Development and characterization of microsatellite markers based on whole-genome sequences and pathogenicity differentiation of Pyrenophora graminea, the causative agent of barley leaf stripe

Abstract

Barley leaf stripe, caused by the fungal pathogen Pyrenophora graminea, is becoming a destructive disease in the Hexi corridor of the Gansu province (China). Knowledge of the genetic diversity, population structure, and specific pathogenicity of P. graminea is essential for barley resistance breeding and efficient control of leaf stripe. In this study, whole-genome sequences of P. graminea were applied to identify microsatellites and develop simple sequence repeat (SSR) markers. These SSR markers were used to examine the genetic diversity and population structure of barley leaf stripe fungus in China. A total of 403 SSR primers were designed and synthesized, and target fragments were amplified by 390 microsatellite markers. These fragments corresponded to 789 alleles (1–5 alleles per locus) in eight strains (average, 2.02 alleles per marker locus). The cluster analysis and population structure were analyzed by means of 100 markers, and a total of 696 alleles were detected from 44 accessions (mean, 6.96 alleles per marker; range, 3–17). The average gene diversity was 0.63, and ranged from 0.11 to 0.92. The polymorphism information content (PIC) ranged from 0.11 to 0.91 with an average of 0.59. The Shannon’s Information Index ranged from 0.29 (for Pgm254 and Pgm337) to 2.64 (for Pgm371), with an average of 1.36. Results of cluster analysis showed that 44 accessions were divided into four clusters. Overall, 44 strains were divided into three subpopulations, comprising 19, 17, and eight strains each. Six isolates were tested for pathogenicity differentiation on 10 barley cultivars. Cultivar Ganpi2 was found to be resistant to all six isolates; nine of 10 cultivars had varied reactions to six isolates. Genome sequencing of P. graminea enabled SSR marker development, and SSR markers are applicable to studies of population genetic structure in P. graminea. We conclude that pathogenicity differentiation exists among the six isolates in the study.