BACKGROUND
the adoption of arabidopsis thaliana as a model plant was suggested as early as  <dig>  yet its prominence in the study of plant genetics and physiology did not emerge until the 1980's with the recognition that its small genome and ease of manipulation offered the opportunity to mutate and study every gene within the genome  <cit> .
the ability to fully realise this objective has been facilitated through the development of an elegantly simple transformation system  <cit>  and the completion of the genome sequence  <cit> .
the most recent annotation of the genome sequence has identified a total of  <dig>  genes comprising  <dig>  protein coding genes,  <dig>  pseudogenes or transposable elements and  <dig>  non coding rnas .
computational biology tools allow the potential function of almost half of these proteins to be inferred, which provides an enormous resource for hypothesis driven research, while the remaining unknown proteins present an intriguing palette for curious researchers.
the development of tools to elucidate the function of the inferred genes is required in order to exploit the potential wealth of information provided by the annotated genome sequence.
large scale random mutagenesis has been utilised to successfully address the knowledge gap between sequence and function in a number of plant species  <cit>  and has been widely applied in a. thaliana  <cit> .
numerous strategies have been employed to saturate the genome, including exposure to chemical mutagens such as ethyl methanesulphonate   <cit> , transposon tagging  <cit> , fast neutron deletion  <cit>  and agrobacterium-mediated t-dna mutagenesis  <cit> .
while ems mutagenesis has the advantages of ease of application, non-biased distribution across the genome and generation of subtle phenotypes, its utility has been somewhat limited by the time-consuming map-based cloning required to verify the underlying gene responsible.
the use of specific dna insertional elements, such as transposons and t-dnas, allows the rapid identification of the point of entry in the genome using pcr based protocols, which have been optimised for high throughput sequencing  <cit> .
the generation of large collections of mutagenised lines and the concurrent sequencing of insertion sites to develop readily searchable databases for these populations has revolutionised gene characterisation by providing 'in silico' access to thousands of mutant alleles.
the arabidopsis community is fortunate that a number of populations are readily available for reverse genetics applications and can be accessed through the arabidopsis information resource .
in total, three publicly available t-dna flanking-sequence tag  databases provide access to over  <dig>  insertion sites; signal, flagdb and gabi-kat  <cit> , which have been estimated to interrupt the transcription of 80% of the annotated protein coding genes  <cit> .
although the utility of t-dna mutagenesis has been enhanced through the use of vectors that can facilitate gene, enhancer or promoter trapping  <cit> , there is an inherent limitation to simple insertional mutagenesis due to functional redundancy within the genome.
approximately 17% of a. thaliana genes are found in direct tandem repeats and 58% of the genome is thought to be duplicated, providing the plant with the ability to compensate for many null mutations  <cit> .
the development of vectors which can generate gain-of-function as well as loss-of-function alleles, so called activation tagging, has led to the discovery of a number of novel alleles controlling important functions in plant development, metabolism and stress responses  <cit> .
activation tagging exploits a tetrameric repeat of the enhancer element of the cauliflower mosaic virus  35s gene to direct the transcription of adjacent genes generating dominant phenotypes  <cit> .
although a number of resources have been developed for a. thaliana using this strategy  <cit> , access to these lines is generally via pooled seed samples or through databases of predetermined visual phenotypes .
in addition, ulker et al   <cit>  recently observed unanticipated activation and anomalous expression events in what would traditionally be considered knock-out populations suggesting that such populations may harbour novel phenotypes.
this study describes the generation of an archived activation tagged t-dna a. thaliana  population derived from almost  <dig>  individual t <dig> lines, where to date at least  <dig>  flanking sequence tags  have been identified to facilitate reverse and forward genetics applications http://aafc-aac.usask.ca/fst.
the distribution of the integration events in the genome was investigated and found to be closely correlated with gene density and not with recombination frequency although a reduction in frequency was observed across all datasets in centromeric regions.
the analyses identified the presence of novel alleles, multiple insertions sites, complex ti plasmid integrations and the somewhat unexpected assimilation of agrobacterium sequences into the genome.
the utility of the described population for identifying new mutations controlling a number of physiological traits is being explored and preliminary phenotypes are presented for trichome development and proanthocyanidin metabolism.
RESULTS
generation of the sk population
an a. thaliana t-dna mutagenised population, named sk, was developed and archived as t <dig> seed derived from  <dig>  individual herbicide resistant t <dig> lines with a t-dna transformation efficiency estimated to be ~ <dig> %.
single seed descent with continued selection was employed to generate a population of  <dig>  t <dig> families that will be enriched for homozygous mutant genotypes.
the number of independent insertion events per line was estimated initially by assessing the segregation ratio for herbicide resistance scored in the progeny from  <dig> t <dig> plants.
this resulted in an estimate of  <dig>  insertion loci/line suggesting the entire population may contain ~ <dig>  independent t-dna integration events.
however, southern analysis of  <dig> lines suggested a greater number of actual integration events  with a high percentage  of the insertion alleles being the result of complex t-dna integrations events .
this was later confirmed through sequence analysis of the dna flanking the t-dna left border , which is in contrast with the lower frequency of t-dna integration reported in previously characterised populations  <cit> .
genomic distribution of flanking sequence tags
tail-pcr was employed as a relatively efficient high-throughput strategy to amplify the sequence flanking the t-dna insertion events  present in the sk mutagenised population  <cit> .
the genetic origin of  <dig>  fst sequences derived from dna flanking the left border of stably inherited t-dna molecules was determined by analysing the sequence from amplification products generated from  <dig>  individual t2lines.
additional sequencing is on-going to characterise further sk lines.
the genomic location of the integrated t-dna molecules was determined by aligning each fst sequence with the five nuclear and two extra-nuclear a. thaliana pseudochromosomes.
the t-dna integration sites were classified based on the available annotation  and the frequency of integration in promoter, 5'-utr, exon, intron, 3'-utr and intergenic regions was determined .
this initial survey revealed integration events in  <dig>   unique gene regions including promoter sequence, with 36% of these insertion events predicted to interrupt exons.
t-dna integration events were observed more frequently in the untranslated sequences  and less frequently in intron and exon sequences  than expected based on their relative proportion of the annotated genome.
a. echr represents the two extra-nuclear genomes.
b. number of independent t-dna integrations.
c. number of independent disrupted genes.
d. number of unique genes with t-dna insertions.
the distribution of t-dna integration sites was not uniform, with many regions of the genome possessing either an over abundance or a dearth of insertion events .
the density of t-dna insertions was compared to both the level of gene expression in carpel tissue and the rate of genetic recombination previously observed for a. thaliana  <cit> .
there was strong correlation between the level of gene expression and the frequency of t-dna integration, but no correlation with recombination frequency along each chromosome; although a stark reduction in gene expression, recombination and t-dna insertion frequency was observed in the centromeric regions .
nearing a mutation saturated arabidopsis thaliana genome
the sk fst data combined with available sequence data from previously established t-dna mutagenised populations of a. thaliana, signal  <cit> , flagdb  <cit> , sail  <cit>  and gabi-kat  <cit> , revealed that the arabidopsis genome is reaching complete saturation with knock-out alleles now available for  <dig>   of the annotated genes .
when considering only those fsts residing in exon sequences, which are the mutations most likely to generate loss of function alleles, this number was reduced to  <dig>  and represented 71% of the annotated genes .
by assessing all populations,  <dig>   genes with multiple independent potentially deleterious alleles were identified, of which  <dig>   genes possessed multiple alleles with interrupted exon sequences.
unique insertion events have been identified in each population in proportion to the depth of fst sequence capture .
in particular, the sk population provides  <dig> novel insertion events in a. thaliana genes and a second allele for  <dig> genes.
a. a. thaliana ecotypes: col – colombia; and ws – wassilewskija.
b. number of fsts assigned to a unique position within the a. thaliana genome.
c. number of recorded fsts.
d. number of unique genes interrupted by an fst.
e. total number of unique genes with an insertion.
characterisation of the a. thaliana genes without insertions
there remain  <dig>  a. thaliana genes with no identified t-dna insertion event when all available populations are considered.
after removing  <dig>  annotated gene codes that were less than  <dig> bp in length , a number of basic characteristics were assessed for each of the remaining genes.
these included gene expression level from carpel tissue, position relative to the centromere, annotated length, and gene copy number .
a significant bias in gene length was observed with the median length for genes with and without an insert being  <dig>  bp and  <dig>  bp, respectively .
the distributions of gene expression levels for genes with and without insertions were also distinct .
the median absolute expression level was seven-fold lower for those genes without an insertion compared to those having a t-dna integration event.
this observation correlated with the position of the genes relative to the centromere, where gene expression is repressed, since those genes lacking an insertion event were found to be demonstrably closer to the centromeric region .
similarly, pseudogenes that are generally not expressed or expressed at low levels were three-fold over-represented among the gene annotations for gene codes with no observed t-dna integration.
identification of complex t-dna and non-ti plasmid integration
based on visual analysis of the fst sequence chromatogram files it was apparent that some of the fst sequences represented multiple amplification products .
further analyses of the fst database identified  <dig> sk lines harbouring two independent t-dna integration events  and an additional  <dig>  lines  with complex t-dna integration events .
figure  <dig> depicts the type and frequency of each complex insertion event observed, 73% of which were back-to-back tandem insertion events, with the majority being found in the left border-right border  orientation.
a portion  of the remaining lines contained a second left border sequence or internal t-dna vector sequence which identified a nested integration event.
in a small percentage of lines imprecise transfer of the t-dna resulted in integration of ti vector backbone sequence adjacent to the left border.
an additional  <dig> sk lines contained segments of agrobacterium tumefaciens genomic sequence, the majority of which  originated from the linear chromosome of a. tumefaciens.
this phenomenon was recently observed by ulker et al   <cit>  and suggests that transfer of bacterial genomic dna occurs at a low but discernable rate during agrobacterium plant transformation.
sk fst data handling and visualisation
the dna sequencing data for each sk line was warehoused using aped .
each fst was aligned to the genome sequence of a. thaliana and the resulting sequence similarity was used to represent the insertion site locations within gbrowse  <cit>  .
the dna sequencing data  as well as the visualization relative to the a. thaliana genome are available http://aafc-aac.usask.ca/fst.
forward genetic screens reveal novel mutations
aberrant morphological variation was observed in individual lines throughout the generation of the sk population and a number of these were confirmed as alleles of previously characterised mutations through the mapping of the fsts.
some examples of these included mutations in apetala <dig> , leafy , and cabbage .
in addition to loss-of-function alleles, gain-of-function mutants should also be discovered since the sk population was developed using a vector carrying multiple enhancer elements.
activation of genes adjacent to the insertion site was confirmed for at least two phenotypic variants, one leading to ectopic expression of a gibberellin oxidase resulting in a dwarf phenotype  <cit>  and the second to activation of an adjacent microrna resulting in enhanced seed carotenoid levels .
to fully realise the potential of this genetic resource, a number of forward genetic screens were initiated to identify lesions in targeted developmental and biochemical pathways.
the preliminary results from two screens dissecting trichome development and proanthocyanidin accumulation in the seed coat are presented.
fifty-one lines were selected by screening  <dig>  t <dig> sk seed lines and  <dig> sk t <dig> seed pools for seed colour variation and proanthocyanidin patterning.
concomitant screening of  <dig>  t <dig> non-activation t-dna lines  did not realise any seed colour variants.
based on visual inspection in comparison to wild type, selected lines were divided into colour categories, ranging from dark brown to yellow .
the seed coat phenotype for most of these lines appeared similar to published transparent testa  or tannin deficient seed  mutants after histochemical staining .
further studies have revealed altered phenotypes  resulting from mutant alleles of seven genes already known to be involved in proanthocyanidin biosynthesis.
in addition, on-going analysis of four proanthocyanidin variants suggests their novel phenotypes are conferred by mutations affecting previously uncharacterised genes, based on diallelic crossing with known mutants and molecular characterization of the insertion sites .
a typical wild-type a. thaliana leaf will have on average 97% of the trichomes with  <dig> branches , 1% two-branched, and 2% with four-branched trichomes as based on our analysis of  <dig> plants.
an initial set of  <dig>  t <dig> sk lines were screened for alterations in trichome morphology, from which thirteen showed variation in cell shape, branch number, or the texture of the cell surface .
sk <dig> produced small trichomes of which approximately 80% lacked aerial extension of the cell similar to glabrous mutants, while the remaining trichomes produced partially or fully extended spikes   <cit> .
sk <dig>  and sk <dig>  developed branchless trichomes, 100% branchless in the sk270; however, the phenotype of sk <dig> showed incomplete penetrance, such that 2–5% of the trichomes maintained two branches.
in three lines, all observed trichomes displayed short stalks with two branches.
in sk <dig> the two branches were of similar thickness; however, in sk <dig> and sk <dig>  one branch was thicker than the other and resembled a thumb and forefinger .
three lines had supernumerary branching phenotypes similar to kaktus  <cit> .
in two of these lines, sk <dig> and sk <dig>  all trichomes showed supernumerary branches , while in sk <dig> at least 90% of the trichomes had 4– <dig> branches and the remaining appeared wild type .
three lines were also identified with distorted trichome phenotypes  similar to the deformed trichomes of crooked and distorted <dig>  <cit> .
the final mutant, sk <dig>  had normal branching, but its mature trichome lacked papillae normally present on the cell surface  and were similar to the trichome birefringence mutant  <cit> .
fst sequences were available for four of the thirteen trichome mutant lines, which confirmed that sk <dig> and sk <dig> possessed alleles of stichel  <cit>  and zwichel  <cit>  respectively, as suggested by their observed trichome morphology.
the other two t-dna insertions were not located near any known trichome genes.
discussion
functional genomics tools are used to elucidate the role each gene plays within an organism.
due to its comparatively small size and the breadth of resources available, a. thaliana was a prime target to attempt a holistic assault on the genome .
the arabidopsis community and indeed related species such as the important crop brassica species have benefited greatly from the ambitious goal of assigning function to each of the ~ <dig>  annotated arabidopsis genes.
a number of t-dna mutagenised populations of a. thaliana have been developed and released into the public domain  <cit> , which greatly facilitate reverse genetic analysis of target genes through the identification of knock-out alleles.
the sk population of almost  <dig>  activation tagged a. thaliana lines was generated and archived as t <dig> seed through single seed descent to provide a resource for forward and reverse genetic screens.
the activity of the enhancer element present within the integrated t-dna was expected to produce novel alleles and to increase the likelihood of affecting phenotypes for genes previously masked through the inherent redundancy in the a. thaliana genome.
the sk lines carried an average of  <dig>  independently segregating insertions per line.
sequencing of dna flanking insertion sites has genetically characterised  <dig>  t-dna integration events in  <dig>  sk lines.
the distribution of insertion sites closely mirrored the gene content and gene expression level observed along the a. thaliana chromosomes, with a dearth of insertions in centromeric regions.
a comparison with previously characterised populations determined that the sk population provides  <dig> unique insertion events in previously untagged a. thaliana genes.
including the sk lines, the available populations provide multiple mutagenic alleles for  <dig>  loci.
since the background mutation rate in such populations has been estimated to be as high as 60%  <cit>  the availability of independent alleles for each gene is essential to confirm functional assignment.
mutagenic saturation of the a. thaliana gene complement has yet to be achieved, since  <dig>  loci still do not have a characterised t-dna insertion event.
an assessment of the loci without insertion events supports the previous analysis which suggested that t-dna integration preferentially targets transcriptionally active regions of the genome  <cit> .
among the genes lacking an insertion event there was a bias towards short loci that lacked introns and were expressed at very low levels in carpel tissue .
this bias could explain the prevalence of transcription factors which were found among the non-mutagenised loci.
single copy genes were not over-represented among the untagged loci, which might have been expected for essential non-redundant loci.
however, it is possible that such loci are being maintained within the populations in the hemizygous state.
the apparent necessity for accessible or open chromatin regions for t-dna integration is in conflict with the observed bias of insertion events to intergenic genomic sequence compared to annotated genic regions .
there is increasing evidence that there are additional unannotated a. thaliana loci present in the genome  <cit>  that could explain the apparent 'intergenic' insertion events.
however, only  <dig> of the  <dig>  intergenic insertion events within the sk population were associated with either the recently described  <dig>  sorfs predicted from whole genome expression tiling arrays or  <dig>  newly annotated proteins determined from extensive peptide sequencing  <cit> .
the observed discrepancy could be accounted for by insufficient annotation of distal regulatory regions, which have been erroneously classified as intergenic sequence.
based on the resolvable fst data, a notable number of the t-dna integration events were found to be complex in nature , predominantly indicating inverted or direct tandem insertion events.
although this implies that single genetic loci are affected, such loci complicate downstream cloning efforts and can potentially lead to additional chromosomal rearrangements  <cit> .
in recent years, collections of arabidopsis mutants  have been identified by screening for alterations in seed coat colour, flavonoid biosynthesis and proanthocyanidin accumulation  <cit> .
these lines have been used to investigate the flavonoid and proanthocyanidin pathways , yet the biochemical characterization of the latter stages of the pathway has been inadequate and the relative functional position of some proteins remains obscure  <cit> .
the poorly characterised steps in flavonoid synthesis could be elucidated further through exploitation of the sk lines.
similarly, questions remaining on the development and regulation of trichome formation  <cit>  could also be addressed using the described genetic resource.
the sk population is the first a. thaliana activation tagged population to be screened for seed coat colour, proanthocyanidin patterning, and trichome variation.
to date, we have recovered a broad assortment of mutants including  <dig> sk trichome variants, seven sk-tt lines defining new alleles of proanthocyanidin genes and four sk-tt lines with as yet uncharacterised proanthocyanidin phenotypes and genes .
these mutant lines will add a wealth of information to our understanding of the flavonoid pathway and trichome development.
the sk population already has proven its value by yielding new phenotypes and genes previously unknown to be involved in proanthocyanidin biosynthesis and regulation .
the population should be an excellent resource for exploring additional key processes controlling a multitude of traits and is currently being screened for mutants affected in abiotic stress tolerance and caretonoid biosynthesis.
CONCLUSIONS
an additional resource of almost  <dig>  t-dna tagged a. thaliana lines has been developed enabling the continuing efforts to assign function to the entire gene complement of a plant http://aafc-aac.usask.ca/fst.
this population can be screened for both loss and gain-of-function phenotypes due to the presence of enhancers on the integrated t-dna molecule.
mapping of the fsts for  <dig>  sk lines has identified insertion events in  <dig> genes with no previously recorded t-dna mutation and a second allele for  <dig> additional genes.
the utility of this population for capturing novel phenotypic variation has been demonstrated through initial screens assaying trichome development and proanthocyanidin accumulation.
the potential of this resource to genetically dissect complex physiological responses is being exploited in the study of the plant's response to abiotic stress.

 the adoption of arabidopsis thaliana as a model plant was suggested as early as yet its prominence in the study of plant genetics and physiology did not emerge until the 1980's with the recognition that its small genome and ease of manipulation offered the opportunity to mutate and study every gene within the genome. the ability to fully realise this objective has been facilitated through the development of an elegantly simple transformation system and the completion of the genome sequence. the most recent annotation of the genome sequence has identified a total of genes comprising protein coding genes, pseudogenes or transposable elements and non coding rnas. computational biology tools allow the potential function of almost half of these proteins to be inferred, which provides an enormous resource for hypothesis driven research, while the remaining unknown proteins present an intriguing palette for curious researchers. the development of tools to elucidate the function of the inferred genes is required in order to exploit the potential wealth of information provided by the annotated genome sequence. large scale random mutagenesis has been utilised to successfully address the knowledge gap between sequence and function in a number of plant species and has been widely applied in a. thaliana. numerous strategies have been employed to saturate the genome, including exposure to chemical mutagens such as ethyl methanesulphonate, transposon tagging, fast neutron deletion and agrobacterium-mediated t-dna mutagenesis. while ems mutagenesis has the advantages of ease of application, non-biased distribution across the genome and generation of subtle phenotypes, its utility has been somewhat limited by the time-consuming map-based cloning required to verify the underlying gene responsible. the use of specific dna insertional elements, such as transposons and t-dnas, allows the rapid identification of the point of entry in the genome using pcr based protocols, which have been optimised for high throughput sequencing. the generation of large collections of mutagenised lines and the concurrent sequencing of insertion sites to develop readily searchable databases for these populations has revolutionised gene characterisation by providing'in silico' access to thousands of mutant alleles. the arabidopsis community is fortunate that a number of populations are readily available for reverse genetics applications and can be accessed through the arabidopsis information resource. in total, three publicly available t-dna flanking-sequence tag databases provide access to over insertion sites; signal, flagdb and gabi-kat, which have been estimated to interrupt the transcription of 80% of the annotated protein coding genes. although the utility of t-dna mutagenesis has been enhanced through the use of vectors that can facilitate gene, enhancer or promoter trapping, there is an inherent limitation to simple insertional mutagenesis due to functional redundancy within the genome. approximately 17% of a. thaliana genes are found in direct tandem repeats and 58% of the genome is thought to be duplicated, providing the plant with the ability to compensate for many null mutations. the development of vectors which can generate gain-of-function as well as loss-of-function alleles, so called activation tagging, has led to the discovery of a number of novel alleles controlling important functions in plant development, metabolism and stress responses. activation tagging exploits a tetrameric repeat of the enhancer element of the cauliflower mosaic virus 35s gene to direct the transcription of adjacent genes generating dominant phenotypes. although a number of resources have been developed for a. thaliana using this strategy, access to these lines is generally via pooled seed samples or through databases of predetermined visual phenotypes. in addition, ulker et al recently observed unanticipated activation and anomalous expression events in what would traditionally be considered knock-out populations suggesting that such populations may harbour novel phenotypes. this study describes the generation of an archived activation tagged t-dna a. thaliana population derived from almost individual t lines, where to date at least flanking sequence tags have been identified to facilitate reverse and forward genetics applications http://aafc-aac.usask.ca/fst. the distribution of the integration events in the genome was investigated and found to be closely correlated with gene density and not with recombination frequency although a reduction in frequency was observed across all datasets in centromeric regions. the analyses identified the presence of novel alleles, multiple insertions sites, complex ti plasmid integrations and the somewhat unexpected assimilation of agrobacterium sequences into the genome. the utility of the described population for identifying new mutations controlling a number of physiological traits is being explored and preliminary phenotypes are presented for trichome development and proanthocyanidin metabolism.  generation of the sk population an a. thaliana t-dna mutagenised population, named sk, was developed and archived as t seed derived from individual herbicide resistant t lines with a t-dna transformation efficiency estimated to be ~ %. single seed descent with continued selection was employed to generate a population of t families that will be enriched for homozygous mutant genotypes. the number of independent insertion events per line was estimated initially by assessing the segregation ratio for herbicide resistance scored in the progeny from t plants. this resulted in an estimate of insertion loci/line suggesting the entire population may contain ~ independent t-dna integration events. however, southern analysis of lines suggested a greater number of actual integration events with a high percentage of the insertion alleles being the result of complex t-dna integrations events. this was later confirmed through sequence analysis of the dna flanking the t-dna left border, which is in contrast with the lower frequency of t-dna integration reported in previously characterised populations. genomic distribution of flanking sequence tags tail-pcr was employed as a relatively efficient high-throughput strategy to amplify the sequence flanking the t-dna insertion events present in the sk mutagenised population. the genetic origin of fst sequences derived from dna flanking the left border of stably inherited t-dna molecules was determined by analysing the sequence from amplification products generated from individual t2lines. additional sequencing is on-going to characterise further sk lines. the genomic location of the integrated t-dna molecules was determined by aligning each fst sequence with the five nuclear and two extra-nuclear a. thaliana pseudochromosomes. the t-dna integration sites were classified based on the available annotation and the frequency of integration in promoter, 5'-utr, exon, intron, 3'-utr and intergenic regions was determined. this initial survey revealed integration events in unique gene regions including promoter sequence, with 36% of these insertion events predicted to interrupt exons. t-dna integration events were observed more frequently in the untranslated sequences and less frequently in intron and exon sequences than expected based on their relative proportion of the annotated genome. a. echr represents the two extra-nuclear genomes. b. number of independent t-dna integrations. c. number of independent disrupted genes. d. number of unique genes with t-dna insertions. the distribution of t-dna integration sites was not uniform, with many regions of the genome possessing either an over abundance or a dearth of insertion events. the density of t-dna insertions was compared to both the level of gene expression in carpel tissue and the rate of genetic recombination previously observed for a. thaliana. there was strong correlation between the level of gene expression and the frequency of t-dna integration, but no correlation with recombination frequency along each chromosome; although a stark reduction in gene expression, recombination and t-dna insertion frequency was observed in the centromeric regions. nearing a mutation saturated arabidopsis thaliana genome the sk fst data combined with available sequence data from previously established t-dna mutagenised populations of a. thaliana, signal, flagdb, sail and gabi-kat, revealed that the arabidopsis genome is reaching complete saturation with knock-out alleles now available for of the annotated genes. when considering only those fsts residing in exon sequences, which are the mutations most likely to generate loss of function alleles, this number was reduced to and represented 71% of the annotated genes. by assessing all populations, genes with multiple independent potentially deleterious alleles were identified, of which genes possessed multiple alleles with interrupted exon sequences. unique insertion events have been identified in each population in proportion to the depth of fst sequence capture. in particular, the sk population provides novel insertion events in a. thaliana genes and a second allele for genes. a. a. thaliana ecotypes: col – colombia; and ws – wassilewskija. b. number of fsts assigned to a unique position within the a. thaliana genome. c. number of recorded fsts. d. number of unique genes interrupted by an fst. e. total number of unique genes with an insertion. characterisation of the a. thaliana genes without insertions there remain a. thaliana genes with no identified t-dna insertion event when all available populations are considered. after removing annotated gene codes that were less than bp in length, a number of basic characteristics were assessed for each of the remaining genes. these included gene expression level from carpel tissue, position relative to the centromere, annotated length, and gene copy number. a significant bias in gene length was observed with the median length for genes with and without an insert being bp and bp, respectively. the distributions of gene expression levels for genes with and without insertions were also distinct. the median absolute expression level was seven-fold lower for those genes without an insertion compared to those having a t-dna integration event. this observation correlated with the position of the genes relative to the centromere, where gene expression is repressed, since those genes lacking an insertion event were found to be demonstrably closer to the centromeric region. similarly, pseudogenes that are generally not expressed or expressed at low levels were three-fold over-represented among the gene annotations for gene codes with no observed t-dna integration. identification of complex t-dna and non-ti plasmid integration based on visual analysis of the fst sequence chromatogram files it was apparent that some of the fst sequences represented multiple amplification products. further analyses of the fst database identified sk lines harbouring two independent t-dna integration events and an additional lines with complex t-dna integration events. figure depicts the type and frequency of each complex insertion event observed, 73% of which were back-to-back tandem insertion events, with the majority being found in the left border-right border orientation. a portion of the remaining lines contained a second left border sequence or internal t-dna vector sequence which identified a nested integration event. in a small percentage of lines imprecise transfer of the t-dna resulted in integration of ti vector backbone sequence adjacent to the left border. an additional sk lines contained segments of agrobacterium tumefaciens genomic sequence, the majority of which originated from the linear chromosome of a. tumefaciens. this phenomenon was recently observed by ulker et al and suggests that transfer of bacterial genomic dna occurs at a low but discernable rate during agrobacterium plant transformation. sk fst data handling and visualisation the dna sequencing data for each sk line was warehoused using aped. each fst was aligned to the genome sequence of a. thaliana and the resulting sequence similarity was used to represent the insertion site locations within gbrowse. the dna sequencing data as well as the visualization relative to the a. thaliana genome are available http://aafc-aac.usask.ca/fst. forward genetic screens reveal novel mutations aberrant morphological variation was observed in individual lines throughout the generation of the sk population and a number of these were confirmed as alleles of previously characterised mutations through the mapping of the fsts. some examples of these included mutations in apetala, leafy, and cabbage. in addition to loss-of-function alleles, gain-of-function mutants should also be discovered since the sk population was developed using a vector carrying multiple enhancer elements. activation of genes adjacent to the insertion site was confirmed for at least two phenotypic variants, one leading to ectopic expression of a gibberellin oxidase resulting in a dwarf phenotype and the second to activation of an adjacent microrna resulting in enhanced seed carotenoid levels. to fully realise the potential of this genetic resource, a number of forward genetic screens were initiated to identify lesions in targeted developmental and biochemical pathways. the preliminary results from two screens dissecting trichome development and proanthocyanidin accumulation in the seed coat are presented. fifty-one lines were selected by screening t sk seed lines and sk t seed pools for seed colour variation and proanthocyanidin patterning. concomitant screening of t non-activation t-dna lines did not realise any seed colour variants. based on visual inspection in comparison to wild type, selected lines were divided into colour categories, ranging from dark brown to yellow. the seed coat phenotype for most of these lines appeared similar to published transparent testa or tannin deficient seed mutants after histochemical staining. further studies have revealed altered phenotypes resulting from mutant alleles of seven genes already known to be involved in proanthocyanidin biosynthesis. in addition, on-going analysis of four proanthocyanidin variants suggests their novel phenotypes are conferred by mutations affecting previously uncharacterised genes, based on diallelic crossing with known mutants and molecular characterization of the insertion sites. a typical wild-type a. thaliana leaf will have on average 97% of the trichomes with branches, 1% two-branched, and 2% with four-branched trichomes as based on our analysis of plants. an initial set of t sk lines were screened for alterations in trichome morphology, from which thirteen showed variation in cell shape, branch number, or the texture of the cell surface. sk produced small trichomes of which approximately 80% lacked aerial extension of the cell similar to glabrous mutants, while the remaining trichomes produced partially or fully extended spikes. sk and sk developed branchless trichomes, 100% branchless in the sk270; however, the phenotype of sk showed incomplete penetrance, such that 2–5% of the trichomes maintained two branches. in three lines, all observed trichomes displayed short stalks with two branches. in sk the two branches were of similar thickness; however, in sk and sk one branch was thicker than the other and resembled a thumb and forefinger. three lines had supernumerary branching phenotypes similar to kaktus. in two of these lines, sk and sk all trichomes showed supernumerary branches, while in sk at least 90% of the trichomes had 4– branches and the remaining appeared wild type. three lines were also identified with distorted trichome phenotypes similar to the deformed trichomes of crooked and distorted. the final mutant, sk had normal branching, but its mature trichome lacked papillae normally present on the cell surface and were similar to the trichome birefringence mutant. fst sequences were available for four of the thirteen trichome mutant lines, which confirmed that sk and sk possessed alleles of stichel and zwichel respectively, as suggested by their observed trichome morphology. the other two t-dna insertions were not located near any known trichome genes. discussion functional genomics tools are used to elucidate the role each gene plays within an organism. due to its comparatively small size and the breadth of resources available, a. thaliana was a prime target to attempt a holistic assault on the genome. the arabidopsis community and indeed related species such as the important crop brassica species have benefited greatly from the ambitious goal of assigning function to each of the ~ annotated arabidopsis genes. a number of t-dna mutagenised populations of a. thaliana have been developed and released into the public domain, which greatly facilitate reverse genetic analysis of target genes through the identification of knock-out alleles. the sk population of almost activation tagged a. thaliana lines was generated and archived as t seed through single seed descent to provide a resource for forward and reverse genetic screens. the activity of the enhancer element present within the integrated t-dna was expected to produce novel alleles and to increase the likelihood of affecting phenotypes for genes previously masked through the inherent redundancy in the a. thaliana genome. the sk lines carried an average of independently segregating insertions per line. sequencing of dna flanking insertion sites has genetically characterised t-dna integration events in sk lines. the distribution of insertion sites closely mirrored the gene content and gene expression level observed along the a. thaliana chromosomes, with a dearth of insertions in centromeric regions. a comparison with previously characterised populations determined that the sk population provides unique insertion events in previously untagged a. thaliana genes. including the sk lines, the available populations provide multiple mutagenic alleles for loci. since the background mutation rate in such populations has been estimated to be as high as 60% the availability of independent alleles for each gene is essential to confirm functional assignment. mutagenic saturation of the a. thaliana gene complement has yet to be achieved, since loci still do not have a characterised t-dna insertion event. an assessment of the loci without insertion events supports the previous analysis which suggested that t-dna integration preferentially targets transcriptionally active regions of the genome. among the genes lacking an insertion event there was a bias towards short loci that lacked introns and were expressed at very low levels in carpel tissue. this bias could explain the prevalence of transcription factors which were found among the non-mutagenised loci. single copy genes were not over-represented among the untagged loci, which might have been expected for essential non-redundant loci. however, it is possible that such loci are being maintained within the populations in the hemizygous state. the apparent necessity for accessible or open chromatin regions for t-dna integration is in conflict with the observed bias of insertion events to intergenic genomic sequence compared to annotated genic regions. there is increasing evidence that there are additional unannotated a. thaliana loci present in the genome that could explain the apparent'intergenic' insertion events. however, only of the intergenic insertion events within the sk population were associated with either the recently described sorfs predicted from whole genome expression tiling arrays or newly annotated proteins determined from extensive peptide sequencing. the observed discrepancy could be accounted for by insufficient annotation of distal regulatory regions, which have been erroneously classified as intergenic sequence. based on the resolvable fst data, a notable number of the t-dna integration events were found to be complex in nature, predominantly indicating inverted or direct tandem insertion events. although this implies that single genetic loci are affected, such loci complicate downstream cloning efforts and can potentially lead to additional chromosomal rearrangements. in recent years, collections of arabidopsis mutants have been identified by screening for alterations in seed coat colour, flavonoid biosynthesis and proanthocyanidin accumulation. these lines have been used to investigate the flavonoid and proanthocyanidin pathways, yet the biochemical characterization of the latter stages of the pathway has been inadequate and the relative functional position of some proteins remains obscure. the poorly characterised steps in flavonoid synthesis could be elucidated further through exploitation of the sk lines. similarly, questions remaining on the development and regulation of trichome formation could also be addressed using the described genetic resource. the sk population is the first a. thaliana activation tagged population to be screened for seed coat colour, proanthocyanidin patterning, and trichome variation. to date, we have recovered a broad assortment of mutants including sk trichome variants, seven sk-tt lines defining new alleles of proanthocyanidin genes and four sk-tt lines with as yet uncharacterised proanthocyanidin phenotypes and genes. these mutant lines will add a wealth of information to our understanding of the flavonoid pathway and trichome development. the sk population already has proven its value by yielding new phenotypes and genes previously unknown to be involved in proanthocyanidin biosynthesis and regulation. the population should be an excellent resource for exploring additional key processes controlling a multitude of traits and is currently being screened for mutants affected in abiotic stress tolerance and caretonoid biosynthesis.  an additional resource of almost t-dna tagged a. thaliana lines has been developed enabling the continuing efforts to assign function to the entire gene complement of a plant http://aafc-aac.usask.ca/fst. this population can be screened for both loss and gain-of-function phenotypes due to the presence of enhancers on the integrated t-dna molecule. mapping of the fsts for sk lines has identified insertion events in genes with no previously recorded t-dna mutation and a second allele for additional genes. the utility of this population for capturing novel phenotypic variation has been demonstrated through initial screens assaying trichome development and proanthocyanidin accumulation. the potential of this resource to genetically dissect complex physiological responses is being exploited in the study of the plant's response to abiotic stress. 
they allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. the population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented. the presence of enhancer elements on the inserted t-dna molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest. we have developed a population of almost activation tagged a. thaliana lines that have been archived as individual lines to the t generation. functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. insertion site sequences have been generated and mapped for lines to enable further application of the population, while providing a clear distribution of t-dna insertions across the genome. the population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants.
 functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. mutagenised plant populations are one such resource that facilitate gene characterisation. they allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. one limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.  we have developed a population of almost activation tagged a. thaliana lines that have been archived as individual lines to the t generation. the population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. insertion site sequences have been generated and mapped for lines to enable further application of the population, while providing a clear distribution of t-dna insertions across the genome. the population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.  this publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the arabidopsis genome sequence http://aafc-aac.usask.ca/fst. the presence of enhancer elements on the inserted t-dna molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest. 