An eQTL analysis of partial resistance to Puccinia hordei in barley

Abstract

Background: Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes.

Methodology/principal findings: We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis.

Conclusions/significance: The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population.

Figure 1. Micrographs viewed under epi-fluorescence microscopy after staining with Uvitex, showing development of P. hordei at different time points post inoculation.

A: overview of germinating urediospores on barley leaves 10 hpi, green spots are inert spores of lycopodium; B, C and D: close-up images showing infection units at10, 18 and 24 hpi, respectively. Solid arrows indicate haustorial mother cells, dotted arrows haustoria. Scale bar = 50 µm.

Figure 2. Number of informative comparisons across the barley genome based on a distant-pair design (see text) with extra weight given to four pQTL regions (shown as grey blocks).

The solid horizontal line at 36 represents the average number of informative comparisons when samples were randomly paired.

Figure 3. Numbers and proportions of eQTL with different LOD scores.

A total of 15685 significant eQTL (p<0.001) was detected with LOD scores ranging from 2.4 to 55.7.

Figure 4. Overview of eQTL mapping results for genes previously mapped by SNP and TDM markers.

The x-axis shows the locations of eQTL associated with transcript abundance from the current experiment. The y-axis shows the location of genes mapped previously as SNPs (253 genes, [29]), TDMs (1066 genes, [31]) or both (63 genes). The 1256 previously mapped genes correspond to 1623 eQTL in the present study. eQTL corresponding to SNP- and TDM- mapped genes are displayed in blue and green respectively. eQTL with LOD score>10 and <10 are distinguished as circles or dots. Circles and dots on the diagonal represent correspondence between the locations observed in the current study with previous work , . Circles or dots off the diagonal represent trans-eQTL. While all eQTL and their corresponding SNP-mapped genes were on the diagonal, 12 eQTL with LOD>10 (highlighted as numbered and red-filled green circles) when compared to TDM-mapped genes were located at distinctly different (>25cM away) positions.

Figure 5. Relationship between eQTL LOD scores and position relative to their corresponding genes.

Numbers on the top of the columns are the total number of genes mapped by SNP (blue) and TDM (black). The colour key represents different eQTL categories assigned according to distance from their corresponding genes.

Figure 6. eQTL density for Ph-responsive genes across the genome.

x-axis, eQTL genomic location on chromosomes. y-axis, eQTL density calculated on a 10 cM sliding window. Chromosomal regions corresponding to the three most significant peaks are named as eQTL hotspots 1, 2 and 3 from left to right. The five pQTL regions were indicated by the grey blocks.