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A TILLING Platform for Functional Genomics in Brachypodium distachyon.

Dalmais M, Antelme S, Ho-Yue-Kuang S, Wang Y, Darracq O, d'Yvoire MB, Cézard L, Légée F, Blondet E, Oria N, Troadec C, Brunaud V, Jouanin L, Höfte H, Bendahmane A, Lapierre C, Sibout R - PLoS ONE (2013)

Bottom Line: Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants.The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line.The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

View Article: PubMed Central - PubMed

Affiliation: URGV, Unité de Recherche en Génomique Végétale, Université d'Evry Val d'Essonne, INRA, Evry, France.

ABSTRACT
The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

No MeSH data available.


Phylogenetic analysis of putative COMT proteins.Phylogeny tree (phylogram) made with OMT proteins from Brachypodium (BdCOMT), rice (OsOMT), maize (ZmOMT) and Arabidopsis (AtOMT). The proteins known to be involved in lignification in ryegrass (LpCOMT), sorghum (SbCOMT), switchgrass (PvCOMT), fescue (FaCOMT) and poplar (PtCOMT) are included in the analysis and shown in red in the phylogram as well as Arabidopsis (AtOMT1) and Maize (ZmCOMT1) proteins. Brachypodium proteins (BdCOMT) are shown in green. Protein sequences are available in Information S1. Bootstrap values indicating the level of support for the displayed representation after re-sampling are shown on each node.
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pone-0065503-g003: Phylogenetic analysis of putative COMT proteins.Phylogeny tree (phylogram) made with OMT proteins from Brachypodium (BdCOMT), rice (OsOMT), maize (ZmOMT) and Arabidopsis (AtOMT). The proteins known to be involved in lignification in ryegrass (LpCOMT), sorghum (SbCOMT), switchgrass (PvCOMT), fescue (FaCOMT) and poplar (PtCOMT) are included in the analysis and shown in red in the phylogram as well as Arabidopsis (AtOMT1) and Maize (ZmCOMT1) proteins. Brachypodium proteins (BdCOMT) are shown in green. Protein sequences are available in Information S1. Bootstrap values indicating the level of support for the displayed representation after re-sampling are shown on each node.

Mentions: To unambiguously identify the B. distachyon COMT gene specifically involved in lignification, protein sequences of orthologs in several species were BLASTed onto the B. distachyon predicted proteome sequence. Eight proteins were identified: BdCOMT1 (Bradi1g14870), BdCOMT2 (Bradi2g02380), BdCOMT3 (Bradi2g02390), BdCOMT4 (Bradi2g19830), BdCOMT5 (Bradi2g19850), BdCOMT6 (Bradi3g16530), BdCOMT7 (Bradi3g55890), BdCOMT8 (Bradi4g20020). We performed a phylogenetic analysis with the most exhaustive list of encoded OMT proteins found in Oryza sativa, Arabidopsis thaliana and Zea mays genomes [45], [46], [47] (Figure 3, Information S1). In addition, we added to the phylogenetic analysis the protein sequences for which biological data (transgenics or mutants) support indisputably a role of the corresponding protein in lignification of several grass (maize, ryegrass, tall fescue, switchgrass, sorghum) and poplar [24], [39], [42], [48], [49], [50], [51]. AtOMT1 was clearly identified by our group as a unique gene involved in sinapyl alcohol biosynthesis in Arabidopsis since the knockout mutant displays a lignin devoid of S units [52]. Therefore, AtOMT1 is a reference model for dicot COMT proteins as well as maize ZmCOMT1 (BM3) is a reference for grasses [49]. The phylogenetic analysis shows that both dicot proteins (AtOMT1, PtOMT) cluster together whereas four members of BdCOMTs (BdCOMT3, BdCOMT2, BdCOMT1 and BdCOMT6) are grouped with the genuine grass COMT proteins, BdCOMT6 being the closest ortholog (figure 3). Consequently, BdCOMT6 was chosen for TILLING. It is worth noting, that despite no comt mutant was identified in rice to our knowledge this analysis suggests strongly that OsOMT1 (Os08g06100) is involved in monolignol formation.


A TILLING Platform for Functional Genomics in Brachypodium distachyon.

Dalmais M, Antelme S, Ho-Yue-Kuang S, Wang Y, Darracq O, d'Yvoire MB, Cézard L, Légée F, Blondet E, Oria N, Troadec C, Brunaud V, Jouanin L, Höfte H, Bendahmane A, Lapierre C, Sibout R - PLoS ONE (2013)

Phylogenetic analysis of putative COMT proteins.Phylogeny tree (phylogram) made with OMT proteins from Brachypodium (BdCOMT), rice (OsOMT), maize (ZmOMT) and Arabidopsis (AtOMT). The proteins known to be involved in lignification in ryegrass (LpCOMT), sorghum (SbCOMT), switchgrass (PvCOMT), fescue (FaCOMT) and poplar (PtCOMT) are included in the analysis and shown in red in the phylogram as well as Arabidopsis (AtOMT1) and Maize (ZmCOMT1) proteins. Brachypodium proteins (BdCOMT) are shown in green. Protein sequences are available in Information S1. Bootstrap values indicating the level of support for the displayed representation after re-sampling are shown on each node.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3686759&req=5

pone-0065503-g003: Phylogenetic analysis of putative COMT proteins.Phylogeny tree (phylogram) made with OMT proteins from Brachypodium (BdCOMT), rice (OsOMT), maize (ZmOMT) and Arabidopsis (AtOMT). The proteins known to be involved in lignification in ryegrass (LpCOMT), sorghum (SbCOMT), switchgrass (PvCOMT), fescue (FaCOMT) and poplar (PtCOMT) are included in the analysis and shown in red in the phylogram as well as Arabidopsis (AtOMT1) and Maize (ZmCOMT1) proteins. Brachypodium proteins (BdCOMT) are shown in green. Protein sequences are available in Information S1. Bootstrap values indicating the level of support for the displayed representation after re-sampling are shown on each node.
Mentions: To unambiguously identify the B. distachyon COMT gene specifically involved in lignification, protein sequences of orthologs in several species were BLASTed onto the B. distachyon predicted proteome sequence. Eight proteins were identified: BdCOMT1 (Bradi1g14870), BdCOMT2 (Bradi2g02380), BdCOMT3 (Bradi2g02390), BdCOMT4 (Bradi2g19830), BdCOMT5 (Bradi2g19850), BdCOMT6 (Bradi3g16530), BdCOMT7 (Bradi3g55890), BdCOMT8 (Bradi4g20020). We performed a phylogenetic analysis with the most exhaustive list of encoded OMT proteins found in Oryza sativa, Arabidopsis thaliana and Zea mays genomes [45], [46], [47] (Figure 3, Information S1). In addition, we added to the phylogenetic analysis the protein sequences for which biological data (transgenics or mutants) support indisputably a role of the corresponding protein in lignification of several grass (maize, ryegrass, tall fescue, switchgrass, sorghum) and poplar [24], [39], [42], [48], [49], [50], [51]. AtOMT1 was clearly identified by our group as a unique gene involved in sinapyl alcohol biosynthesis in Arabidopsis since the knockout mutant displays a lignin devoid of S units [52]. Therefore, AtOMT1 is a reference model for dicot COMT proteins as well as maize ZmCOMT1 (BM3) is a reference for grasses [49]. The phylogenetic analysis shows that both dicot proteins (AtOMT1, PtOMT) cluster together whereas four members of BdCOMTs (BdCOMT3, BdCOMT2, BdCOMT1 and BdCOMT6) are grouped with the genuine grass COMT proteins, BdCOMT6 being the closest ortholog (figure 3). Consequently, BdCOMT6 was chosen for TILLING. It is worth noting, that despite no comt mutant was identified in rice to our knowledge this analysis suggests strongly that OsOMT1 (Os08g06100) is involved in monolignol formation.

Bottom Line: Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants.The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line.The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

View Article: PubMed Central - PubMed

Affiliation: URGV, Unité de Recherche en Génomique Végétale, Université d'Evry Val d'Essonne, INRA, Evry, France.

ABSTRACT
The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

No MeSH data available.