Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates

Abstract

Background: Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an economically important disease of barley that also infects wheat. Using genetic analysis to characterize loci in Ptm genomes associated with virulence or avirulence is an important step to identify pathogen effectors that determine compatible (virulent) or incompatible (avirulent) interactions with cereal hosts. Association mapping (AM) is a powerful tool for detecting virulence loci utilizing phenotyping and genotyping data generated for natural populations of plant pathogenic fungi.

Results: Restriction-site associated DNA genotyping-by-sequencing (RAD-GBS) was used to generate 4,836 single nucleotide polymorphism (SNP) markers for a natural population of 103 Ptm isolates collected from Idaho, Montana and North Dakota. Association mapping analyses were performed utilizing the genotyping and infection type data generated for each isolate when challenged on barley seedlings of thirty SFNB differential barley lines. A total of 39 marker trait associations (MTAs) were detected across the 20 barley lines corresponding to 30 quantitative trait loci (QTL); 26 novel QTL and four that were previously mapped in Ptm biparental populations. These results using diverse US isolates and barley lines showed numerous barley-Ptm genetic interactions with seven of the 30 Ptm virulence/avirulence loci falling on chromosome 3, suggesting that it is a reservoir of diverse virulence effectors. One of the loci exhibited reciprocal virulence/avirulence with one haplotype predominantly present in isolates collected from Idaho increasing virulence on barley line MXB468 and the alternative haplotype predominantly present in isolates collected from North Dakota and Montana increasing virulence on barley line CI9819.

Conclusions: Association mapping provided novel insight into the host pathogen genetic interactions occurring in the barley-Ptm pathosystem. The analysis suggests that chromosome 3 of Ptm serves as an effector reservoir in concordance with previous reports for Pyrenophora teres f. teres, the causal agent of the closely related disease net form net blotch. Additionally, these analyses identified the first reported case of a reciprocal pathogen virulence locus. However, further investigation of the pathosystem is required to determine if multiple genes or alleles of the same gene are responsible for this genetic phenomenon.

Keywords: Association mapping; Barley; Pyrenophora teres f. maculata; Reciprocal virulence/avirulence.

Fig. 1

Violin plot showing the phenotypic distribution of Pyrenophora teres f. maculata subpopulations across barley lines and grouped by location. Generated using ggplot2 3.3.2 [38] in R 3.6.3

Fig. 2

A Manhattan plot for barley lines where the standard BLINK model utilizing the 1–5 Ptm phenotyping scale was identified as the optimal model. Bonferroni correction threshold is indicated by the solid (α-level 0.05) and dashed (α-level 0.01) red lines. SNP density is indicated along the bottom of the plot with the corresponding heat scale shown to the left and the 12 Pyrenophora teres f. maculata chromosomes (Chr) designated below. The QTL designations are given below each chromosome. B QQ plots for corresponding lines within the standard BLINK model Manhattan plot with 95% confidence interval shown by the shaded color. The Manhattan and QQ plots were generated using CMplot [39] in R 3.6.3

Fig. 3

A Manhattan plot for barley lines where the binary BLINK model was the identified as the optimal model. Bonferroni correction threshold is indicated by the solid (α-level 0.05) and dashed (α-level 0.01) red lines. SNP density is indicated along the bottom of the plot with the corresponding heat scale shown to the left and the 12 Pyrenophora teres f. maculata chromosomes (Chr) designated below. The QTL designations are given below each chromosome. B QQ plots for corresponding lines within the binary BLINK model Manhattan plot with 95% confidence interval shown by the shaded color. The Manhattan and QQ plots were generated using CMplot [39] in R 3.6.3

Fig. 4

A Manhattan plot for barley lines where the binary BLINK model accounting for population structure (PC4 or PC15) was identified as the optimal model. Bonferroni correction threshold is indicated by the solid (α-level 0.05) and dashed (α-level 0.01) red lines. SNP density is indicated along the bottom of the plot with the corresponding heat scale shown to the left and the 12 Pyrenophora teres f. maculata chromosomes (Chr) designated below. The QTL designations are given below each chromosome. B QQ plots for corresponding lines within the binary BLINK account for population structure model Manhattan plot with 95% confidence interval shown by the shaded color. The Manhattan and QQ plots were generated using CMplot [39] in R 3.6.3

Fig. 5

A Manhattan plot for barley lines where the standard BLINK model utilizing the 1–5 Ptm scale phenotyping and accounting for population structure (PC4 or PC15) was identified as the optimal model. Bonferroni correction threshold is indicated by the solid (α-level 0.05) and dashed (α-level 0.01) red lines. SNP density is indicated along the bottom of the plot with the corresponding heat scale shown to the left and the 12 Pyrenophora teres f. maculata chromosomes (Chr) designated below. The QTL designations are given below each chromosome. B QQ plots for corresponding lines within the binary BLINK model Manhattan plot with 95% confidence interval shown by the shaded color. The Manhattan and QQ plots were generated using CMplot [39] in R 3.6.3

Fig. 6

Jitter genotype by phenotype plot for alleles A and B of the marker 3_630965 on barley line (A) MXB468 and (B) CI9819 using the 1–5 Ptm phenotyping scale [37] identified as the potential reciprocal virulence locus. Phenotypic scores are color coded based on isolate origin and standard error is displayed in blue for allele A and red for allele B