  <dig> carbon units to  <dig> carbon fatty acid coa conjugates, resulting in very long chain fatty acids  <cit> .
manipulation of genes affecting traits of interest requires knowledge of their duplication status.
whole genome duplication is particularly relevant because it is common in plants, and because in the case of allopolyploidy it results in two or three independent copies of each gene.
allopolyploidy, such as found in wheat, cotton and peanut, is defined by the concurrent presence and maintenance in the same nucleus of two or more diploid genomes.
in an allopolyploid, each chromosome pairs specifically to its own homolog, and not to any homoeolog, resulting in diploid inheritance  <cit> .
allopolyploids are usually formed by interspecific hybridization concurrent to genome duplication, but could also result from diploidization and divergence of genomic sets in an autopolyploid  <cit> .
once formed, allopolyploids are relatively stable.
gene duplicates slowly decay over millions of years back to diploidy.
for example, a distinct but partial duplication pattern still detectable in the arabidopsis genome is thought to result from an approximately  <dig> million year old polyploidization event  <cit> .
the genomes of maize and soybean display widespread, but not universal duplication and are estimated to be  <dig> million year old polyploids  <cit> .
polyploids in which gene loss has advanced so far that duplication is no longer universal have been defined "paleopolyploids" although this term carries no precise temporal definition and could be extended to all known sequenced diploid angiosperms.
gene duplication is thus universal in a recent polypoid and becomes less and less pervasive in older polyploids as duplicates decay back to singletons.
for a set of nearly  <dig> genes the singleton pattern can be confirmed in all major sequenced diploid species  <cit> .
we report the sequences of three copies of both fae <dig> and fad <dig> recovered from c. sativa.
we used southern blots to determine whether the recovered copies are allelic or if they represent multiple loci.
moreover, we performed phylogenetic analyses to infer the evolutionary history of the copies, and quantitative pcr  to explore whether there is evidence of functional divergence among them.
to better understand the c. sativa genome and to determine whether the multiple copies recovered are the result of polyploidization, we analyzed the genome sizes of c. sativa and its closest relatives in the genus camelina by flow cytometry.
finally, we used next generation rna sequencing data to demonstrate that well-characterized single-copy genes are present in triplicates.
collectively our results indicate that c. sativa is a hexaploid whose oil composition is likely influenced by more than one functional copy of fae <dig> and fad <dig>  thus in c. sativa, oil composition as well as other traits are likely to be determined by multiple copies of causative genes.
RESULTS
southern blot hybridizations show multiple copies of genes in camelina sativa
as a first step to characterize genes involved in fatty acid biosynthesis, we determined the copy number of fad <dig> and fae <dig> by southern blot analysis.
since c. sativa is closely related to arabidopsis thaliana  <cit> , we designed primers based on arabidopsis genomic sequence that amplified conserved regions of fad <dig> and fae <dig> .
using these primers, we pcr amplified products of  <dig> base pairs   and  <dig> bp  from arabidopsis and from c. sativa.
the c. sativa products were cloned, sequenced, and compared with arabidopsis fad <dig> and fae <dig> sequences  <cit>  to confirm their identities.
we used the c. sativa fragments as probes in southern blot experiments .
results of the southern blots revealed three bands in c. sativa for both fad <dig>  and fae <dig> , whereas hybridization revealed only a single band in arabidopsis for both genes .
these results suggest that fad <dig> and fae <dig> occur in at least three copies in c. sativa, while they are single copy in arabidopsis  <cit> .
fatty acid genes can be multi-copy in many species, including soybean  <cit> , brassica napus  <cit> , olive   <cit> , maize  <cit> , and sunflower  <cit> .
therefore, we designed a probe for southern blot hybridization of the gene leafy , which is known to be single copy in a wide variety of species from several plant families  <cit> .
three bands were observed following hybridization with the lfy probe of the same blot as was used for fad <dig> and fae <dig>  suggesting lfy also exists as three copies in c. sativa .
copies of c. sativa fad <dig> and fae <dig> are highly similar to each other and to their putative orthologs from arabidopsis
we cloned and sequenced the full length genomic and cdna sequences of c. sativa fad <dig> and fae <dig>  using primers designed from arabidopsis fad <dig> and crambe abyssinica fae <dig> , we pcr amplified a band of approximately  <dig>  kb for fad <dig> and  <dig>  kb for fae <dig> from c. sativa.
for each gene, we sequenced more than  <dig> clones.
three different versions of both csfad <dig> and csfae <dig> were recovered and designated a, b, and c. it should be noted that the a, b, and c copies were named independently for csfad <dig> and csfae <dig>  and thus are not associated with a particular genome.
the three copies of c. sativa fad <dig> are  <dig> bp long, lack introns in the coding regions, are 97% identical at the nucleotide level, and encode proteins that are 99% identical in sequence .
one of the csfad <dig> copies, csfad <dig> a, contains a bamhi site , and thus this copy likely produced the smallest fragment in the southern blot hybridization of fad <dig> .
the c. sativa nucleotide sequences of fad <dig> are greater than 93% identical to arabidopsis fad <dig>  and the putative encoded proteins from the two species share greater than 96% identity .
*nucleotide identity is in coding region only.
the 5' untranslated region  was recovered for all three copies of csfad <dig> by rapid amplification of cdna ends  pcr.
we then used primers designed from the 5' utr sequence  to amplify an approximately  <dig>  kb intron found within the 5' utr from all three copies of c. sativa fad <dig>  a similarly sized intron is present in arabidopsis  <cit>  and in sesamum indicum  where it has been shown to be involved in regulating fad <dig> expression  <cit> .
all three copies of fae <dig> in c. sativa are  <dig> bp long and lack introns.
when the nucleotide sequences and the putative encoded proteins of the three copies are compared they are more than 96% identical .
in comparison to arabidopsis, the nucleotide sequences are more than 90% identical, while the encoded proteins are more than 91% identical .
thus, the three copies of c. sativa fad <dig> and the three copies of fae <dig> are highly similar to each other and to their putative orthologs from arabidopsis.
alignments of fad <dig> and fae <dig> protein sequences from several species reveal conserved and non-conserved domains
we aligned translated amino acid sequences from the three copies of c. sativa fad <dig> with the fad <dig> protein sequences from arabidopsis; brassica rapa, an agronomically important member of the brassicaceae family; glycine max, an agronomically important dicot; and zea mays, an agronomically important monocot .
all three copies of c. sativa fad <dig> have the three conserved his boxes found in all membrane-bound desaturases  <cit>  as well as the er localization signal described by mccartney et al  <cit> .
furthermore, the conserved amino acids identified in an alignment of the fad <dig> sequences from  <dig> different species  <cit>  are also present in c. sativa with the exception of a positively-charged histidine at position number  <dig>  which is substituted by a polar, uncharged glutamine in c. sativa.
when we amplified the fad <dig> gene from several camelina and outgroup species and aligned the translated amino acid sequences, we found that the fad <dig> proteins from capsella rubella, camelina microcarpa, camelina laxa, and one copy from camelina rumelica contain a glutamine at amino acid position  <dig>  while the fad <dig> proteins from arabidopsis lyrata, camelina hispida, and a second copy from camelina rumelica contained a histidine .
we aligned the translated amino acid sequences from the three copies of c. sativa fae <dig> with the seed-specific fae <dig> proteins from arabidopsis, crambe abyssinica, a high and low erucic acid brassica rapa, limnanthes alba, and tropaeolum majus .
l. alba and t. majus are both in the order brassicales and their seeds accumulate high levels of very long chain fatty acids  <cit> .
four conserved histidine residues and six conserved cysteine residues, including the active site at cysteine  <dig>  as well as an asparagine residue at  <dig> required for fae <dig> acitivity were previously identified by ghanevati and jaworski  <cit> .
all conserved residues were found to be present in all three copies of c. sativa fae <dig>  more differences were apparent between the three c. sativa fae <dig> sequences and the other fae <dig> sequences than observed in the fad <dig> comparison , an observation consistent with the level of amino acid identity seen between arabidopsis and c. sativa fad <dig> versus fae <dig> .
all three copies of fad <dig> and fae <dig> are expressed in developing seeds of c. sativa
the conservation of amino acids as well as the presence of the 5' regulatory intron in csfad <dig> suggests that all three copies of csfad <dig> and csfae <dig> could be functional.
to determine whether these genes are also expressed, we first evaluated total csfad <dig> and csfae <dig> gene expression in developing seeds and in seedling tissue using real time quantitative pcr  with primer/probe combinations designed to detect all three copies of each gene .
csfad <dig> expression in seedling tissue is present but minimal ), while csfae <dig> expression could not be detected in seedlings .
in developing seeds, both csfad <dig> and csfae <dig> expression peaks at  <dig> dpa and is reduced by  <dig> dpa .
in arabidopsis, fad <dig> peaks earlier and decreases sooner than fae <dig>  <cit> .
we wondered whether the expression of each of the fad <dig> and fae <dig> copies present in c. sativa are equally or differentially expressed in the seed.
duplicated genes are frequently silenced either throughout the plant or in a tissue-specific manner  <cit> ; hence we hypothesized that one or more of the copies of each gene could be significantly down-regulated.
we used the sequenom massarray™ method for determining allele-specific expression of a gene  <cit>  to evaluate the relative expression of each of the copies of csfad <dig> and csfae <dig>  we identified at least three single nucleotide polymorphisms  specific to each of the csfad <dig> a, b, and c and the csfae <dig> a, b, and c copies  and then calculated the frequency of each snp in seed cdna.
controls consisting of the cloned csfae <dig> a, b, and c copies combined to known frequencies showed that the method is greater than 80% accurate .
no evidence of silencing of any particular copy of either csfae <dig> or csfad <dig> was discovered.
we did observe differential expression, especially of csfae <dig> a, which accounts for approximately 40-50% of csfae <dig> expression in seeds at 20- <dig> dpa .
characterization of sequences upstream of c. sativa fae <dig> and downstream of c. sativa fad <dig> suggests colinearity with arabidopsis
to investigate whether the different copies of c. sativa fad <dig> and fae <dig> are the result of allelic variation or are in fact independent loci, we obtained sequence from the region upstream of csfae <dig> and downstream of csfad <dig>  assuming colinearity between c. sativa and arabidopsis for the region around fae <dig>  we pcr amplified the region 5' to csfae <dig> using a forward primer for the upstream gene kcs <dig> with reverse primers for c. sativa fae <dig> .
the resulting sequences we obtained for the putative c. sativa kcs <dig> were highly similar to the last  <dig> bp of arabidopsis kcs <dig>  suggesting that we had in fact amplified the orthologous c. sativa region upstream of fae <dig>  confirming colinearity between the two species.
we then used a dot plot  <cit>  to compare the three c. sativa upstream sequences to each other and to arabidopsis with parameters set for perfect match on a sliding window of  <dig> bases .
the coordinates from the dot plot were used to define blocks of homology between arabidopsis and the three c. sativa copies .
the results show a variable intergenic region containing potentially related blocks common to two or more genomes.
colinearity with arabidopsis was also found for a region downstream of fad <dig> containing the actin <dig>  gene for two out of the three c. sativa copies .
for the third copy, the region downstream of csfad <dig> a could have been missed if the length of the amplified product was too large.
alternatively, the region downstream of csfad <dig> a might not exhibit colinearity with arabidopsis and the possibility remains that two of the copies of csfad <dig> result from a tandem gene duplication.
deep sequencing of camelina sativa developing seed transcriptome reveals three expressed haplotypes for predicted single-copy genes
to further explore the c. sativa genome, we determined the haplotype number of predicted single-copy genes in a  <dig> sequencing data set of cdna expressed in  <dig> dpa c. sativa seeds.
the reads were aligned to  <dig> genes identified by duarte et al.
<cit>  as single-copy genes shared in flowering plants.
the six genes with the highest coverage  were selected for further evaluation.
remarkably, all  <dig> genes examined showed expression of three clear haplotypes  as exemplified by the agmatine deiminase gene , indicating that the triplication of the genes in the c. sativa genome is common and not limited to fad <dig>  fae <dig>  and lfy.
when the genomic status of the same  <dig> genes was examined in the genomes of paleopolyploids such as maize and soybean, whose genome duplication is about  <dig> million years old  <cit> , only a subset of these genes was retained as duplicates .
this lack of duplication in maize and soybean contrasted with the consistent pattern of triplication in c. sativa.
<dig> arabidopsis genome sequencing project
2expressed cdna, this study
3maize genome sequencing project
4soybean genome sequencing project
the genomes of c. sativa, c. alyssum, and c. microcarpa are larger than the genomes of other camelina species
we calculated dna content in several accessions of c. sativa and related species from flow cytometry analyses using propidium iodide-stained nuclei.
we used arabidopsis accession col- <dig>  and its tetraploid  derivative as genome size standards.
c. sativa, c. alyssum, and c. microcarpa diploid  genomes had a haploid content between  <dig> and  <dig> mb .
c. sativa accessions uniformly displayed a genome size close to  <dig> mb.
north american isolates of c. sativa, c. alyssum, and c. microcarpa have reported chromosome counts of n =  <dig>  <cit> .
the genomes of c. rumelica , c. hispida  and c. laxa  are smaller than those of c. sativa, c. alyssum, and c. microcarpa.
chromosome counts of both n =  <dig>  <cit>  and n =  <dig>  <cit>  have been recorded for c. rumelica, while only a single count of n =  <dig> exists for c. hispida  <cit> .
to our knowledge, no published counts exist for c. laxa.
phylogenetic analysis of fad <dig> and fae <dig> indicate that c. sativa and c. microcarpa are closely related
to understand the duplication history of the multiple fad <dig> and fae <dig> copies recovered from c. sativa, we amplified the fad <dig> and fae <dig> genes from several camelina species and outgroup species, and inferred phylogeny for each gene.
the sampling of taxa chosen allowed us to test whether fad <dig> and fae <dig> duplication events occurred after camelina diverged from its closest relatives or within the genus.
results from the evaluation of  <dig> different models of sequence evolution using modeltest  <dig>   <cit>  indicated that the fad <dig> sequence data are best described by the tvm+i+Γ model, while the fae <dig> data are best described by the hky+i+Γ model.
likelihood phylogenetic analyses in paup*  <dig> b  <cit>  produced a single fad <dig> tree , and a single fae <dig> tree .
phylogenies inferred from fad <dig> and fae <dig> data indicate a history of duplication for both markers.
both c. microcarpa and c. sativa have three distinct copies of fad <dig> and fae <dig>  moreover, for fad <dig>  the a and c copies from these two species are monophyletic with strong  bootstrap support ; for fae <dig> the a and b copies from these species are strongly monophyletic .
in contrast, neither the fad <dig> b copies of c. sativa and c. microcarpa, nor the fae <dig> c copies of these species form a monophyletic group with each other.
instead, our results indicate that c. rumelica has two distinct copies of fad <dig> and that one of these copies  is strongly monophyletic with c. microcarpa fad <dig> b. we recovered only a single fad <dig> copy for c. laxa and c. hispida.
in contrast, we recovered at least two distinct copies of fae <dig> from all sampled camelina species.
the fae1- <dig> copy of c. laxa, c. hispida, and c. rumelica form a monophyletic group , with the former two species sister to one another with strong support .
similar to the results from fad <dig>  c. rumelica fae1- <dig> is sister to one of the c. microcarpa copies .
neither the c. sativa fad <dig> b copy, nor the c. sativa fae <dig> c copy, shows a well supported sister relationship to other fad <dig> or fae <dig> sequences.
however, in the fae <dig> tree, c. sativa fae <dig> c is very weakly supported as sister to c. hispida fae1- <dig> .
finally, all recovered fad <dig> and fae <dig> copies from species of the genus camelina are monophyletic and sister to other sampled members of the tribe camelineae, consistent with phylogenies based on other markers  <cit> .
discussion
camelina sativa is a re-emerging oilseed with tremendous potential as an alternative biofuel crop and for which genomic information is becoming increasingly available.
we have obtained molecular data for nine genes, characterized in detail two genes encoding fatty acid biosynthesis enzymes and, in the process, have discovered unexpected complexity in the c. sativa genome.
the close relationship between c. sativa and the model plant arabidopsis thaliana  <cit>  facilitates the manipulation of known pathways, such as the one regulating fatty acid biosynthesis.
c. sativa seed oil is high in both polyunsaturated and long chain fatty acids  <cit> , suggesting that both csfad <dig> and csfae <dig> are present and active.
three copies each of the fad <dig> and fae <dig> genes were isolated from an agronomic accession of c. sativa using primers designed from a. thaliana or crambe abyssinica sequence.
previously identified conserved sites in csfad <dig>  <cit>  and csfae <dig>  <cit>  are present in all three copies of each gene and a 5' intron shown to be important in regulating fad <dig> expression in sesame  <cit>  was identified in all three csfad <dig> copies.
real time qpcr data and sequenom massarray snp analysis of the csfad <dig> and csfae <dig> cdna showed that all three copies of each gene are expressed in developing seeds.
thus, it seems likely that all three copies of fad <dig> and fae <dig> in c. sativa are functional.
the cloning of three copies of fad <dig> and fae <dig> from the c. sativa genome, as well as the observation of three lfy hybridization signals by southern analysis and three expressed haplotypes for  <dig> more predicted single-copy genes in developing seeds, could be explained by at least two possible scenarios: segmental duplications of selected regions within a diploid genome either through tandem duplications or through transpositions, or whole genome duplications resulting from polyploidization.
segmental duplications or transpositions affecting all nine examined loci are improbable compared with the explanation of polyploidy.
furthermore, no evidence of recent segmental duplication involving multiple genes has been observed in sequenced plant genomes  <cit> .
triplication of the c. sativa genome therefore likely occurred through whole genome duplication, either through autopolyploidization or through allopolyploidization.
an autopolyploidy event might have triplicated a single diploid genome resulting in an autohexaploid with a haploid genome of  <dig>   <dig>  or  <dig> chromosomes.
given that c. sativa has a chromosome count of n =  <dig>  chromosome splitting or fusion could then have increased the chromosomes from  <dig> to  <dig>  or decreased the chromosomes from  <dig> or  <dig> to  <dig>
alternatively, triplication of the c. sativa genome might have resulted from two allopolyploidy events, resulting in first a tetraploid then a hexaploid, similar to the origin of cultivated wheat.
according to this hypothesis, the three copies of each gene diverged in different diploid genomes before converging through polyploidy events.
taking into consideration the reported chromosome counts of various camelina species, the basal chromosome number of the diploid parental species contributing to the c. sativa haploid genome of  <dig> chromosomes could be 7+7+ <dig> or 8+6+ <dig>  the allopolyploid hypothesis is supported by the observation that c. sativa demonstrates diploid inheritance  <cit> , as would be expected for an allopolyploid  <cit> .
a hexaploid c. sativa could also be derived from the combination of an autotetraploid and a diploid species if, in an autopolyploidized genome, homologous chromosomes differentiated so that the subsequent chromosome-specific pairing mimicked an allopolyploid genome in its diploid inheritance patterns.
regardless of its evolutionary path, the c. sativa genome appears organized in three redundant and differentiated copies and can be formally considered to be an allohexaploid.
results from our phylogenetic analyses support a history of duplication for both fad <dig> and fae <dig> in camelina.
for fad <dig>  duplications were only recovered for c. sativa, c. microcarpa, and c. rumelica.
these data are consistent with genome size data, which indicate that all three genomes are larger than c. laxa and c. hispida, from which only a single fad <dig> copy was recovered.
taken together, the results suggest that c. sativa, c. microcarpa, and c. rumelica are likely polyploids.
given the slightly smaller genome size of c. rumelica, and the fact that we recovered only two fad <dig> copies from it, the c. rumelica sampled may be tetraploid while c. sativa and c. microcarpa are hexaploid.
interestingly, in both the fad <dig> and fae <dig> trees, one copy each of c. rumelica and c. microcarpa are strongly supported as sister.
thus, trees from these genes indicate that c. rumelica and c. microcarpa are closely related.
the various placement of c. microcarpa fad <dig> and fae <dig> copies can be explained if c. microcarpa is the result of a hybridization event between c. rumelica and a currently unsampled, and thus unidentified species of camelina.
two of the three copies of both fad <dig> and fae <dig> are identical, or nearly identical, in c. sativa and c. microcarpa, suggesting that c. sativa and c. microcarpa share a parental genome.
thus, we suggest that a camelina species we did not sample contributed its genome to the hybrid formation of both c. sativa and c. microcarpa.
in the case of c. microcarpa, the hybridization event likely involved c. rumelica.
given the chromosome count of n =  <dig> for c. rumelica, we expect the other putative parent to have an x =  <dig> genome, and furthermore to be tetraploid at n =  <dig>  such a cross would result in the observed c. microcarpa genome, with chromosome count n =  <dig>  interestingly, c. hispida is the only species we sampled with a chromosome count of n =  <dig>  however no strong relationship between c. hispida and c. microcarpa is inferred in either gene tree.
however, we do infer a weak relationship between c. sativa and c. hispida in the fae <dig> tree, and thus the possibility that c. hispida is involved in the polyploid formation of c. sativa should be explored further.
what is the age of the polyploidization events likely to have formed the c. sativa genome?
a complete answer will require a better understanding of its genome, but two findings suggest a recent origin.
first, the chromosome number of c. sativa is inconsistent with extensive karyotype evolution and likely represents the sum of the ancestral contributions.
second, paleopolyploids such as soybean and maize display duplication of many, but not all genes as a sizeable number have decayed to singleton state.
in contrast, the presence of triplicates for nine test genes of c. sativa is consistent with high retention of duplicates, as expected in recent polyploids.
the likely allohexaploid nature of the camelina sativa genome has multiple implications.
its vigor and adaptability to marginal growth conditions may result at least in part from polyploidy.
polyploids are thought to be more adaptable to new or harsh environments, with the ability to expand into broader niches than either progenitor  <cit> .
indeed, c. hispida and c. laxa, both of which are likely diploids, are found only in turkey, iran, armenia, and azerbaijan, while c. microcarpa and c. sativa are distributed throughout asia, europe, and north africa and are naturalized in north america  <cit> .
the mechanisms behind this increased adaptability are not completely understood, but have been attributed to heterosis, genetic and regulatory network redundancies, and epigenetic factors  <cit> .
allohexaploidy might also affect any potential manipulations of the c. sativa genome, such as introgression of germplasm or induced mutations.
introgression of an exotic germplasm could be facilitated by the type of polyploidy-dependent manipulations that are possible in wheat, a potentially comparable allohexaploid  <cit> .
in addition, polyploids have displayed excellent response to reverse genomics approaches such as targeting induced local lesions ingenomes   <cit> .
as in wheat, any recessive induced mutations could be masked by redundant homoeologous loci that have maintained function  <cit> .
this mutation masking implies that multiple knockout alleles at different homoeologous sites can be combined to achieve partial or complete suppression of a targeted function  <cit> .
we also expect that single locus traits, whether transgenic or not, will display diploid inheritance due to preferential intragenomic pairing.
in a hexaploid oilseed crop such as c. sativa, manipulations of oil composition and/or yield should therefore be possible through transgenic or reverse genetic approaches, or through other genome manipulations similar to those performed in wheat.
for example, the characterization of fad <dig> and fae <dig> in c. sativa could enable the use of tilling techniques to isolate c. sativa plants with mutations in each of the three identified copies of both genes.
we expect these mutations to result in plants with reduced levels of polyunsaturated fatty acids or long chain fatty acids, possibly in a dosage dependent manner.
this will allow us to manipulate the seed oil composition of c. sativa, potentially creating a broad spectrum of c. sativa varieties possessing useful biodiesel properties, thereby further increasing the utility of this emerging biofuel crop.
CONCLUSIONS
the discovery of triplication and divergence of genes that in known diploids are present in single copy, the cytometrically determined genome size of camelina species, the pattern of relationship and inferred duplication history in the gene trees, together with the previously known chromosome counts for this taxon, indicate a likely allohexaploid genomic constitution.
the characterization of genes encoding key functions of fatty acid biosynthesis lays the foundation for future manipulations of this pathway in camelina sativa.
targeted manipulations of oil composition and general development of this crop, however, need to consider the implications of polyploidy and when possible take advantage of this common condition in crop plants.

 interest in biofuels has prompted researchers to critically evaluate alternative feedstocks for biofuel production. one important, emerging biofuel crop is camelina sativa l. cranz, commonly referred to as "false flax" or "gold-of-pleasure". renewed interest in c. sativa as a biofuel feedstock is due in part to its drought tolerance and minimal requirements for supplemental nitrogen and other agricultural inputs. similar to other non-traditional, renewable oilseed feedstocks such as jatropha curcas l., c. sativa grows on marginal land. unlike jatropha, which is a tropical and subtropical shrub, c. sativa is native to europe and is naturalized in north america, where it grows well in the northern united states and southern canada. in addition to its drought tolerance and broad distribution, several other aspects of c. sativa biology make it well suited for development as an oilseed crop. first, c. sativa is a member of the family brassicaceae, and thus is a relative of both the genetic model organism arabidopsis thaliana and the oilseed crop brassica napus. the close relationship between c. sativa and arabidopsis makes the arabidopsis genome an ideal reference point for the development of genetic and genomic tools in c. sativa. second, the oil content of c. sativa seeds is comparable to that of b. napus, ranging from to 40%, suggesting that agronomic lessons from the cultivation of b. napus are applicable to c. sativa cultivation. finally, the properties of c. sativa biodiesel are already well described, and both seed oil and biodiesel from c. sativa were used as fuel in engine trials with promising results. notwithstanding its potential for oil production, there is limited molecular and genomic information on this crop. published studies detailing the biology of c. sativa and its closest relatives in the genus camelina are few. however, several important findings can be drawn from the literature. taxonomic treatments describe species in the genus with a center of diversity in eurasia, although c. sativa, c. rumelica, c. microcarpa, and c. alyssum are naturalized weeds with broad distributions. camelina species can be annual or biennial, with some species requiring vernalization to induce flowering. chromosome counts range from n = in c. rumelica, or n = in c. hispida, upwards to n = in c. sativa, c. microcarpa, and c. alyssum. some camelina species are interfertile; crosses of c. sativa with c. alyssum, and c. sativa with c. microcarpa, produce viable seed. in addition to these studies, a limited amount of molecular and sequence information is available for c. sativa. understanding the camelina sativa genome is essential if agronomic properties are to be improved through molecular assisted breeding, mutation breeding, and/or genetic manipulation. for example, modification of the oil composition for superior biodiesel is a natural goal for this oilseed crop. c. sativa is high in polyunsaturated fatty acids such as linoleic acid and alpha-linolenic acid as well as very long chain fatty acids such as 11-eicosenoic acid, while an ideal biodiesel blend is high in oleic acid. target genes for modification could therefore include fatty acid desaturase, a membrane bound delta-12-desaturase which converts oleic acid to linoleic acid, and fatty acid elongase which sequentially adds carbon units to carbon fatty acid coa conjugates, resulting in very long chain fatty acids. manipulation of genes affecting traits of interest requires knowledge of their duplication status. whole genome duplication is particularly relevant because it is common in plants, and because in the case of allopolyploidy it results in two or three independent copies of each gene. allopolyploidy, such as found in wheat, cotton and peanut, is defined by the concurrent presence and maintenance in the same nucleus of two or more diploid genomes. in an allopolyploid, each chromosome pairs specifically to its own homolog, and not to any homoeolog, resulting in diploid inheritance. allopolyploids are usually formed by interspecific hybridization concurrent to genome duplication, but could also result from diploidization and divergence of genomic sets in an autopolyploid. once formed, allopolyploids are relatively stable. gene duplicates slowly decay over millions of years back to diploidy. for example, a distinct but partial duplication pattern still detectable in the arabidopsis genome is thought to result from an approximately million year old polyploidization event. the genomes of maize and soybean display widespread, but not universal duplication and are estimated to be million year old polyploids. polyploids in which gene loss has advanced so far that duplication is no longer universal have been defined "paleopolyploids" although this term carries no precise temporal definition and could be extended to all known sequenced diploid angiosperms. gene duplication is thus universal in a recent polypoid and becomes less and less pervasive in older polyploids as duplicates decay back to singletons. for a set of nearly genes the singleton pattern can be confirmed in all major sequenced diploid species. we report the sequences of three copies of both fae and fad recovered from c. sativa. we used southern blots to determine whether the recovered copies are allelic or if they represent multiple loci. moreover, we performed phylogenetic analyses to infer the evolutionary history of the copies, and quantitative pcr to explore whether there is evidence of functional divergence among them. to better understand the c. sativa genome and to determine whether the multiple copies recovered are the result of polyploidization, we analyzed the genome sizes of c. sativa and its closest relatives in the genus camelina by flow cytometry. finally, we used next generation rna sequencing data to demonstrate that well-characterized single-copy genes are present in triplicates. collectively our results indicate that c. sativa is a hexaploid whose oil composition is likely influenced by more than one functional copy of fae and fad thus in c. sativa, oil composition as well as other traits are likely to be determined by multiple copies of causative genes.  southern blot hybridizations show multiple copies of genes in camelina sativa as a first step to characterize genes involved in fatty acid biosynthesis, we determined the copy number of fad and fae by southern blot analysis. since c. sativa is closely related to arabidopsis thaliana, we designed primers based on arabidopsis genomic sequence that amplified conserved regions of fad and fae. using these primers, we pcr amplified products of base pairs and bp from arabidopsis and from c. sativa. the c. sativa products were cloned, sequenced, and compared with arabidopsis fad and fae sequences to confirm their identities. we used the c. sativa fragments as probes in southern blot experiments. results of the southern blots revealed three bands in c. sativa for both fad and fae, whereas hybridization revealed only a single band in arabidopsis for both genes. these results suggest that fad and fae occur in at least three copies in c. sativa, while they are single copy in arabidopsis. fatty acid genes can be multi-copy in many species, including soybean, brassica napus, olive, maize, and sunflower. therefore, we designed a probe for southern blot hybridization of the gene leafy, which is known to be single copy in a wide variety of species from several plant families. three bands were observed following hybridization with the lfy probe of the same blot as was used for fad and fae suggesting lfy also exists as three copies in c. sativa. copies of c. sativa fad and fae are highly similar to each other and to their putative orthologs from arabidopsis we cloned and sequenced the full length genomic and cdna sequences of c. sativa fad and fae using primers designed from arabidopsis fad and crambe abyssinica fae, we pcr amplified a band of approximately kb for fad and kb for fae from c. sativa. for each gene, we sequenced more than clones. three different versions of both csfad and csfae were recovered and designated a, b, and c. it should be noted that the a, b, and c copies were named independently for csfad and csfae and thus are not associated with a particular genome. the three copies of c. sativa fad are bp long, lack introns in the coding regions, are 97% identical at the nucleotide level, and encode proteins that are 99% identical in sequence. one of the csfad copies, csfad a, contains a bamhi site, and thus this copy likely produced the smallest fragment in the southern blot hybridization of fad. the c. sativa nucleotide sequences of fad are greater than 93% identical to arabidopsis fad and the putative encoded proteins from the two species share greater than 96% identity. *nucleotide identity is in coding region only. the 5' untranslated region was recovered for all three copies of csfad by rapid amplification of cdna ends pcr. we then used primers designed from the 5' utr sequence to amplify an approximately kb intron found within the 5' utr from all three copies of c. sativa fad a similarly sized intron is present in arabidopsis and in sesamum indicum where it has been shown to be involved in regulating fad expression. all three copies of fae in c. sativa are bp long and lack introns. when the nucleotide sequences and the putative encoded proteins of the three copies are compared they are more than 96% identical. in comparison to arabidopsis, the nucleotide sequences are more than 90% identical, while the encoded proteins are more than 91% identical. thus, the three copies of c. sativa fad and the three copies of fae are highly similar to each other and to their putative orthologs from arabidopsis. alignments of fad and fae protein sequences from several species reveal conserved and non-conserved domains we aligned translated amino acid sequences from the three copies of c. sativa fad with the fad protein sequences from arabidopsis; brassica rapa, an agronomically important member of the brassicaceae family; glycine max, an agronomically important dicot; and zea mays, an agronomically important monocot. all three copies of c. sativa fad have the three conserved his boxes found in all membrane-bound desaturases as well as the er localization signal described by mccartney et al. furthermore, the conserved amino acids identified in an alignment of the fad sequences from different species are also present in c. sativa with the exception of a positively-charged histidine at position number which is substituted by a polar, uncharged glutamine in c. sativa. when we amplified the fad gene from several camelina and outgroup species and aligned the translated amino acid sequences, we found that the fad proteins from capsella rubella, camelina microcarpa, camelina laxa, and one copy from camelina rumelica contain a glutamine at amino acid position while the fad proteins from arabidopsis lyrata, camelina hispida, and a second copy from camelina rumelica contained a histidine. we aligned the translated amino acid sequences from the three copies of c. sativa fae with the seed-specific fae proteins from arabidopsis, crambe abyssinica, a high and low erucic acid brassica rapa, limnanthes alba, and tropaeolum majus. l. alba and t. majus are both in the order brassicales and their seeds accumulate high levels of very long chain fatty acids. four conserved histidine residues and six conserved cysteine residues, including the active site at cysteine as well as an asparagine residue at required for fae acitivity were previously identified by ghanevati and jaworski. all conserved residues were found to be present in all three copies of c. sativa fae more differences were apparent between the three c. sativa fae sequences and the other fae sequences than observed in the fad comparison, an observation consistent with the level of amino acid identity seen between arabidopsis and c. sativa fad versus fae. all three copies of fad and fae are expressed in developing seeds of c. sativa the conservation of amino acids as well as the presence of the 5' regulatory intron in csfad suggests that all three copies of csfad and csfae could be functional. to determine whether these genes are also expressed, we first evaluated total csfad and csfae gene expression in developing seeds and in seedling tissue using real time quantitative pcr with primer/probe combinations designed to detect all three copies of each gene. csfad expression in seedling tissue is present but minimal ), while csfae expression could not be detected in seedlings. in developing seeds, both csfad and csfae expression peaks at dpa and is reduced by dpa. in arabidopsis, fad peaks earlier and decreases sooner than fae. we wondered whether the expression of each of the fad and fae copies present in c. sativa are equally or differentially expressed in the seed. duplicated genes are frequently silenced either throughout the plant or in a tissue-specific manner; hence we hypothesized that one or more of the copies of each gene could be significantly down-regulated. we used the sequenom massarray™ method for determining allele-specific expression of a gene to evaluate the relative expression of each of the copies of csfad and csfae we identified at least three single nucleotide polymorphisms specific to each of the csfad a, b, and c and the csfae a, b, and c copies and then calculated the frequency of each snp in seed cdna. controls consisting of the cloned csfae a, b, and c copies combined to known frequencies showed that the method is greater than 80% accurate. no evidence of silencing of any particular copy of either csfae or csfad was discovered. we did observe differential expression, especially of csfae a, which accounts for approximately 40-50% of csfae expression in seeds at 20- dpa. characterization of sequences upstream of c. sativa fae and downstream of c. sativa fad suggests colinearity with arabidopsis to investigate whether the different copies of c. sativa fad and fae are the result of allelic variation or are in fact independent loci, we obtained sequence from the region upstream of csfae and downstream of csfad assuming colinearity between c. sativa and arabidopsis for the region around fae we pcr amplified the region 5' to csfae using a forward primer for the upstream gene kcs with reverse primers for c. sativa fae. the resulting sequences we obtained for the putative c. sativa kcs were highly similar to the last bp of arabidopsis kcs suggesting that we had in fact amplified the orthologous c. sativa region upstream of fae confirming colinearity between the two species. we then used a dot plot to compare the three c. sativa upstream sequences to each other and to arabidopsis with parameters set for perfect match on a sliding window of bases. the coordinates from the dot plot were used to define blocks of homology between arabidopsis and the three c. sativa copies. the results show a variable intergenic region containing potentially related blocks common to two or more genomes. colinearity with arabidopsis was also found for a region downstream of fad containing the actin gene for two out of the three c. sativa copies. for the third copy, the region downstream of csfad a could have been missed if the length of the amplified product was too large. alternatively, the region downstream of csfad a might not exhibit colinearity with arabidopsis and the possibility remains that two of the copies of csfad result from a tandem gene duplication. deep sequencing of camelina sativa developing seed transcriptome reveals three expressed haplotypes for predicted single-copy genes to further explore the c. sativa genome, we determined the haplotype number of predicted single-copy genes in a sequencing data set of cdna expressed in dpa c. sativa seeds. the reads were aligned to genes identified by duarte et al. as single-copy genes shared in flowering plants. the six genes with the highest coverage were selected for further evaluation. remarkably, all genes examined showed expression of three clear haplotypes as exemplified by the agmatine deiminase gene, indicating that the triplication of the genes in the c. sativa genome is common and not limited to fad fae and lfy. when the genomic status of the same genes was examined in the genomes of paleopolyploids such as maize and soybean, whose genome duplication is about million years old, only a subset of these genes was retained as duplicates. this lack of duplication in maize and soybean contrasted with the consistent pattern of triplication in c. sativa. arabidopsis genome sequencing project 2expressed cdna, this study 3maize genome sequencing project 4soybean genome sequencing project the genomes of c. sativa, c. alyssum, and c. microcarpa are larger than the genomes of other camelina species we calculated dna content in several accessions of c. sativa and related species from flow cytometry analyses using propidium iodide-stained nuclei. we used arabidopsis accession col- and its tetraploid derivative as genome size standards. c. sativa, c. alyssum, and c. microcarpa diploid genomes had a haploid content between and mb. c. sativa accessions uniformly displayed a genome size close to mb. north american isolates of c. sativa, c. alyssum, and c. microcarpa have reported chromosome counts of n =. the genomes of c. rumelica, c. hispida and c. laxa are smaller than those of c. sativa, c. alyssum, and c. microcarpa. chromosome counts of both n = and n = have been recorded for c. rumelica, while only a single count of n = exists for c. hispida. to our knowledge, no published counts exist for c. laxa. phylogenetic analysis of fad and fae indicate that c. sativa and c. microcarpa are closely related to understand the duplication history of the multiple fad and fae copies recovered from c. sativa, we amplified the fad and fae genes from several camelina species and outgroup species, and inferred phylogeny for each gene. the sampling of taxa chosen allowed us to test whether fad and fae duplication events occurred after camelina diverged from its closest relatives or within the genus. results from the evaluation of different models of sequence evolution using modeltest indicated that the fad sequence data are best described by the tvm+i+Γ model, while the fae data are best described by the hky+i+Γ model. likelihood phylogenetic analyses in paup* b produced a single fad tree, and a single fae tree. phylogenies inferred from fad and fae data indicate a history of duplication for both markers. both c. microcarpa and c. sativa have three distinct copies of fad and fae moreover, for fad the a and c copies from these two species are monophyletic with strong bootstrap support; for fae the a and b copies from these species are strongly monophyletic. in contrast, neither the fad b copies of c. sativa and c. microcarpa, nor the fae c copies of these species form a monophyletic group with each other. instead, our results indicate that c. rumelica has two distinct copies of fad and that one of these copies is strongly monophyletic with c. microcarpa fad b. we recovered only a single fad copy for c. laxa and c. hispida. in contrast, we recovered at least two distinct copies of fae from all sampled camelina species. the fae1- copy of c. laxa, c. hispida, and c. rumelica form a monophyletic group, with the former two species sister to one another with strong support. similar to the results from fad c. rumelica fae1- is sister to one of the c. microcarpa copies. neither the c. sativa fad b copy, nor the c. sativa fae c copy, shows a well supported sister relationship to other fad or fae sequences. however, in the fae tree, c. sativa fae c is very weakly supported as sister to c. hispida fae1-. finally, all recovered fad and fae copies from species of the genus camelina are monophyletic and sister to other sampled members of the tribe camelineae, consistent with phylogenies based on other markers. discussion camelina sativa is a re-emerging oilseed with tremendous potential as an alternative biofuel crop and for which genomic information is becoming increasingly available. we have obtained molecular data for nine genes, characterized in detail two genes encoding fatty acid biosynthesis enzymes and, in the process, have discovered unexpected complexity in the c. sativa genome. the close relationship between c. sativa and the model plant arabidopsis thaliana facilitates the manipulation of known pathways, such as the one regulating fatty acid biosynthesis. c. sativa seed oil is high in both polyunsaturated and long chain fatty acids, suggesting that both csfad and csfae are present and active. three copies each of the fad and fae genes were isolated from an agronomic accession of c. sativa using primers designed from a. thaliana or crambe abyssinica sequence. previously identified conserved sites in csfad and csfae are present in all three copies of each gene and a 5' intron shown to be important in regulating fad expression in sesame was identified in all three csfad copies. real time qpcr data and sequenom massarray snp analysis of the csfad and csfae cdna showed that all three copies of each gene are expressed in developing seeds. thus, it seems likely that all three copies of fad and fae in c. sativa are functional. the cloning of three copies of fad and fae from the c. sativa genome, as well as the observation of three lfy hybridization signals by southern analysis and three expressed haplotypes for more predicted single-copy genes in developing seeds, could be explained by at least two possible scenarios: segmental duplications of selected regions within a diploid genome either through tandem duplications or through transpositions, or whole genome duplications resulting from polyploidization. segmental duplications or transpositions affecting all nine examined loci are improbable compared with the explanation of polyploidy. furthermore, no evidence of recent segmental duplication involving multiple genes has been observed in sequenced plant genomes. triplication of the c. sativa genome therefore likely occurred through whole genome duplication, either through autopolyploidization or through allopolyploidization. an autopolyploidy event might have triplicated a single diploid genome resulting in an autohexaploid with a haploid genome of or chromosomes. given that c. sativa has a chromosome count of n = chromosome splitting or fusion could then have increased the chromosomes from to or decreased the chromosomes from or to alternatively, triplication of the c. sativa genome might have resulted from two allopolyploidy events, resulting in first a tetraploid then a hexaploid, similar to the origin of cultivated wheat. according to this hypothesis, the three copies of each gene diverged in different diploid genomes before converging through polyploidy events. taking into consideration the reported chromosome counts of various camelina species, the basal chromosome number of the diploid parental species contributing to the c. sativa haploid genome of chromosomes could be 7+7+ or 8+6+ the allopolyploid hypothesis is supported by the observation that c. sativa demonstrates diploid inheritance, as would be expected for an allopolyploid. a hexaploid c. sativa could also be derived from the combination of an autotetraploid and a diploid species if, in an autopolyploidized genome, homologous chromosomes differentiated so that the subsequent chromosome-specific pairing mimicked an allopolyploid genome in its diploid inheritance patterns. regardless of its evolutionary path, the c. sativa genome appears organized in three redundant and differentiated copies and can be formally considered to be an allohexaploid. results from our phylogenetic analyses support a history of duplication for both fad and fae in camelina. for fad duplications were only recovered for c. sativa, c. microcarpa, and c. rumelica. these data are consistent with genome size data, which indicate that all three genomes are larger than c. laxa and c. hispida, from which only a single fad copy was recovered. taken together, the results suggest that c. sativa, c. microcarpa, and c. rumelica are likely polyploids. given the slightly smaller genome size of c. rumelica, and the fact that we recovered only two fad copies from it, the c. rumelica sampled may be tetraploid while c. sativa and c. microcarpa are hexaploid. interestingly, in both the fad and fae trees, one copy each of c. rumelica and c. microcarpa are strongly supported as sister. thus, trees from these genes indicate that c. rumelica and c. microcarpa are closely related. the various placement of c. microcarpa fad and fae copies can be explained if c. microcarpa is the result of a hybridization event between c. rumelica and a currently unsampled, and thus unidentified species of camelina. two of the three copies of both fad and fae are identical, or nearly identical, in c. sativa and c. microcarpa, suggesting that c. sativa and c. microcarpa share a parental genome. thus, we suggest that a camelina species we did not sample contributed its genome to the hybrid formation of both c. sativa and c. microcarpa. in the case of c. microcarpa, the hybridization event likely involved c. rumelica. given the chromosome count of n = for c. rumelica, we expect the other putative parent to have an x = genome, and furthermore to be tetraploid at n = such a cross would result in the observed c. microcarpa genome, with chromosome count n = interestingly, c. hispida is the only species we sampled with a chromosome count of n = however no strong relationship between c. hispida and c. microcarpa is inferred in either gene tree. however, we do infer a weak relationship between c. sativa and c. hispida in the fae tree, and thus the possibility that c. hispida is involved in the polyploid formation of c. sativa should be explored further. what is the age of the polyploidization events likely to have formed the c. sativa genome? a complete answer will require a better understanding of its genome, but two findings suggest a recent origin. first, the chromosome number of c. sativa is inconsistent with extensive karyotype evolution and likely represents the sum of the ancestral contributions. second, paleopolyploids such as soybean and maize display duplication of many, but not all genes as a sizeable number have decayed to singleton state. in contrast, the presence of triplicates for nine test genes of c. sativa is consistent with high retention of duplicates, as expected in recent polyploids. the likely allohexaploid nature of the camelina sativa genome has multiple implications. its vigor and adaptability to marginal growth conditions may result at least in part from polyploidy. polyploids are thought to be more adaptable to new or harsh environments, with the ability to expand into broader niches than either progenitor. indeed, c. hispida and c. laxa, both of which are likely diploids, are found only in turkey, iran, armenia, and azerbaijan, while c. microcarpa and c. sativa are distributed throughout asia, europe, and north africa and are naturalized in north america. the mechanisms behind this increased adaptability are not completely understood, but have been attributed to heterosis, genetic and regulatory network redundancies, and epigenetic factors. allohexaploidy might also affect any potential manipulations of the c. sativa genome, such as introgression of germplasm or induced mutations. introgression of an exotic germplasm could be facilitated by the type of polyploidy-dependent manipulations that are possible in wheat, a potentially comparable allohexaploid. in addition, polyploids have displayed excellent response to reverse genomics approaches such as targeting induced local lesions ingenomes. as in wheat, any recessive induced mutations could be masked by redundant homoeologous loci that have maintained function. this mutation masking implies that multiple knockout alleles at different homoeologous sites can be combined to achieve partial or complete suppression of a targeted function. we also expect that single locus traits, whether transgenic or not, will display diploid inheritance due to preferential intragenomic pairing. in a hexaploid oilseed crop such as c. sativa, manipulations of oil composition and/or yield should therefore be possible through transgenic or reverse genetic approaches, or through other genome manipulations similar to those performed in wheat. for example, the characterization of fad and fae in c. sativa could enable the use of tilling techniques to isolate c. sativa plants with mutations in each of the three identified copies of both genes. we expect these mutations to result in plants with reduced levels of polyunsaturated fatty acids or long chain fatty acids, possibly in a dosage dependent manner. this will allow us to manipulate the seed oil composition of c. sativa, potentially creating a broad spectrum of c. sativa varieties possessing useful biodiesel properties, thereby further increasing the utility of this emerging biofuel crop.  the discovery of triplication and divergence of genes that in known diploids are present in single copy, the cytometrically determined genome size of camelina species, the pattern of relationship and inferred duplication history in the gene trees, together with the previously known chromosome counts for this taxon, indicate a likely allohexaploid genomic constitution. the characterization of genes encoding key functions of fatty acid biosynthesis lays the foundation for future manipulations of this pathway in camelina sativa. targeted manipulations of oil composition and general development of this crop, however, need to consider the implications of polyploidy and when possible take advantage of this common condition in crop plants. 
in addition, three expressed haplotypes were observed for six predicted single-copy genes in sequencing analysis and results from flow cytometry indicate that the dna content of c. sativa is approximately three-fold that of diploid camelina relatives. there is compelling evidence for triplication of the c. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other camelina relatives, three isolated copies of fad fae and the kcs17-fae intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. based on these results, we propose that c. sativa be considered an allohexaploid. phylogenetic analyses further support a history of duplication and indicate that c. sativa and c. microcarpa might share a parental genome. in c. sativa, southern analysis indicates the presence of three copies of both fad and fae as well as lfy, a known single copy gene in other species. camelina sativa, an oilseed crop in the brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. the characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of c. sativa should consider and, when possible take advantage of, the implications of polyploidy. a study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase and fatty acid elongase which revealed unexpected complexity in the c. sativa genome. little is understood of the nature of the c. sativa genome, however. the regions downstream of csfad and upstream of csfae demonstrate co-linearity with the arabidopsis genome.
 camelina sativa, an oilseed crop in the brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. little is understood of the nature of the c. sativa genome, however. a study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase and fatty acid elongase which revealed unexpected complexity in the c. sativa genome.  in c. sativa, southern analysis indicates the presence of three copies of both fad and fae as well as lfy, a known single copy gene in other species. all three copies of both csfad and csfae are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. the regions downstream of csfad and upstream of csfae demonstrate co-linearity with the arabidopsis genome. in addition, three expressed haplotypes were observed for six predicted single-copy genes in sequencing analysis and results from flow cytometry indicate that the dna content of c. sativa is approximately three-fold that of diploid camelina relatives. phylogenetic analyses further support a history of duplication and indicate that c. sativa and c. microcarpa might share a parental genome.  there is compelling evidence for triplication of the c. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other camelina relatives, three isolated copies of fad fae and the kcs17-fae intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. based on these results, we propose that c. sativa be considered an allohexaploid. the characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of c. sativa should consider and, when possible take advantage of, the implications of polyploidy. 