Comparative and Evolutionary Analysis of Grass Pollen Allergens Using <i>Brachypodium distachyon</i> as a Model System

<div><p>Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from <i>Brachypodium distachyon</i> as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in <i>B</i>. <i>distachyon</i>. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific <i>cis</i>-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.</p></div

Phylogenetic analysis of pollen allergen protein families in <i>B</i>. <i>distachyon</i> and representative member of each group from Ryegrass and Timothy grass.

<p>The protein sequences were aligned by Clustal X2.0 and unrooted phylogenetic tree was constructed by neighbour-joining method with 100 bootstrap replicates. Branches with less than 50% bootstrap support were collapsed. The tree was divided into eight phylogenetic clusters. The members were distinctly coloured to represent respective protein family. Lol p 1 (P14946) for group 1, Lol p 2 (P14947) for group 2/3, Lol p 4 (Q5TIW3) for group 4, Lol p 5a (Q40240) for group 5, Phl p 7 (O82040) for group 7, Lol p 11 (Q7M1X5) for group 11, Phl p 12 (P35079) for group 12 and Phl p 13 (CAB42886) for group 13 were chosen as representative members respectively.</p

Expression profile and evolutionary pattern of pollen allergens.

<p>The heat map shows the relative abundances of putative allergen-encoding genes identified in <i>B</i>. <i>distachyon</i>. The level of expression for a gene across different samples are represented as percentage of the maximum expression level in colour code from 0% (white) to 100% (black). Heat map was plotted against the phylogenetic tree constructed using <i>B</i>. <i>distachyon</i> protein sequences. The diagram also shows relative expression values in anther tissue of <i>B</i>. <i>distachyon</i> based on RNA-Seq data analysis obtained from nine developmental stages. Inflo1 is for pre-emergence flowers stage, Inflo2 is for post-emergence flowers stage, Seed1 is for whole seed at 5days after pollination, Seed2 is for whole seed at 10 days after pollination and Endosp is for endosperm respectively.</p

Duplication frequency of 14 most abundant <i>cis</i>-regulatory elements (CREs) in putative allergen-encoding genes in <i>B</i>. <i>distachyon</i>.

<p>Duplication frequency of 14 most abundant <i>cis</i>-regulatory elements (CREs) in putative allergen-encoding genes in <i>B</i>. <i>distachyon</i>.</p

Predicted structures of putative pollen allergen proteins in <i>B</i>. <i>distachyon</i>.

<p>The structure of 24 putative allergen-encoding proteins with >90% confidence level were shown.</p

Phylogenetic tree and domain architecture of pollen allergens.

<p>Neighbour-Joining phylogenetic tree is constructed based on protein alignments of putative allergen-encoding proteins of <i>B</i>. <i>distachyon</i> and known pollen allergens from other grasses using ClustalX2. Bootstrap values greater than 50% are shown at the nodes. The schematic diagrams show the domain organization of these proteins as obtained from SMART and PFAM searches. Different domains are indicated by the use of different colours as shown at the bottom of the figure. The accession numbers for the sequences used in the alignment are also listed in the figure.</p

Boxplots of abundance of transcripts in six developmental stages.

<p>Boxplots of RPKM values in log₁₀-scale in different tissues showing the distribution of these values about the median.</p

Effect of query sequence length on the percentage of sequences for which significant matches were found.

<p>The proportion of sequences with matches (e-value cut off 1e-05) in NCBI embryophytes nr database is greater among longer assembled transcripts.</p

Comparison of <i>de novo</i> assembly of VM (vegetative thallus male) data set using Velvet program for different <i>k-mer</i> lengths.

<p>The bars indicate number of contigs obtained with different <i>k-mer</i> lengths used in the assembly program (left axis). The lines indicate N50 length (rectangles) and average contig length (triangles) in bp (right axis).</p

Gene Ontology categorization of <i>M. polymorpha</i> transcripts.

<p>A bar chart of GOSlim term assigned to the <i>Marchantia</i> transcripts in different categories of biological process, molecular function and cellular component.</p