Abstract
The aim of this study was to identify candidate genes involved in stem rust resistance, to develop stem rust resistance specific molecular markers and to characterize crown rust resistances in the highly heterozygous forage crop L. perenne.
The NGS-based transcriptome profiling technique massive analysis of cDNA ends (MACE) in combination with bulked segregant analysis was used. To identify highly resistant and susceptible genotypes for BSA, the mapping population was phenotyped for stem rust resistance with four stem rust field isolates from different regions. For transcription profiling by MACE, leaf segments of 20 fully resistant and 20 highly susceptible genotypes from the mapping population were selected. Leaf segments for transcription profiling were harvested right before inoculation and at 4, 8, 18, and 24 h after inoculation (hai). Leaves from 4 and 8 hai and 18 and 24 hai were bulked, giving a total of six different samples for MACE. Genexpression profiles of stem rust resistant and susceptible bulks were compared and exclusively expressed resistance specific and highly differentially expressed transcripts and SNPs in resistant bulks were identified. Based on these sequences, we were able to develop stem rust resistance specific molecular markers with strong linkage to the LpPg1 resistance locus. Two markers flanked and one co-segregated with the LpPg1 locus. To predict the genomic location of the closely linked markers and resistance specific transcripts on L. perenne linkage groups, an in silico-mapping in combination with the perennial ryegrass GenomeZipper was used. Most of them were localized on Lolium LG 2 and 7. Sequence of the co-segregating marker LpETR_18 showed homology to scaffold_3494_ref0018849 at 78 cM on Lolium LG2, assuming the location of LpPg1 in this region. In addition, MACE also enabled us to investigate how the LpPg1 gene orchestrates gene expression of the efficient resistance mechanism. Most informative was the comparison of gene expression between all infected and non-inoculated bulks. Here, the early resistant bulk revealed the highest induction of plant defense processes, based on gene expression of antifungal proteins and phytoalexins.
In addition, the same mapping population carrying the stem rust resistance locus LpPg1 were phenotyped for crown rust resistance with three different field isolates. The population showed different levels of resistance: most of the individuals were fully resistant, some were moderate resistant and very few were highly susceptible to P. coronata. Histological analyses were conducted to characterize fungal development and resistance reactions in a fully resistant, moderate resistant, highly susceptible genotype and in addition the resistant parent and the highly susceptible variety “Aurora”. Two types of crown rust disease resistance responses were observed. In the fully resistant genotype and the resistant parent crown rust development stopped at haustorial mother cell formation at 36 hours post inoculation. Cell autofluorescence was seen in mesophyll cells in direct contact with primary haustorial mother cells at 60 hours post inoculation, indicating a hypersensitive cell death in both genotypes. In the moderate resistant genotype, a delayed haustorial mother cell formation in comparison to the highly susceptible genotype and “Aurora” was observed. Gene expression analyses revealed crown rust resistance specific gene expression profiles which can separate fully resistant and moderate resistant genotypes. Compared to the susceptible Lolium genotype, in the fully resistant genotype and the resistant parent expression of genes involved in hypersensitive cell death were identified, which was evidenced by autofluorescence of mesophyll cells 60 hours post inoculation. In the moderate resistant and fully resistant genotype numerous genes were up-regulated coding for antifungal proteins. Similarities between the fully resistant and moderate resistant, and also between the fully resistant and resistant parent indicating both crown rust resistances are present in the fully resistant genotype.
The phenotyping of stem and crown rust resistance in the mapping population was not correlated and a comparison of the gene expression of stem rust resistant bulks with crown-rust-resistant genotypes identified 11 identical transcripts involved in unspecific defense reactions. In summary, there is no correlated inheritance between the stem and crown rust resistances.