Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.)

Additional File 1:

Degenerate oligonucleotide primers used for NBS domain-containing sequence amplification. Sequence information for primer synthesis was obtained from published data specific to barley, sorghum and perennial ryegrass.

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Additional File 2:

Bioinformatic (BLASTX and wuBLASTX) annotation of cloned and sequenced primary and secondary perennial ryegrass R gene templates to both GenBank and UniProt databases within the Bioinformatic Advanced Scientific Computing (BASC) database (as of June 2007 release). BASC is linked to the rice Ensemble Browser and Uniprot databases and employs known gene ontology and Pfam domain analysis to assign putative function to candidate sequences. Nomenclature of paralogous sequences is based on the unique identifier for the primary template sequence (e.g., LpESTe11_14) followed by a numerical suffix (.1, .2 etc.), e.g. LpESTe11_14rg.1.

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Additional File 3:

Summary details for specific R gene-directed degenerate primer pair combinations, as described in Additional File 1, along with primer pair code, numbers of amplification products and corresponding R gene templates.

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Additional File 4:

Functional characterisation of predicted translation products from perennial ryegrass candidate R genes. All information was derived from Pfam links within the best-available Uniprot wuBLASTX hits in BASC. Pfam information was used to obtain the probable location of candidate sequences, protein size, position of NBS sequence and number of LRR repeats.

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Additional File 5:

Major protein sequence motifs in predicted Lolium NBS domains. ^aMotifs listed in the order of occurrence in the NBS domain of putative perennial ryegrass R genes. Perennial ryegrass motifs were named in accordance with descriptions obtained from both rice and A. thaliana [30,31,66]; ^bBioinformatic analysis using Pfam on putative R gene sequences identified all to be CC-NBS types (CNL denotes CC-NBS-LRR and TNL denotes TIR-NBS-LRR); ^cConsensus amino acid sequences for Lolium NBS sequences were derived from MEME, while those for wheat were derived from [58].

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Additional File 6:

Summary information of amino acid structure for NBS domain protein sequence motifs numbered in Fig. 2, based on matching using the MEME program.

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Additional File 7:

Reference information for sequences corresponding to individual clusters identified during phylogenetic analysis for the complete NBS domain (P-Loop-GLPL), as depicted in Additional File 8.

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Additional File 8:

NJ dendrograms based on amino acid alignment of the full-length (P-Loop – GLPL) regions of NBS protein domains encoded by Lolium R genes. Bootstrap values are displayed as percentages of 1000 neighbour joining bootstrap replications. Bootstrap values at or greater than 65% are shown. Bars at the right of the dendrograms represent R gene sub-classes.

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Additional File 9:

Reference information for sequences corresponding to individual clusters identified during phylogenetic analysis for the Kin-2A-GLPL region of the NBS domain, as depicted in Additional File 10.

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Additional File 10:

NJ dendrograms based on amino acid alignment of the partial (Kin-2A -GLPL) regions of NBS protein domains encoded by Lolium R genes. Details are as described in the legend for Additional File 8.

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Additional File 11:

Summary information for LAP and SNuPe primers used for predicted R gene SNP validation. Information on segregation structure, parental polymorphism, SNP variant and successful genetic map assignment is included. All LAP PCRs and SNuPe reactions were designed for operating annealing temperatures of 55°C and 50°C, respectively.

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Additional File 12:

Comparative chromosomal positions of predicted putative orthologous R genes between perennial ryegrass and barley: Lps-217 (coded as xlprg50-464ca) on p150/112 LG2 compared to Hvs-217 at the bottom of chromosome 2H. qLr represents a QTL for barley leaf rust resistance.

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Additional File 13:

Comparative chromosomal positions of predicted putative orthologous R genes between perennial ryegrass and barley: Lps-L8 (coded as xlprg54-688ag) on NA₆-LG3 compared to Hvs-L8 at the bottom of chromosome 3H. qMIL represents a major QTL for powdery mildew resistance in barley.

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