Global transcriptome changes in perennial ryegrass during early infection by pink snow mould

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.publishedVersio

Transcriptome anlysis of perennial ryegrass (LOLIUM PERENNE L.) during an early stage infection to pink snow mould

Microdochium nivale causes pink snow mould disease that damages forage grasses (especially perennial ryegrass) covered by snow, particularly in late winter. This makes the survival of perennial ryegrass (Lolium perenne L.), which is an economically important forage grass in Europe, difficult. Resistance mechanisms against pink snow mould disease is not fully understood. Thus, to gain a better understanding of resistance mechanisms in perennial ryegrass, we investigated variation in resistance among L. perenne genotypes, measured as relative regrowth after inoculation with several M. nivale isolates, and conducted a global transcriptome study of resistant and susceptible genotypes using an RNA sequencing approach. Ten genotypes, 8 from the Norwegian cultivar genotypes Fagerlin and 2 from the European cultivar Picaro, were used in this study. Clones of ten genotypes were inoculated with M. nivale isolates and incubated in darkness at 2°C for 5-12 weeks. Relative regrowth was calculated as the ratio of the regrowth of inoculated and non-inoculated plants after 5, 8 and 12 weeks of incubation. Based on relative regrowth one ‘resistant’ genotype (M) and one ‘susceptible’ genotype (F) were selected for transcriptome studies. Total RNA was extracted from the leaf blade tissue of plants exposed to three different treatments: non-inoculated and non-incubated plants (non-incubated control), non-inoculated plants after 4 days of incubation (incubated control), and inoculated plants after 4 days of incubation (inoculated). There were two biological replicates of each genotype and treatment (totally 12 samples). cDNA libraries were prepared and paired-end sequencing was performed using Illumina Hiseq 2000. Before sequencing, qPCR was performed with the candidate gene PR-5 (thaumatin-like protein), a gene which is expressed in response to pathogen attack. The results indicate that there is significant variation among M and F genotypes during 0 day and the 4th day. Higher transcript levels of PR-5 were observed in both non-incubated control (0 day) and inoculated (4th day) plants of the ‘resistant’ M genotype than in the ‘susceptible’ F genotype. This variation in the expression of PR-5 indicates that the two selected genotypes are different in respect to resistance against pink snow mould, and that they are suitable for being used for global RNA sequencing. De-novo assembly was done using the Trinity software platform. A total number of 188,355 and 261,978 assembled contigs with N50 values of 1,672 and 1,784 bp were generated from the “M” and “F” genotypes, respectively. Mapping was done by aligning the reads back to the assemble contigs using the Bowtie program and then transcript abundance was estimated by RSEM. Using EdgeR, differential expression analysis of read counts was done. A total number of 7,282 and 19,055 significantly differentially expressed (DE) genes were detected between non-incubated control and inoculated (incubated at 2°C), 6,227 and 19,832 DE genes were detected between non-incubated control and non-inoculated plants, and 275 and 83 DE genes were detected between inoculated and non-inoculated plants of the “M” and “F” genotypes, respectively. In this study we mainly focused on gene expression in inoculated and non-inoculated incubated plants as they were treated under the same conditions, i.e. incubation in darkness. DE genes from the ‘resistant’ (M) genotype and from the ‘susceptible’ (F) genotype were blasted and annotated against the NCBI non-redundant database using Blast2go program. The annotation results gave us about various genes that are differentially expressed at various stress factors. Further detailed studies on these genes will help us to understand the interactions between perennial ryegrass plants and M. nivale and also provied information that can be used to develop cultivars with improved resistance against pink snow mould by studying expressions of various defence related genes

Global transcriptome changes in perennial ryegrass during early infection by pink snow mould

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould

Data from: Global transcriptome changes in perennial ryegrass during early infection by pink snow mould

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould

significant_GO_Terms_fisherstest

Sequences of the genes under significant gene ontology (GO) terms which are over represented in snow mould resistant (R) genotype

EST_candidategenes_snowmould

Sequences of potential candidate genes for snow mould resistance detected in this stud

Snowmould_Susceptible_genotype_Trinity_transcriptome

Snow mould susceptible genotype of Lolium perenne de novo assembly generated by Trinity progra

Snowmould_Resistant_genotype_Trinity_transcriptome

Snow mould resistant genotype of Lolium perenne denovo assembly by Trinity progra