Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation
© Czaban et al.; licensee BioMed Central. 2015
Received: 2 December 2014
Accepted: 6 March 2015
Published: 28 March 2015
The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf fescues, both belonging to the subfamily Pooideae. This subfamily also includes wheat, barley, oat and rye, making it extremely important to world agriculture. Species within the Lolium-Festuca complex show very diverse phenotypes, and many of them are related to agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity.
We have generated de novo transcriptome assemblies for four species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some of the differences between the more stress tolerant Festuca, and the less stress tolerant Lolium species.
Our data presents a comprehensive transcriptome sequence comparison between species from the Lolium-Festuca complex, with the identification of potential candidate genes underlying some important phenotypical differences within the complex (such as VRN2). The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.
KeywordsLolium-Festuca complex RNAseq Comparative transcriptomics Gene families
Next Generation Sequencing is a valuable tool for the analysis and study of transcriptomes of non model species , especially when resources are limited and a complete re-sequencing of the genome is not practical. Transcriptome sequencing allows us to overcome some of the challenges associated with sequencing complex, highly repetitive and large plant genomes.
The Lolium-Festuca complex is a common name for the grasses belonging to both the Lolium genus and broad leaved fescues from the Schedonorus subgenus of Festuca. Both genus are part of the Poaceae family , but their exact taxonomic relationship is unclear, with reports of a shared common ancestor [3,4] or the Lolium diverging from Festuca around 2 million years ago . The Poaceae family also includes species such as wheat, barley, bamboo, rice, sorghum and sugarcane making it one of the most important plant families from an agricultural, economic and ecological point of view . The Lolium genus contains ten species  all of which are exclusively diploid in nature , whereas the Festuca genus comprises 600 species and the ploidy numbers range from diploid up to dodecaploid . The species belonging to the Lolium-Festuca complex are thought to be closely related and interspecific crosses between some of them occur naturally in the wild. In fact, Festuloliums, which are a cross between Loliums and Fescues are very well established as agriculturally important plants [10-12]. This has led to many discussions as to the exact taxonomy of the complex, as one can find over 500 names for the few Lolium species . However, despite such a close relationship and being universally distributed around the globe, the plants within the complex exhibit significant diversity for agriculturally important traits  such as growth speed, root length, forage quality, resistance to biotic and abiotic stresses, annuality and perenniality. The Lolium species generally have a good nutrient content and are highly palatable . L.perenne can withstand heavy grazing, and L. multiflorum is characterized by rapid establishment . All these traits make them a very good choice for animal fodder. On the other hand, F. pratensis exhibits higher persistence, with a better developed root system allowing it to grow on lower quality soils. It also exhibits resistance to extreme abiotic conditions, such as drought and cold stress, being found as far north as within the Arctic Circle. Introgression of specific traits within the complex are possible and natural hydrids can be found in north-western Europe .
Numerous studies have succesfully introduced important traits from F. pratensis into Loliums, including crown rust resistance in L. perenne  and L. multiflorum [19-21], freezing tolerance  and drought tolerance in L. multiflorum . Species from the Lolium-Festuca complex provide a large pool of genetic variation, both within single species, as well as within the complex. This makes it possible to breed forage and turf vartieties suited for use under a range of environments. Not suprisingly, Poacea, to which both Festuca and Lolium belong has been proposed as a model clade for comparative genomics . Currently well-annotated genomes for this clade are Brachypodium distachyon , and Oryza sativa  with ongoing research into Hordeum vulgare .
The aims of the study were to; (1) reconstruct the transcriptomes of four species within the Lolium-Festuca complex: F. pratensis, L. multiforum, L. m. westerwoldicum and L. temulentum. This would complement the already published Lolium perenne transcriptome [28,29], (2) establish the phylogeny of the species based on orthologous protein sequences, (3) identify and compare gene families across the analyzed transcriptomes, and (4) identify genes under positive selection between the very resistant to biotic and abiotic stresses F. pratensis, and more susceptible Lolium species.
Results and discussion
De-novo assembly of transcriptomes from the Lolium-Festuca complex
Statistics of the filtered de-novo assemblies
Number of sequences
Total nucleotide count
Max. transcript length
Average transcript length
L. m. westerwoldicum
Full length transcripts analysis
Template protein dataset
> 80% coverage
> 20% coverage
Results of CEGMA analysis
Out of 248
% of fully represented
% of at least partially represented
average number of orthologs per CEG
% of detected CEGs with more than 1 ortholog
Overview of functional annotation output of the four species transcriptomes
L. m. westerwoldicum
Comparative gene family analysis
One way of understanding differences between related species on a genome-wide scale is to compare and find contrasts in the entire gene complement of each species. Best reciprocal BLAST hits between genes within a single species suggests the genes are paralogs. Best reciprocal BLAST hits between genes from different species suggests the genes are orthologs, and this strategy is widely used to generate orthologous pairs . We used OrthoMCL  in order to compute orthologous clusters for all of our predicted proteins from the four species. We filtered proteins for the longest peptide predicted from a single representative transcript per locus, in order to avoid bias in the creation of the orthologous groups. We generated 15,930 clusters, assigning 57,822 (76,59%) to clusters of sizes from 2 to 176 proteins. The number of proteins contained in all clusters for each species varied between 14,161 and 14,835.
Annotation of the species-unique proteins identified
Selected protein homologs
ZCCT2-A2 VRN2 homologue [T. urartu]
Serine/threonine-protein kinase SMG1 [A. tauschii]
serine/threonine-protein kinase GSO1 [A. tauschii]
Hydroxyisourate hydrolase [A. tauschii]
Disease resistance protein RPM1 [T. urartu]
Putative disease resistance protein RGA4 [T. urartu]
ABC transporter G family member 37 [T. urartu]
60S ribosomal protein L28-1 [A. tauschii]
Ribosomal L1 domain-containing protein 1 [T. urartu]
ABC transporter C family member 3 [T. urartu]
Coatomer subunit beta’-2 [T. urartu]
Kinesin-like protein KIF15 [T. urartu]
Kinesin-like protein KIF15 [A. tauschii]
GRF zinc finger family protein [O. sativa Japonica Group]
DNA mismatch repair protein mutS [A. tauschii]
Splicing factor 3A subunit 3 [T. urartu]
DNA mismatch repair protein Mlh1 [A. tauschii]
ubiquitin [S. aucuparia]
calcineurin B-like protein 4 [T. aestivum]
fasciclin-like protein FLA14 [T aestivum]
anthranilate N-benzoyltransferase protein 1 [A. tauschii]
calcineurin B-like protein 4 [T. aestivum]
cyclopropane-fatty-acyl-phospholipid synthase [T. urartu]
B3 domain-containing protein [A tauschii]
Analysis of clusters with high and low sequence similarity
The average identity of sequences in the OrthoMCL groups indicates the level of similarity among proteins belonging to that group. The combined average sequence identity (referred to as %id) of all protein families was 91.61%. 747 families contained highly conserved proteins, and their %id was equal to 100. 2,056 families have a %id below 80%, constituting less conserved groups. Using the DAVID database  we have analyzed which functional annotation terms are overrepresented in the groups with different levels of percent sequence identity. GO Biological Process, INTERPROSCAN, and KEGG Pathway terms have been used for the annotation. Out of the proteins from groups having 100% identity 513 sequences could be matched in the DAVID database. They have been grouped into 45 clusters enriched for GO Biological Process terms. The most abundant classes of enriched terms include response to abiotic stress, ubiquitination, phosphorus metabolism, electron transport chain, protein localization, response to organic and hormone stimulus, positive regulation of transcription, carbohydrate metabolism, cell cycle, and meiotic cell cycle. Enriched KEGG pathway terms included purine and pirimidyne metabolism, pyruvate metabolism, glycolisis/gluconeogenesis, carbon fixation, biosynthesis of plant hormones, terpenoids, steroids and alkaloids, and citrate cycle. Enriched INTERPRO domains were related to ubiquitin, protein kinases, GTPases, ATPases, EF hands, and DNA/RNA helicases. Genes responsible for terms like basic metabolic processes related to biosynthesis and degradation, transcriptional and translational activity, protein synthesis and destination and signal transduction are amongst the most conserved in plants . The same is true for genes involved in basic cell cycle machinery .
The families with a low %id represent proteins with less restrained sequence conservation, with possible multiple copies allowing for more relaxed selection. For the families having below 80%id we have identified 1,548 IDs using DAVID, which group into 90 clusters enriched for GO Biological Process terms. Clusters with the highest enrichment scores consisted of proteins related to phosphorylation, enzyme linked receptor protein signalling pathway, response to radiation, light and abiotic stimulus, protein ubiquitination, proteolysys and protein catabolic processes, response to organic and hormone stimuli, ion transport, root development, nucleotide metabolic processes and response to hormone stimulus. Three clusters were identified for enriched KEGG pathways, related to metabolism of methane, cyanoamino acid and glycine, serine and threonine, phenylopropandoid biosynthesis, and gluconeogenesis, biosynthesis of alkaloids and terpenoids. 64 clusters have been enriched for INTERPRO domains, with ten highest containing protein kinases, ABC transporters, ubiquitin, ATPases, zinc fingers, sulfphate ion transporters, DNA/RNA helicases, EF-hands, EGF-like domains, and PAS domains. Full overview of the GO Biological Process annotation is available in Additional file 5: Table S5 and Additional file 6: Table S6.
Phylogenetic analysis based on orthologous gene families
The exact taxonomy of the Lolium-Festuca complex species is complicated and historically not completely agreed upon, with questions raised about the relationship between different Loliums as well as the origin of the species. The genus Festuca is considered to be ancestral to the genus Lolium, as it incorporates far more species and contains natural polyploids [4,13,50]. Evidence exists for both (i) the evolution of Loliums from a perennial Festuca subgenus Schedonorus ancestor , and (ii) a common ancestral form for both Lolium and Festuca [3,4]. Some reports are in favor of classifying the genus Lolium as part of the Schedonorus [52,53]. In general, the Lolium genus can be separated based on self-pollinating or out-pollinating behaviour. The most recent and complete analyses of the Lolium-Festuca complex reports the crown age of the Lolium - Festuca complex to be 8.97 +- 1.5 Ma. It also reports the F. pratensis to have originated in the Southwest Asia around 2 million years ago, and the Loliums to have first diversified in the eastern Mediterranean region around 4.1 Ma .
Genes under positive selection pressure in Lolium species compared to F. pratensis
Because F. pratenesis and L. perenne are perennial plants, and L. multiflorum, L. m. westerwoldicum and L. temulentum have a bi-annual or annual growth cycle, protein types present in every type of comparison except for F. pratensis - L.perenne have been closely investigated. One example of such proteins are cyclins, family of conserved proteins responsible for the control of cell-cycle progression . Cyclin T1-1, has been identified in all comparisons except for the comparison with L.perenne. Other cyclins, T1-4 and T1-5, and Cyclin-dependent kinase F-4 have been identified in pairwise comparisons with L. temulentum E3 ubiquitin ligases have also been identified in every comparison apart from L.perenne - RNF128 in L. multiflorum, RFWD3 in L. m. westerwoldicum and RNF25 in L. temulentum. Additionally, multiple diverse transcription factors have been identified in non - L.perenne comparisons. These proteins constitute a group worth investigation of the perenniality/annuality trait genetic background.
When analyzing PFAM domains, the most abundant classes in every comparison were Leucine Rich Repeats, AAA domains and Tetratricopepdide repeats. All three of those protein domains can be found in proteins involved in diverse functions - such as protein-protein interactions, transcription factors, protein degradation and signal transduction. The full list of annotated proteins and PFAM domains is available in Additional file 11.
Apart from the pairwise comparisons of Festuca to Lolium species, we have also performed a comparison of L. multiflorum and L. m. westerwoldicum, assuming that a large amount of changes on the molecular level might have been caused by human influence . It is an interesting comparison as L. m. westerwoldicum was developed by selecting L. multiflorum plants for annuality. A very high number of positively selected orthologous pairs - 235 - has been identified for these two species (Additional file 12: Table S11). As the main difference between the species is the strictly annual habit of L. m. westerwoldicum, apart from the basic metabolism and disease resistance we were also interested in proteins related to development and perenniality-annuality cycle. Annotations extracted from the previously created annotation files (Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4) included multiple ubiquitin protein ligases as well as Cyclin-T1-1. Multiple disease resistance proteins have been identified: two RGA2 proteins, 1 RPM1 and one RPP13 protein. Among the pfam domain annotations we have found one that is related to seed dormancy control [PF14144.1], and two genes with a anaphase-promoting complex subunit [PF12861.2]. We have also identified multiple domains associated with sugar metabolism, such as fructose-1-6-bisphosphatase [PF00316.15], sugar efflux transporter for intercellular exchange [PF03083.11], MFS/sugar transport protein [PF13347.1] and sugar transporter [PF00083.19]. Another interesting category of domains included drought induced 19 protein (Di19) [PF05605.7] and Arabidopsis broad-spectrum mildew resistance protein [PF05659.6]. In spite of the extremely close phylogenetic distance, the amount and diversity of proteins under putative positive selection between those two species is very high, likely reflecting the intense selection pressure applied during the breeding of L. m. westerwoldicum from L. multiflorum.
Many of the enriched terms identified as being positively selected in this study share functions comparable to the ones in similar analyses [61,67,68]. Terms associated with protein kinases, protein phospthatases, transcription regulation and glycotransferases are linked to disease resistance , which are one of the fastest evolving and critical proteins in plant evolution. Terms related to stress response were present in almost every comparison, which is not suprising given the phenotypic background of the plants. The VRN2 gene has been identified as being important for determining spring or winter wheat varieties . We have often observed terms related to reproductive structure development. Seeds and fruit size are one of the most distinct differences between wild and domesticated plants. L. temulentum is considered to be a mimic weed of wheat, and as such it has been involuntarily domesticated alongside that species . Breeding of perennial grasses has a much shorter history, with the earliest records of it starting around 90 years ago . However, given the intensity of modern breeding programs and the fact that F. pratensis, L. multiflorum and L. m. westerwoldicum plants used in our study are a result of a directed breeding effort, it might be worth investigating if some of the observed variation could be related to domestication like processes.
This study presents the first de-novo transcriptome assemblies for four species from the Lolium-Festuca complex, and uses them to perform comparative transcritpomics. The orthologous genes between the species have a very high sequence similarities (91,61%), and the majority of gene families were shared for all of them. A consensus phylogenetic tree based on our large set of one-to-one orthologous genes is in agreement with the most recent study that was based on nuclear ribosomal intergenic spacer and plastid trnT-L and trnL-F regions. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.
In order to capture a broad sequence diversity, we have chosen four Lolium-Festuca species that differ highly between each other with regard to phenotypic traits. The seeds were obtained from the breeding company DLF-Trifolium, and three of the species were commercial breeding varieties; F. pratensis - “Laura”, L.multiflorum “Lemtal”, L. m. westerwoldicum “Nerissa”. L. temulentum was a wild type. The seeds have been germinated and grown in the greenhouse under standard conditions. The RNA has been isolated on the 16th of April 2013 from mature leaf samples from single genotype of each of the four species using the RNeasy plant mini kit from Qiagen according to the manufacturers protocol.
cDNA preparation and sequencing
cDNA library preparation has been done using the TruSeq kit, generating paired-end libraries with insert size of 300 bp. Sequencing has been carried out by the Beijing Genomics Institute, using Illumina Hi-Seq platform (91 bp paired-end sequencing), as well as the Ion Proton platform for a subset (25,5 milion reads) of L. temulentum sequences (91 bp single-end sequencing). The adapters were removed and reads were quality trimmed by BGI.
The original reads have been corrected for sequencing errors using the Allpaths-LG software (version 44837) built-in error correction tool , with default parameters. The tool is based on an algorithm eliminating exceptions from an overwhelming consensus read stack . This process has reduced the total amount of paired-end reads by between 92.4 and 94.8%.
The samples from Illimuna and Ion Proton platforms have been merged and used together in the assembly. Trinity software  (version r2013_08_14) has been used for the generation of independent de-novo transcriptome assemblies, using the following parameters: –JM 20G –min_contig_ 200 –full_cleanup –min_kmer_cov 2. This has resulted a total number of 96,710 assembled transcripts for F. pratensis, 69,651 for L.multiflorum, 63,112 for L. m. westerwoldicum and 76,751 for L. temulentum. The reads have been mapped back to their assembly using RSEM (version 2013-02-16)  in order to filter out transcripts with low support.
Transdecoder (version r2013_08_14)  has been used to de novo predict putative coding regions and protein sequences using filtered transcripts as input. 39,981 proteins were predicted for F. pratensis, 30,940 for L. multiflorum, 30,182 for L. m. westerwoldicum and 35,005 for L. temulentum. CEGMA  pipeline was used to assess the completion of the transcriptome assemblies.
The trinotate (release 2014.07.08)  pipeline was used for the annotation of the protein dataset. Both transcripts and proteins have been aligned using blast software  (version 2.2.28+), blastx and blastp respectively, using swissprot protein database (release 09_07_2014) as the target. HMMER  (version 3.1b1) with Pfam-A database  (version 27.0) has been used to identify protein domains, signalP  (version 4.1) was used to predict signal peptides and tmHMM  (version 2.0c) was used to predict transmembrane helices. The resulting information has been loaded into SQLite database and wrapped up by the Trinotate report script, creating a comprehensive annotation.
Validation of assembly completion by CEGMA
CEGMA software  (version 2.4.010312) has been used to assess the completion of the transcript dataset. The software was run with default parameters with the included reference dataset.
Full-length transcript analysis
We have used high confidence protein datasets from T. aestivum, B. distachyon and O. sativa, downloaded from MIPS PlantsDB database (30.07.2014) . NCBI blastx was used (-evalue 1e-20 -max_target_seqs 1 -outfmt 6) to align each of our four filtered transcriptome assemblies to each of the protein databases separately. Afterwards we have used the analyze_blastPlus_topHit_coverage.pl script from the Trinity package to identify unique top matching proteins that align across certain length thresholds of the template sequence.
Orthologous group assignment
Predicted protein sequences have been clustered into orthologous groups using the OrthoMCL software . The input protein dataset predicted by transdecoder has been filtered to contain only the proteins predicted from the longest, unique transcript splice variants, giving 19,863, 17,718, 18,095, and 19,817 proteins, respectively. A custom perl script was used in order to get information about the number of clusters shared. A set of custom python scripts has been used to get information about groups with over- and underrepresented sequences and assesing a group wide pfam domain classification. Sequences from the species-unique groups have been aligned online with the NCBI protein database for manual functional annotation. Sequences having %id hits below 40 have been discarded. Sequences having hits to putative or predicted proteins without any assigned function have not been considered for further analysis.
Identification of putative orthologs
For the identification of putative orthologs between two species, we applied bi-directional blastp  where two sequences are considered as orthologous if they satisfy a sequence identity cut-off over the length of amino acids > 30 . We have used the F. pratensis protein dataset predicted earlier for the identification of proteins under positive selection.
Estimation of synonymous and non-synonymous substitution rate
Orthologous gene pairs were aligned using CLUSTALW . The maximum likelihood estimation of synonymous (Ks) and non-synonymous (Ka) substitution rate was estimated using the yn00 module of the PAML4 suite .
Analysis of evolutionary conserved and relaxed groups
For every sequence pair showing a Ka/Ks ratio higher than 1 and below 10, a representative sequence was chosen. It was belonging to the F. pratensis for all but one case. L. multiflorum representative was chosen for the L. multiflorum and L. m. westerwoldicum pair. The choice of one of the two representatives did not change the final outcome of the analysis. Matching uniprot identifiers have been identified by aligning the representative sequences with A. thaliana uniprot sequences (accessed 16.09.2014). The results have been filtered to contain only hits to plant species. The uniprot identifiers have then been used af input for DAVID. Clustering was done using the default parameters, with the KEGG pathway, INTERPRO domain and Biological Process GO terms used for annotation and A. thaliana sequences as a background for enrichment study.
OrthoMCL clusters have been filtered in search of clusters containing a single representative from each species. 4022 groups have been selected and used to infer gene trees using PAML4 suite . The resulting trees have been which were then clustered using the consensus tree program, version 3.69 of the Phylip package  to infer a consensus tree.
Availability of supporting data
The error corrected transcriptome reads have been deposited in the SRA database under the following accession numbers: SRR1648382 (F.pratensis), SRR1648406 (L. multiflorum), SRR1648407 (L.m. westerwoldicum), SRR1648408, SRR1648409 and SRR1648494 (L. temulentum). The Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession numbers GBXZ00000000 (F.pratensis), GBXX00000000 (L. multiflorum), GBXY00000000 (L.m. westerwoldicum), and GBXW00000000 (L. temulentum). The versions described in this paper are the first versions, GBXZ01000000, GBXX01000000, GBXY01000000, and GBXW01000000 respectively.
We would like to thank Mr. Stephan Hentrup for his excellent technical support. This project was financed by the GUDP (Grønt Udviklings- og Demonstrationsprogram - Green Development and Demonstration Program) (3405-11-0241).
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