BACKGROUND
loose silky bentgrass  beauv.
poaceae: pooidea) occurs in europe, central asia, north-west africa, caucasus, turkey, and north-west iran  <cit> .
it is the most serious agricultural weed in eastern and central europe, infesting many crops particularly winter wheat and winter barley .
it is an annual, dioecious, outcrossing species with bisexual flowers.
its life cycle consists of seed germination in autumn, then overwintering at the 2- <dig> leaf stage.
it resumes growth in early spring becoming taller than most crop species.
it flowers in mid-summer and finally sheds seeds around july  <cit> .
one plant can produce up to  <dig>  seeds which enables it to infest whole fields within a few generations  <cit> .
at a density of  <dig> plants per m <dig>  a. spica-venti decreases the yield of winter wheat up to 30% while another study  <cit>  estimates the reduction in crop yield to be proportional to the quantity of the weed  <cit> .
the european populations have shown a high degree of morphological and genetic variation  <cit>  allowing it to adapt quickly to changing agricultural landscapes.
control of apera spica-venti has been, and still is, heavily relying upon herbicides, specifically acetolactate synthase  and acetyl-coa  inhibitors  <cit> .
however, the continuous use of herbicides favored the evolution of resistance in a. spica-venti populations.
resistance to als herbicides was reported for the first time in switzerland in  <dig>  and has since been found in  <dig> other european countries  <cit> .
the study of resistance mechanisms is dependent on previous knowledge of genetic information such as gene identification, abundance, and nucleotide sequences.
information on other genes not related to herbicide resistance, as well as genetic information from susceptible genotypes, are, however, necessary for proper investigations  <cit> .
transcriptome data have been used in different weed species to study the origin of polyploidy events  <cit> , to understand genetics and biology of weeds , and to investigate herbicide resistance .
the majority of transcriptomic studies have focused on dictoyledonous weed species and only four grass weeds have been investigated at the transcriptomic level: lolium rigidum  <cit> , eleusine indica  <cit> , poa annua  <cit> , and echinocloa cruss-galli  <cit> .
the investigation into evolutionary and molecular processes in these weeds is therefore facilitated by the availability of molecular data.
publicly available genetic information on the diploid species a. spica-venti is very limited: only  <dig> nucleotide sequences from five genes  are available in the ncbi database  <cit> .
eight of these come from phylogenetic studies while the four remaining sequences are from herbicide resistance studies .
the aim of this study was to establish a de novo assembled comprehensive reference transcriptome of a. spica-venti from different tissues  and early phenological growth stages bbch <dig>   <dig>   <dig>  and  <dig>  we used multiple individuals from various herbicide susceptible populations in order to represent a precise and accurate reference transcriptome for this species which would contain the genetic variation within this outcrossing species.
each dataset, as well as the combined dataset, were de novo assembled and compared among themselves and to similar dataset from the grass weed lolium rigidum and the model organism brachypodium distachyon.
known herbicide resistance gene families, cytochrome p <dig> monooxygenase , glutathione s-transferase , atp-binding cassette transporters , elongation initiation factor , transcription factor  and glycosyltransferase , were identified and transcript abundance was quantified in each tissue.
RESULTS
transcriptome assembly
a de novo assembly approach was used in order to obtain a comprehensive reference transcriptome of a. spica-venti.
the illumina sequencing resulted in eight libraries having between  <dig> to 80 ×  <dig> reads each with an average read length of 145 bp for a total dataset of  <dig>  mbp.
on average, >80% of the raw reads passed the quality control and normalization process with an average percentage of bases quality score > <dig>  of 88%, an average phred score of  <dig> with mean base call accuracy of  <dig> %.
after trimming, quality control and normalization,  <dig> to 18 ×  <dig> high quality paired-end reads remained in the different libraries.
these were assembled into contigs ranging from  <dig>  to  <dig>  .
a de novo assembly using trinity software resulted in a contig number between  <dig>  and  <dig>  .
these were passed to the redundancy reduction steps .
the combined assembly show transcript lengths between  <dig> and 200 bp, while  <dig> transcripts are longer than  <dig>  bp .
finally, between  <dig>  and  <dig>  unigenes were identified in the eight individual non-redundant assemblies.
of these, >95% were annotated to uniprotkb and to brachypodium distachyon coding sequences .
some assembly statistics are lower for the combined assembly  compared to the individual assemblies, some statistics are higher or similar .table  <dig> assembly and annotation statistics for the combined assembly containing all three tissues and four growth stages along with individual assemblies for the seven tissue/growth stages of a. spica-venti
transcript number before rra
transcripts number after rra
hit % b. distachyon
arr; redundancy reduction steps .
surviving paired reads represents paired reads remaining after the quality control and normalization steps
similar to the individual assemblies, the combined dataset has  <dig>  sequences that have an enzyme code representing 12% of the total transcript number.
the e-values distribution of the combined assembly shows the 78% of transcripts with an e-value of <1e− <dig>  indicating high confidence in the annotation performed
transcriptome assembly quality
the eight assemblies show a high percentage of completeness , both when comparing them to all plants using benchmarking universal single-copy orthologs  and reads mapped back to the transcriptome  where paired-end reads are aligned to their respective transcriptome assembly.
for example, the combined assembly is 83% complete and 90% of its reads mapped back to its own transcripts.
this indicates that these assemblies are properly assembled and are of good quality.
the percentage of reads from each library that are properly paired when mapped to the combined assembly  is greater than the percentage properly paired when mapped to the assembly from that library  indicating that our combined assembly represent well the individual assemblies.
intermediate levels of duplication within assemblies  were found in the busco analysis .table  <dig> quality assessment of the assemblies using benchmarking universal single-copy orthologs  and reads mapped back to the transcriptome  of themselves and the combined assembly
this observed level of redundancy led us to create a zero-redundancy protein id list for each assembly in order to compare the similarity between the different assemblies without the redundancy.
we wanted to ensure that the level of sequence duplication was not introducing a bias in the comparative analysis of the assemblies.
the comparison of zero-redundant protein ids with their respective original assemblies reveals a  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 18% of redundancy in seed bbch <dig>   <dig>   <dig>   <dig>  leaves, root and stem, respectively.
these are similar levels of redundancy as was observed with busco.
the zero-redundant protein lists were further used to compare the number of shared proteins between tissues and growth stages .
the four germinated seed stages share 25% of proteins, while the three tissues share 27% of proteins .
the percentage of shared proteins between the  <dig> seed stages are lower  indicating that each growth stage is genetically distinct.
the assemblies for roots and stems are more similar than to the leaves assembly.
each tissue/stage has a large number of unique proteins.
root assembly has the highest proportion of unique proteins with  <dig>  sequences representing 36% of all its non-redundant expressed proteins recovered.fig.
<dig> venn diagram of non-redundant proteins.
left, between the four seed growth stages bbch <dig>   <dig>   <dig>  and  <dig>  right, between the three tissues leaves, roots, and stems
functional annotation
the lack of functional molecular data regarding a. spica-venti prompted us to perform a functional annotation on all eight assemblies.
similar to the individual assemblies, the combined dataset has  <dig>  sequences that have an enzyme code representing 12% of the total transcript number.
the e-values distribution of the combined assembly shows that 78% of the transcripts were annotated with an e-value of <1e− <dig>  indicating high confidence in the annotation performed.
the combined assembly has  <dig>  sequences belonging to the gene ontology  category of biological process,  <dig>  to metabolic function, and  <dig>  to cellular component for a total of  <dig>  go annotations .
the “organic substance metabolic process” was the most represented within the category biological process  with  <dig>  transcripts, then “ion binding” for the metabolic function  category with  <dig> , and “intracellular function” for the cellular component  with  <dig>  transcripts.
the comparison of functional annotations between individual assemblies shows that these are rather similar in their go annotations .
there does not appear to be any disproportionally represented go categories  between the different tissues and seed growth stages .fig.
<dig> comparison of gene ontology terms found in the seven individual and combined assemblies.
bp; biological processes, mf; metabolic function, cc; cellular component
kyoto encyclopedia of genes and genomes  analysis can be used to identify potential pathways most represented in each tissue/growth stages of a. spica-venti.
the top ten kegg pathways, based on sequences number, were compared between each assembly.
purine metabolism was the pathway with the most sequences in each individual assembly with  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  sequences in seeds bbch <dig>   <dig>   <dig>   <dig>  leaves, stem, root and combined assembly, respectively.
carbon fixation in photosynthetic organisms was also observed in the top ten for seeds at bbch <dig>   <dig>   <dig>  and in leaves with  <dig>   <dig>   <dig>  and  <dig> sequences, respectively.
all assemblies have similar pathways in their top ten such as important metabolisms pathways  and sugar pathways .
these most represented pathways are important basic pathways necessary for cell life and therefore found in high amounts in all plants.
in the top ten kegg pathways, biosynthesis of antibiotics was recovered in all assemblies while the plant secondary metabolite pathway was not recovered in any assembly.
in order to assess for fungal or bacterial contamination in our assemblies, every homologue species recovered from the annotation was verified and confirmed as either algae or green plant organisms.
secondly, the annotation of twenty random sequences that were categorized as part of the biosynthesis of antibiotics was verified.
the twenty sequences were all annotated to grass plants with a similarity percentage greater than 70%.
we believe that many plant secondary metabolites were most likely defined as antibiotics  in the kegg database.
our results indicate that plant antibiotics are produced to a large extent in all tissues of a. spica-venti.
the go enrichment analysis revealed no significantly enriched go category between individual assemblies and the combined assembly.
this result is consistent with figs.
<dig> and  <dig>  indicating that the individual assemblies are well represented within the combined and that the individual assemblies are similar to each other.
comparison to other grass species
comparative approaches are effective for finding differences and analogies between molecular dataset.
sequence conservation of the a. spica-venti assembly to phylogenetically related grasses and weeds was thus performed.
following annotation to uniprotkb, the highest numbers of homologues for the combined assembly were found in aegilops tauschii with  <dig>  transcripts representing 12% of the species distribution, followed by triticum aestivum with  <dig>  transcripts for 11% of transcripts .
overall, the top twenty species account for 77% of homologues in the combined assembly.
the individual assemblies showed a similar species distribution .
a comparison of non-redundant protein id was used  between a. spica-venti, lolium spp.
and b. distachyon.
the lolium spp.
sequences used come from schliesky et al.
<dig>  <cit>  and originate from a concatenation of three species .
the annotation of the lolium spp.
transcriptome yielded  <dig>  annotated protein sequences of which  <dig>   were non-redundant.
brachypodium distachyon yielded  <dig>  protein transcripts of which  <dig>   were non-redundant.
apera spica-venti had  <dig>   non-redundant protein annotations.
these three species share 21% proteins .
apera has a higher percentage of protein similarity to lolium  than to b. distachyon .
apera also has a similar annotation redundancy percentage to lolium spp with 42% and 44%, respectively.
twentytwo percentage of annotations in b. distachyon was redundant.
this lower number is expected considering that this species is a model organism with better annotated data and also because the sequences used here are ab initio predicted from cdna extracted from genomic studies.fig.
<dig> venn diagram of non redundant proteins between a. spica-venti, lolium, and b. distachyon
herbicide resistance gene family
genes involved in herbicide resistance mechanisms were identified in varying number in the different tissues of a. spica venti.
glycosyltransferase is the most abundant  in each tissue and growth stage.
seed growth stages had the highest number of gt  with an average of  <dig> sequences versus  <dig> sequences for the three tissues .
the majority of gt sequences found were homologues of barley .
the gst transcripts were found in low amount in a. spica-venti overall .
five sequences identified as gstf <dig> in apera were identified from seeds bbch <dig>  bbch <dig>  bbch <dig>  root and stem.
sequences identified as gstf <dig> were aligned with the amgstf <dig> identified in blackgrass  as this specific sequence was shown to endow herbicide resistance  <cit> .
these had a 38-66% similarity to the a. myosuroides amgstf <dig> sequence.
this large range of percentage of similarity is attributable to length differences as no snps were identified between the different apera gstf <dig> sequences.
however, a three amino acids insertion was found in the blackgrass sequence.
the three amino acids are asparagine, glutamine and valine  at position  <dig> to 106 bp .
both alleles are functional.
these amino acids were not present in the apera neither in the triticum uratu sequence.
it is possible that this insertion in a. myosuroides is actually a deletion in a. spica-venti and t. uratu.
a gst <dig> identified in e. crus-galli also responsible for herbicide resistance  <cit>  was also identified in apera.fig.
<dig> transcript abundance for major herbicide resistance gene families in the seven different tissues and growth stages from a. spica-venti.
p450; cytochrome p <dig>  gst; glutathione s-transferase, gt; glycosyltransferase, abc; abc transporters, eif; elongation initiation factor, tf; transcription factor
the cytochrome p450s transcripts were found in high numbers across a. spica-venti tissues and growth stages .
we identified a total of  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> cytochrome p <dig> sequences in seeds bbch  <dig>   <dig>   <dig>   <dig>  leaves, root and stem respectively .
a total of  <dig> different cytochrome p450s were identified throughout the seven individuals assemblies.
many sequences were identified as p450s but did not have a subfamily identification and therefore were not counted in this number.
the most abundant p <dig> was from subfamily cyp <dig>  with  <dig> sequences identified as such in the combined assembly .
this specific p <dig> was also the most abundant of the p450s in the seed stages and in leaves with  <dig>   <dig>   <dig>   <dig> and  <dig> sequences respectively.
the second most abundant p <dig> is cyp89a <dig> .
sequences identified as cyp89a <dig> in a. spica-venti were aligned with the cyp89a <dig> identified in l. rigidum  shown to confer herbicide resistance  <cit> .
multiple cyp89a <dig> sequences  were identified in each tissue, with the highest number  found in seeds at bbch  <dig>  overall,  <dig> sequences of p <dig> cyp89a <dig> were found.
a large variation in length was observed with the shortest being  <dig> amino acid long and the longest  <dig> amino acids .
the alignment of all cyp89a <dig> sequences show a 14-40% sequence similarity to lolium spp, with the highest score belonging to sequences that were annotated to the gene triur3_ <dig>  from t. uratu.
this demonstrates high sequence polymorphism among the four grass species for cyp89a <dig>  the p <dig> family was further compared based on protein identification between the seven tissues and growth stages.
overall, the four seed growth stages express 18% of the same p <dig> in optimal growing conditions .
seeds at the bbch  <dig> had the highest number of uniquely expressed p450s .
the three tissues have almost 20% of identical p450s expressed while root have the highest number of uniquely expressed p450s.fig.
<dig> top  <dig> most abundant cytochrome p450s in a. spica-venti
fig.
<dig> venn diagram of shared p <dig> proteins.
left, between germinated seeds at bbch stage  <dig>   <dig>   <dig>  and  <dig>  right, between leaf, root, and stem
discussion
the aim of this study was to enhance genetic resources for a. spica-venti and through the provision of a reference transcriptome creating a platform to decipher, among others, the development of herbicide resistance.
in this study, through a de novo assembly and redundancy reduction strategy,  <dig>  unigenes were found in the combined assembly of a. spica-venti which showed a high level of completeness, as well as in the seven individual assemblies.
the combined assembly is expected to have assembly statistics differences with the individual assemblies since it contains much more reads and has a 7-fold duplication of many transcripts.
the assembly and homology search statistics are all congruent with previous transcriptome studies in poaceae.
the observed n <dig> for a. spica-venti are similar to the values from lolium spp de novo transcriptome , l. rigidum , leymus chinensis  and phragmites australis , but are lower when compared to zea mays , and arundo donax , poa annua , l. perenne , and eleusine indica .
the number of transcript after redundancy reduction is slightly lower than the number found after filtering in lolium spp.
<cit> .
the percentage of functionally annotated protein sequence is also in accordance with lolium spp.
results .
the observed top go terms in each category  are also found in top positions in e. indica  <cit> , l. chinensis  <cit> , and e. crus-galli  <cit> .
the percentage of properly paired reads to their respective assembly  is in line with similar results from a. donax  <cit> .
overall, the similarity found between the a. spica-venti transcriptome and other grass transcriptomes indicate a properly assembled and annotated dataset presented here.
although a strict redundancy reduction strategy was used during the assembly, the a. spica-venti transcriptome still shows an intermediate level of transcript redundancy.
this indicates that many reconstructed transcript were similar enough to annotate to the same description, yet different enough not to be combined during the assembly.
several biological and technical biases could explain this.
biologically, different gene copy number, alternative splicing, heterozygosity and transcripts from large gene families could explain intermediate levels of redundancy.
alternatively, library preparation such as the amplification step and rna fragmentation steps, as well as fragmented contigs could also be an explanation.
this redundancy can be seen in the alignment of cyp89a <dig> , where there is a large variation in sequence similarity and length.
both a. spica-venti and lolium spp showed similar levels of redundancy.
this high level of sequence heterogeneity most likely stems from the heterogeneous genetic background of multiple individuals from different populations used in this study to create the transcriptome representing natural variation within a. spica-venti.
transcript redundancy is a biological artifact that complicates de novo transcriptome assembly when using heterogeneous data  <cit> .
experimentally, contig redundancy could be minimized by performing several generational crosses between individuals from the same population.
the level of redundancy in a. spica-venti is consistent across tissues and growth stages and can be identified by multiple methods presented here  and most importantly, does not affect the levels of similarity between a. spica-venti assemblies.
with and without the redundancy, the a. spica-venti transcriptome has high levels of similarity with phylogenetically related grass species such as b. distachyon and lolium spp.
our study shows that a reference transcriptome can be successfully created from multiple genetically heterogeneous individuals.
using extra steps after the initial de novo assembly can reduce redundancy drastically.
the absence of go enrichment between the different tissues and growth stages is unsurprising considering the large sample size being compared and the fact that the assemblies were constructed from material under the same biotic and abiotic conditions.
this lack of enrichment indicates also that the combined assembly represents well the variation within each tissue and growth stages.
the fact that the a. spica-venti transcriptome is more similar to lolium spp.
than to b. distachyon, despite b. distachyon being a much better annotated reference, is most likely caused by the close phylogenetic relationship or could indicate a genetic similarity between grass weeds.
the genera apera, lolium, alopecurus and poa are all part of the large monophyletic core pooideae while brachypodium is part of the brachypodieae  <cit> .
many populations of a. spica-venti are resistant to herbicides and some have been identified as metabolic resistance cases, with a strong suspicion on the cytochrome p450s family, but never confirmed  <cit> .
we find a higher number of total sequences corresponding to some key herbicide resistance gene families in a. spica-venti than compared to lolium rigidum reference, which contained  <dig> gst and  <dig> gt sequences  <cit> .
the total number of different p450s recovered in this study  is in line with the  <dig> recovered in the transcriptome of lolium rigidum  <cit> .
however, this number is far below the number of p450s identified in other poaceae species such as rice  and sorghum , respectively  <cit> .
transcriptome studies do not recover the total number of genes present in an organism.
this tells us that herbicide susceptible populations of a. spica-venti in denmark express a low number of p450s in optimal growing conditions.
this is consistent with the knowledge of p450s as being involved in stress and xenobioticide defense in plants.
the fact that the roots presented the largest number of unique p450s could indicate they were under some undetermined stress and more likely to indicate higher basal p <dig> expression.
roots in zea mays and in arabidopsis are known to express p450s  <cit>  and some p450s are expressed predominantly in roots  <cit> , but there is no evidence that this is a general trend for the p <dig> family.
the most abundant p <dig>  cyp71d <dig>  has been shown to be implicated in glycoalkaloid and ginsenosides biosynthesis, stress-induced secondary metabolites, as well as green tissue regeneration, especially in shoot development stages, in many crops .
interestingly, cyp71d <dig> is also highly upregulated in blight-resistant potato  <cit> .
this hints at a potential role in herbicide resistance in a. spica-venti, but remains to be investigated.
the large sequence variation in cyp89a <dig> between lolium, apera, and triticum, could indicate that these sequences are not highly conserved as they are implicated in environmental adaptation.
the a. spica-venti and a. myosuroides gstf <dig> sequences are similar indicating a potential for similar metabolic activity in the presence of herbicide as was demonstrated in cummins et al  <cit> .
these two known resistance genes  were recovered in the majority of tissues of this reference transcriptome of a. spica-venti indicate that these are constitutively expressed at many early stages and overall in the plant.
gst are known to be expressed at every stage of plant development in every tissue  <cit> .
many of the known herbicide resistant p450s, gsts, and eifs identified in other weeds species  were not recovered.
CONCLUSIONS
in the present study, we have described the construction and functional annotation of a de novo reference transcriptome assembly for apera spica-venti with emphasis on gene families involved in herbicide detoxification.
these transcriptomes represents a considerable addition to the molecular knowledge available for this important weed species.
moreover, go and kegg analysis were conducted and all unigenes were classified into functional categories with the aim to understand their role and metabolic pathways.
this data can be used to develop oligo-nucleotide microarray to study gene expression studies at large scale.
we have characterized the major gene families responsible for herbicide resistance and shown strong potential of two specific genes to have a role in herbicide resistance in a. spica-venti.
finally, this study can serve as a reference to unravel genes and pathways involved in metabolic herbicide resistance mechanisms, weed adaption, grass genetics and trait evolution in the future.

 loose silky bentgrass beauv. poaceae: pooidea) occurs in europe, central asia, north-west africa, caucasus, turkey, and north-west iran. it is the most serious agricultural weed in eastern and central europe, infesting many crops particularly winter wheat and winter barley. it is an annual, dioecious, outcrossing species with bisexual flowers. its life cycle consists of seed germination in autumn, then overwintering at the 2- leaf stage. it resumes growth in early spring becoming taller than most crop species. it flowers in mid-summer and finally sheds seeds around july. one plant can produce up to seeds which enables it to infest whole fields within a few generations. at a density of plants per m a. spica-venti decreases the yield of winter wheat up to 30% while another study estimates the reduction in crop yield to be proportional to the quantity of the weed. the european populations have shown a high degree of morphological and genetic variation allowing it to adapt quickly to changing agricultural landscapes. control of apera spica-venti has been, and still is, heavily relying upon herbicides, specifically acetolactate synthase and acetyl-coa inhibitors. however, the continuous use of herbicides favored the evolution of resistance in a. spica-venti populations. resistance to als herbicides was reported for the first time in switzerland in and has since been found in other european countries. the study of resistance mechanisms is dependent on previous knowledge of genetic information such as gene identification, abundance, and nucleotide sequences. information on other genes not related to herbicide resistance, as well as genetic information from susceptible genotypes, are, however, necessary for proper investigations. transcriptome data have been used in different weed species to study the origin of polyploidy events, to understand genetics and biology of weeds, and to investigate herbicide resistance. the majority of transcriptomic studies have focused on dictoyledonous weed species and only four grass weeds have been investigated at the transcriptomic level: lolium rigidum, eleusine indica, poa annua, and echinocloa cruss-galli. the investigation into evolutionary and molecular processes in these weeds is therefore facilitated by the availability of molecular data. publicly available genetic information on the diploid species a. spica-venti is very limited: only nucleotide sequences from five genes are available in the ncbi database. eight of these come from phylogenetic studies while the four remaining sequences are from herbicide resistance studies. the aim of this study was to establish a de novo assembled comprehensive reference transcriptome of a. spica-venti from different tissues and early phenological growth stages bbch and we used multiple individuals from various herbicide susceptible populations in order to represent a precise and accurate reference transcriptome for this species which would contain the genetic variation within this outcrossing species. each dataset, as well as the combined dataset, were de novo assembled and compared among themselves and to similar dataset from the grass weed lolium rigidum and the model organism brachypodium distachyon. known herbicide resistance gene families, cytochrome p monooxygenase, glutathione s-transferase, atp-binding cassette transporters, elongation initiation factor, transcription factor and glycosyltransferase, were identified and transcript abundance was quantified in each tissue.  transcriptome assembly a de novo assembly approach was used in order to obtain a comprehensive reference transcriptome of a. spica-venti. the illumina sequencing resulted in eight libraries having between to 80 ×  reads each with an average read length of 145 bp for a total dataset of mbp. on average, >80% of the raw reads passed the quality control and normalization process with an average percentage of bases quality score > of 88%, an average phred score of with mean base call accuracy of %. after trimming, quality control and normalization, to 18 ×  high quality paired-end reads remained in the different libraries. these were assembled into contigs ranging from to. a de novo assembly using trinity software resulted in a contig number between and. these were passed to the redundancy reduction steps. the combined assembly show transcript lengths between and 200 bp, while transcripts are longer than  bp. finally, between and unigenes were identified in the eight individual non-redundant assemblies. of these, >95% were annotated to uniprotkb and to brachypodium distachyon coding sequences. some assembly statistics are lower for the combined assembly compared to the individual assemblies, some statistics are higher or similar.table assembly and annotation statistics for the combined assembly containing all three tissues and four growth stages along with individual assemblies for the seven tissue/growth stages of a. spica-venti transcript number before rra transcripts number after rra  hit % b. distachyon arr; redundancy reduction steps. surviving paired reads represents paired reads remaining after the quality control and normalization steps similar to the individual assemblies, the combined dataset has sequences that have an enzyme code representing 12% of the total transcript number. the e-values distribution of the combined assembly shows the 78% of transcripts with an e-value of <1e− indicating high confidence in the annotation performed transcriptome assembly quality the eight assemblies show a high percentage of completeness, both when comparing them to all plants using benchmarking universal single-copy orthologs and reads mapped back to the transcriptome where paired-end reads are aligned to their respective transcriptome assembly. for example, the combined assembly is 83% complete and 90% of its reads mapped back to its own transcripts. this indicates that these assemblies are properly assembled and are of good quality. the percentage of reads from each library that are properly paired when mapped to the combined assembly is greater than the percentage properly paired when mapped to the assembly from that library indicating that our combined assembly represent well the individual assemblies. intermediate levels of duplication within assemblies were found in the busco analysis.table quality assessment of the assemblies using benchmarking universal single-copy orthologs and reads mapped back to the transcriptome of themselves and the combined assembly this observed level of redundancy led us to create a zero-redundancy protein id list for each assembly in order to compare the similarity between the different assemblies without the redundancy. we wanted to ensure that the level of sequence duplication was not introducing a bias in the comparative analysis of the assemblies. the comparison of zero-redundant protein ids with their respective original assemblies reveals a and 18% of redundancy in seed bbch leaves, root and stem, respectively. these are similar levels of redundancy as was observed with busco. the zero-redundant protein lists were further used to compare the number of shared proteins between tissues and growth stages. the four germinated seed stages share 25% of proteins, while the three tissues share 27% of proteins. the percentage of shared proteins between the seed stages are lower indicating that each growth stage is genetically distinct. the assemblies for roots and stems are more similar than to the leaves assembly. each tissue/stage has a large number of unique proteins. root assembly has the highest proportion of unique proteins with sequences representing 36% of all its non-redundant expressed proteins recovered.fig. venn diagram of non-redundant proteins. left, between the four seed growth stages bbch and right, between the three tissues leaves, roots, and stems functional annotation the lack of functional molecular data regarding a. spica-venti prompted us to perform a functional annotation on all eight assemblies. similar to the individual assemblies, the combined dataset has sequences that have an enzyme code representing 12% of the total transcript number. the e-values distribution of the combined assembly shows that 78% of the transcripts were annotated with an e-value of <1e− indicating high confidence in the annotation performed. the combined assembly has sequences belonging to the gene ontology category of biological process, to metabolic function, and to cellular component for a total of go annotations. the “organic substance metabolic process” was the most represented within the category biological process with transcripts, then “ion binding” for the metabolic function category with, and “intracellular function” for the cellular component with transcripts. the comparison of functional annotations between individual assemblies shows that these are rather similar in their go annotations. there does not appear to be any disproportionally represented go categories between the different tissues and seed growth stages.fig. comparison of gene ontology terms found in the seven individual and combined assemblies. bp; biological processes, mf; metabolic function, cc; cellular component kyoto encyclopedia of genes and genomes analysis can be used to identify potential pathways most represented in each tissue/growth stages of a. spica-venti. the top ten kegg pathways, based on sequences number, were compared between each assembly. purine metabolism was the pathway with the most sequences in each individual assembly with,,,,,, and sequences in seeds bbch leaves, stem, root and combined assembly, respectively. carbon fixation in photosynthetic organisms was also observed in the top ten for seeds at bbch and in leaves with and sequences, respectively. all assemblies have similar pathways in their top ten such as important metabolisms pathways and sugar pathways. these most represented pathways are important basic pathways necessary for cell life and therefore found in high amounts in all plants. in the top ten kegg pathways, biosynthesis of antibiotics was recovered in all assemblies while the plant secondary metabolite pathway was not recovered in any assembly. in order to assess for fungal or bacterial contamination in our assemblies, every homologue species recovered from the annotation was verified and confirmed as either algae or green plant organisms. secondly, the annotation of twenty random sequences that were categorized as part of the biosynthesis of antibiotics was verified. the twenty sequences were all annotated to grass plants with a similarity percentage greater than 70%. we believe that many plant secondary metabolites were most likely defined as antibiotics in the kegg database. our results indicate that plant antibiotics are produced to a large extent in all tissues of a. spica-venti. the go enrichment analysis revealed no significantly enriched go category between individual assemblies and the combined assembly. this result is consistent with figs.  and indicating that the individual assemblies are well represented within the combined and that the individual assemblies are similar to each other. comparison to other grass species comparative approaches are effective for finding differences and analogies between molecular dataset. sequence conservation of the a. spica-venti assembly to phylogenetically related grasses and weeds was thus performed. following annotation to uniprotkb, the highest numbers of homologues for the combined assembly were found in aegilops tauschii with transcripts representing 12% of the species distribution, followed by triticum aestivum with transcripts for 11% of transcripts. overall, the top twenty species account for 77% of homologues in the combined assembly. the individual assemblies showed a similar species distribution. a comparison of non-redundant protein id was used between a. spica-venti, lolium spp. and b. distachyon. the lolium spp. sequences used come from schliesky et al. and originate from a concatenation of three species. the annotation of the lolium spp. transcriptome yielded annotated protein sequences of which were non-redundant. brachypodium distachyon yielded protein transcripts of which were non-redundant. apera spica-venti had non-redundant protein annotations. these three species share 21% proteins. apera has a higher percentage of protein similarity to lolium than to b. distachyon. apera also has a similar annotation redundancy percentage to lolium spp with 42% and 44%, respectively. twentytwo percentage of annotations in b. distachyon was redundant. this lower number is expected considering that this species is a model organism with better annotated data and also because the sequences used here are ab initio predicted from cdna extracted from genomic studies.fig. venn diagram of non redundant proteins between a. spica-venti, lolium, and b. distachyon herbicide resistance gene family genes involved in herbicide resistance mechanisms were identified in varying number in the different tissues of a. spica venti. glycosyltransferase is the most abundant in each tissue and growth stage. seed growth stages had the highest number of gt with an average of sequences versus sequences for the three tissues. the majority of gt sequences found were homologues of barley. the gst transcripts were found in low amount in a. spica-venti overall. five sequences identified as gstf in apera were identified from seeds bbch bbch bbch root and stem. sequences identified as gstf were aligned with the amgstf identified in blackgrass as this specific sequence was shown to endow herbicide resistance. these had a 38-66% similarity to the a. myosuroides amgstf sequence. this large range of percentage of similarity is attributable to length differences as no snps were identified between the different apera gstf sequences. however, a three amino acids insertion was found in the blackgrass sequence. the three amino acids are asparagine, glutamine and valine at position to 106 bp. both alleles are functional. these amino acids were not present in the apera neither in the triticum uratu sequence. it is possible that this insertion in a. myosuroides is actually a deletion in a. spica-venti and t. uratu. a gst identified in e. crus-galli also responsible for herbicide resistance was also identified in apera.fig. transcript abundance for major herbicide resistance gene families in the seven different tissues and growth stages from a. spica-venti. p450; cytochrome p gst; glutathione s-transferase, gt; glycosyltransferase, abc; abc transporters, eif; elongation initiation factor, tf; transcription factor the cytochrome p450s transcripts were found in high numbers across a. spica-venti tissues and growth stages. we identified a total of and cytochrome p sequences in seeds bbch leaves, root and stem respectively. a total of different cytochrome p450s were identified throughout the seven individuals assemblies. many sequences were identified as p450s but did not have a subfamily identification and therefore were not counted in this number. the most abundant p was from subfamily cyp with sequences identified as such in the combined assembly. this specific p was also the most abundant of the p450s in the seed stages and in leaves with and sequences respectively. the second most abundant p is cyp89a. sequences identified as cyp89a in a. spica-venti were aligned with the cyp89a identified in l. rigidum shown to confer herbicide resistance. multiple cyp89a sequences were identified in each tissue, with the highest number found in seeds at bbch overall, sequences of p cyp89a were found. a large variation in length was observed with the shortest being amino acid long and the longest amino acids. the alignment of all cyp89a sequences show a 14-40% sequence similarity to lolium spp, with the highest score belonging to sequences that were annotated to the gene triur3_ from t. uratu. this demonstrates high sequence polymorphism among the four grass species for cyp89a the p family was further compared based on protein identification between the seven tissues and growth stages. overall, the four seed growth stages express 18% of the same p in optimal growing conditions. seeds at the bbch had the highest number of uniquely expressed p450s. the three tissues have almost 20% of identical p450s expressed while root have the highest number of uniquely expressed p450s.fig. top most abundant cytochrome p450s in a. spica-venti fig. venn diagram of shared p proteins. left, between germinated seeds at bbch stage and right, between leaf, root, and stem discussion the aim of this study was to enhance genetic resources for a. spica-venti and through the provision of a reference transcriptome creating a platform to decipher, among others, the development of herbicide resistance. in this study, through a de novo assembly and redundancy reduction strategy, unigenes were found in the combined assembly of a. spica-venti which showed a high level of completeness, as well as in the seven individual assemblies. the combined assembly is expected to have assembly statistics differences with the individual assemblies since it contains much more reads and has a 7-fold duplication of many transcripts. the assembly and homology search statistics are all congruent with previous transcriptome studies in poaceae. the observed n for a. spica-venti are similar to the values from lolium spp de novo transcriptome, l. rigidum, leymus chinensis and phragmites australis, but are lower when compared to zea mays, and arundo donax, poa annua, l. perenne, and eleusine indica. the number of transcript after redundancy reduction is slightly lower than the number found after filtering in lolium spp.. the percentage of functionally annotated protein sequence is also in accordance with lolium spp. results. the observed top go terms in each category are also found in top positions in e. indica, l. chinensis, and e. crus-galli. the percentage of properly paired reads to their respective assembly is in line with similar results from a. donax. overall, the similarity found between the a. spica-venti transcriptome and other grass transcriptomes indicate a properly assembled and annotated dataset presented here. although a strict redundancy reduction strategy was used during the assembly, the a. spica-venti transcriptome still shows an intermediate level of transcript redundancy. this indicates that many reconstructed transcript were similar enough to annotate to the same description, yet different enough not to be combined during the assembly. several biological and technical biases could explain this. biologically, different gene copy number, alternative splicing, heterozygosity and transcripts from large gene families could explain intermediate levels of redundancy. alternatively, library preparation such as the amplification step and rna fragmentation steps, as well as fragmented contigs could also be an explanation. this redundancy can be seen in the alignment of cyp89a, where there is a large variation in sequence similarity and length. both a. spica-venti and lolium spp showed similar levels of redundancy. this high level of sequence heterogeneity most likely stems from the heterogeneous genetic background of multiple individuals from different populations used in this study to create the transcriptome representing natural variation within a. spica-venti. transcript redundancy is a biological artifact that complicates de novo transcriptome assembly when using heterogeneous data. experimentally, contig redundancy could be minimized by performing several generational crosses between individuals from the same population. the level of redundancy in a. spica-venti is consistent across tissues and growth stages and can be identified by multiple methods presented here and most importantly, does not affect the levels of similarity between a. spica-venti assemblies. with and without the redundancy, the a. spica-venti transcriptome has high levels of similarity with phylogenetically related grass species such as b. distachyon and lolium spp. our study shows that a reference transcriptome can be successfully created from multiple genetically heterogeneous individuals. using extra steps after the initial de novo assembly can reduce redundancy drastically. the absence of go enrichment between the different tissues and growth stages is unsurprising considering the large sample size being compared and the fact that the assemblies were constructed from material under the same biotic and abiotic conditions. this lack of enrichment indicates also that the combined assembly represents well the variation within each tissue and growth stages. the fact that the a. spica-venti transcriptome is more similar to lolium spp. than to b. distachyon, despite b. distachyon being a much better annotated reference, is most likely caused by the close phylogenetic relationship or could indicate a genetic similarity between grass weeds. the genera apera, lolium, alopecurus and poa are all part of the large monophyletic core pooideae while brachypodium is part of the brachypodieae. many populations of a. spica-venti are resistant to herbicides and some have been identified as metabolic resistance cases, with a strong suspicion on the cytochrome p450s family, but never confirmed. we find a higher number of total sequences corresponding to some key herbicide resistance gene families in a. spica-venti than compared to lolium rigidum reference, which contained gst and gt sequences. the total number of different p450s recovered in this study is in line with the recovered in the transcriptome of lolium rigidum. however, this number is far below the number of p450s identified in other poaceae species such as rice and sorghum, respectively. transcriptome studies do not recover the total number of genes present in an organism. this tells us that herbicide susceptible populations of a. spica-venti in denmark express a low number of p450s in optimal growing conditions. this is consistent with the knowledge of p450s as being involved in stress and xenobioticide defense in plants. the fact that the roots presented the largest number of unique p450s could indicate they were under some undetermined stress and more likely to indicate higher basal p expression. roots in zea mays and in arabidopsis are known to express p450s and some p450s are expressed predominantly in roots, but there is no evidence that this is a general trend for the p family. the most abundant p cyp71d has been shown to be implicated in glycoalkaloid and ginsenosides biosynthesis, stress-induced secondary metabolites, as well as green tissue regeneration, especially in shoot development stages, in many crops. interestingly, cyp71d is also highly upregulated in blight-resistant potato. this hints at a potential role in herbicide resistance in a. spica-venti, but remains to be investigated. the large sequence variation in cyp89a between lolium, apera, and triticum, could indicate that these sequences are not highly conserved as they are implicated in environmental adaptation. the a. spica-venti and a. myosuroides gstf sequences are similar indicating a potential for similar metabolic activity in the presence of herbicide as was demonstrated in cummins et al. these two known resistance genes were recovered in the majority of tissues of this reference transcriptome of a. spica-venti indicate that these are constitutively expressed at many early stages and overall in the plant. gst are known to be expressed at every stage of plant development in every tissue. many of the known herbicide resistant p450s, gsts, and eifs identified in other weeds species were not recovered.  in the present study, we have described the construction and functional annotation of a de novo reference transcriptome assembly for apera spica-venti with emphasis on gene families involved in herbicide detoxification. these transcriptomes represents a considerable addition to the molecular knowledge available for this important weed species. moreover, go and kegg analysis were conducted and all unigenes were classified into functional categories with the aim to understand their role and metabolic pathways. this data can be used to develop oligo-nucleotide microarray to study gene expression studies at large scale. we have characterized the major gene families responsible for herbicide resistance and shown strong potential of two specific genes to have a role in herbicide resistance in a. spica-venti. finally, this study can serve as a reference to unravel genes and pathways involved in metabolic herbicide resistance mechanisms, weed adaption, grass genetics and trait evolution in the future. 
individual transcripts and gene families involved in metabolic based herbicide resistance were identified. the combined transcriptome assembly consists of contigs with an n and average contig length of and 658 bp, respectively. de novo transcriptome assembly enabled the generation of the first reference transcriptome of a. spica-venti. compared with other grass species, a. spica-venti has 26% proteins in common to brachypodium distachyon, and 41% to lolium spp. the lack of genetic information about this noxious weed limits its biological understanding such as growth, reproduction, genetic variation, molecular ecology and metabolic herbicide resistance. this study produced a reference transcriptome for a. spica-venti from different tissues and various growth stages. the gstf and cyp89a transcripts were recovered from the majority of tissues and aligned at a maximum of and 30% to proven herbicide resistant allele from alopecurus myosuroides and lolium rigidum, respectively. the de novo assembly was performed on individual and combined dataset followed by functional annotations. loose silky bentgrass is an important weed in europe with a recent increase in herbicide resistance cases. this can serve as stepping stone for understanding the metabolic herbicide resistance as well as the general biology of this problematic weed. cytochrome p450glutathione s-transferaseherbicide resistanceloose silky bentgrassweed transcriptomicsinnovation fund denmark0603-00516bkristensen michael issue-copyright-statement© the author 2017
 loose silky bentgrass is an important weed in europe with a recent increase in herbicide resistance cases. the lack of genetic information about this noxious weed limits its biological understanding such as growth, reproduction, genetic variation, molecular ecology and metabolic herbicide resistance. this study produced a reference transcriptome for a. spica-venti from different tissues and various growth stages. the de novo assembly was performed on individual and combined dataset followed by functional annotations. individual transcripts and gene families involved in metabolic based herbicide resistance were identified.  eight separate transcriptome assemblies were performed and compared. the combined transcriptome assembly consists of contigs with an n and average contig length of and 658 bp, respectively. this dataset contains transcripts consisting of total,111 bp. among them 94% had a homologue to uniprotkb, 73% retrieved a go mapping, and 50% were functionally annotated. compared with other grass species, a. spica-venti has 26% proteins in common to brachypodium distachyon, and 41% to lolium spp. glycosyltransferases had the highest number of transcripts in each tissue followed by the cytochrome p450s. the gstf and cyp89a transcripts were recovered from the majority of tissues and aligned at a maximum of and 30% to proven herbicide resistant allele from alopecurus myosuroides and lolium rigidum, respectively.  de novo transcriptome assembly enabled the generation of the first reference transcriptome of a. spica-venti. this can serve as stepping stone for understanding the metabolic herbicide resistance as well as the general biology of this problematic weed. furthermore, this large-scale sequence data is a valuable scientific resource for comparative transcriptome analysis for poaceae grasses. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cytochrome p450glutathione s-transferaseherbicide resistanceloose silky bentgrassweed transcriptomicsinnovation fund denmark0603-00516bkristensen michael issue-copyright-statement© the author 2017 