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Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation.

Czaban A, Sharma S, Byrne SL, Spannagl M, Mayer KF, Asp T - BMC Genomics (2015)

Bottom Line: Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, Slagelse, 4200, Denmark. Adrian.Czaban@mbg.au.dk.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Orthologous groups distribution. The Venn diagram shows the distribution of shared and divergent orthologous groups from an OrthoMCL analysis of Lolium-Festuca complex proteomes, based on non-redundant dataset. The numbers in each division show the amount of groups for each combination.
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Fig2: Orthologous groups distribution. The Venn diagram shows the distribution of shared and divergent orthologous groups from an OrthoMCL analysis of Lolium-Festuca complex proteomes, based on non-redundant dataset. The numbers in each division show the amount of groups for each combination.

Mentions: Most of the proteins are found in clusters containing genes from at least two species, with 8,644 gene families shared between all four species (Figure 2). The number of unique (species-specific) clusters is relatively low, which is not surprising considering that the analyzed species are seperated by very small evolutionary distances. L. multiflorum and L. m. westerwoldicum have the smallest number of species - specific proteins, and many protein sequences that are shared only between these two. Again, this is not surprising because L. m. westerwoldicum is a ‘species’ derived from L. multiflorum through selective breeding for annuality [37]. Out of the gene families identified as unique, two predicted proteins from F. pratensis are showing high sequence identity with a ZCCT2-A2 VRN2 homologue from T. urartu [B8X8J1]. VRN2 has an important role in the vernalization/flowering pathway, by preventing the flowering of the plant unless it has experienced a period of cold temperatures and/or short days [38]. If there is a cold period, VRN2 becomes downregulated and allows the expression of the FT1 gene, which promotes flowering [39,40]. F. pratensis is a perennial species with predominantly a strong vernalization requirement [41]. All of the other species analyzed are of bi-annual or annual type and have a facultative (L. multiflorum) or no vernalization requirement (L. m. westerwoldicum and L. temulentum [42]). Samples for RNA-seq were taken from non vernalised plants, and it is therefore not surprising that VRN2 has been identified in the transcrtipome assembly of F. pratensis. The Lolium species without a vernalisation requirement do not have the VRN2 transcript present in their assemblies. A blastp alignment of the identified Festuca protein against the other transcriptomes revealed no significant hits. None of the original reads from the other species align back to the predicted VRN2 transcript, confiming that the VRN2 transcript is not present in the RNA-seq data sets of non-perennial species. We know from other studies that VRN2 is expressed in a L. perenne which does have a strong vernalization requirement [43]. The absence of VRN2 expression has been proven to enable FT induction and flowering in the closely related cereals [44,45]. Loss of function of VRN2 in wheat results in plants that do not require vernalization to flower, and it is the genetic locus responsible for distinguishing spring and winter wheat types [46]. Our results suggest that VRN2 is a key gene for differentiating vernalisation and non-vernalisation requiring species withinin the Lolium-Festuca complex. Other proteins identified as being species-unique included disease resistance proteins for F. pratensis, ABC transporter C for L. multiflorum, part of a ubiquitin ligase complex for L. m. westerwoldicum and ubiquin for L. temulentum (Table 5).Figure 2


Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation.

Czaban A, Sharma S, Byrne SL, Spannagl M, Mayer KF, Asp T - BMC Genomics (2015)

Orthologous groups distribution. The Venn diagram shows the distribution of shared and divergent orthologous groups from an OrthoMCL analysis of Lolium-Festuca complex proteomes, based on non-redundant dataset. The numbers in each division show the amount of groups for each combination.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389671&req=5

Fig2: Orthologous groups distribution. The Venn diagram shows the distribution of shared and divergent orthologous groups from an OrthoMCL analysis of Lolium-Festuca complex proteomes, based on non-redundant dataset. The numbers in each division show the amount of groups for each combination.
Mentions: Most of the proteins are found in clusters containing genes from at least two species, with 8,644 gene families shared between all four species (Figure 2). The number of unique (species-specific) clusters is relatively low, which is not surprising considering that the analyzed species are seperated by very small evolutionary distances. L. multiflorum and L. m. westerwoldicum have the smallest number of species - specific proteins, and many protein sequences that are shared only between these two. Again, this is not surprising because L. m. westerwoldicum is a ‘species’ derived from L. multiflorum through selective breeding for annuality [37]. Out of the gene families identified as unique, two predicted proteins from F. pratensis are showing high sequence identity with a ZCCT2-A2 VRN2 homologue from T. urartu [B8X8J1]. VRN2 has an important role in the vernalization/flowering pathway, by preventing the flowering of the plant unless it has experienced a period of cold temperatures and/or short days [38]. If there is a cold period, VRN2 becomes downregulated and allows the expression of the FT1 gene, which promotes flowering [39,40]. F. pratensis is a perennial species with predominantly a strong vernalization requirement [41]. All of the other species analyzed are of bi-annual or annual type and have a facultative (L. multiflorum) or no vernalization requirement (L. m. westerwoldicum and L. temulentum [42]). Samples for RNA-seq were taken from non vernalised plants, and it is therefore not surprising that VRN2 has been identified in the transcrtipome assembly of F. pratensis. The Lolium species without a vernalisation requirement do not have the VRN2 transcript present in their assemblies. A blastp alignment of the identified Festuca protein against the other transcriptomes revealed no significant hits. None of the original reads from the other species align back to the predicted VRN2 transcript, confiming that the VRN2 transcript is not present in the RNA-seq data sets of non-perennial species. We know from other studies that VRN2 is expressed in a L. perenne which does have a strong vernalization requirement [43]. The absence of VRN2 expression has been proven to enable FT induction and flowering in the closely related cereals [44,45]. Loss of function of VRN2 in wheat results in plants that do not require vernalization to flower, and it is the genetic locus responsible for distinguishing spring and winter wheat types [46]. Our results suggest that VRN2 is a key gene for differentiating vernalisation and non-vernalisation requiring species withinin the Lolium-Festuca complex. Other proteins identified as being species-unique included disease resistance proteins for F. pratensis, ABC transporter C for L. multiflorum, part of a ubiquitin ligase complex for L. m. westerwoldicum and ubiquin for L. temulentum (Table 5).Figure 2

Bottom Line: Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, Slagelse, 4200, Denmark. Adrian.Czaban@mbg.au.dk.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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