Screening of stable resistant accessions and identification of resistance loci to Barley yellow mosaic virus disease

Abstract

Background: The disease caused by Barley yellow mosaic virus (BaYMV) infection is a serious threat to autumn-sown barley (Hordeum vulgare L.) production in Europe, East Asia and Iran. Due to the rapid diversification of BaYMV strains, it is urgent to discover novel germplasm and genes to assist breeding new varieties with resistance to different BaYMV strains, thus minimizing the effect of BaYMV disease on barley cropping.

Methods: A natural population consisting of 181 barley accessions with different levels of resistance to BaYMV disease was selected for field resistance identification in two separate locations (Yangzhou and Yancheng, Jiangsu Province, China). Additive main effects and multiplicative interaction (AMMI) analysis was used to identify accessions with stable resistance. Genome-wide association study (GWAS) of BaYMV disease resistance was broadly performed by combining both single nucleotide polymorphisms (SNPs) and specific molecular markers associated with the reported BaYMV disease resistance genes. Furthermore, the viral protein genome linked (VPg) sequences of the virus were amplified and analyzed to assess the differences between the BaYMV strains sourced from the different experimental sites.

Results: Seven barley accessions with lower standardized Area Under the Disease Progress Steps (sAUDPS) index in every environment were identified and shown to have stable resistance to BaYMV disease in each assessed location. Apart from the reported BaYMV disease resistance genes rym4 and rym5, one novel resistance locus explaining 24.21% of the phenotypic variation was identified at the Yangzhou testing site, while two other novel resistance loci that contributed 19.23% and 19.79% of the phenotypic variation were identified at the Yancheng testing site, respectively. Further analysis regarding the difference in the VPg sequence of the predominant strain of BaYMV collected from these two testing sites may explain the difference of resistance loci differentially identified under geographically distinct regions. Our research provides novel genetic resources and resistance loci for breeding barley varieties for BaMYV disease resistance.

Keywords: Additive main effects and multiplicative interaction (AMMI); Barley (Hordeum vulgare L.); Barley yellow mosaic disease (BYMD); Genome-wide association study (GWAS); Stable resistant accessions.

Figure 1. BaYMV disease grade under natural disease nursery.

BaYMV disease levels were divided into different levels of severity, 0 means healthy leaves with no symptoms of disease, 1 represents typical chlorotic spots, 2 represents a quarter of the leaves displaying a yellow mosaic pattern, 3 represents more than half of the leaves displaying a yellow mosaic pattern, and 4 represents more than three-quarters of the leaves displaying a yellow mosaic pattern.

Figure 2. Distribution of molecular markers used in this study and known BaYMV disease resistance genes on barley chromosomes.

Rym represents a dominant gene, while rym represents a recessive gene. The genes in the color blue represent genes that have been assigned a chromosome position, and genes in the color purple represent genes that have been cloned.

Figure 3. LD decay estimated by the squared allele frequency correlation (r²) against the pairwise distance between 3,839 molecular markers used in this study.

The black scatter points represent the average r² value of each distance segment, the red curve is the fitting of the scatter points by the OriginPro 2021b software and the blue lines represent an r² cutoff of 0.2 that was chosen to define the extent of LD in the population.

Figure 4. Sequence analysis of BaYMV.

(A) Identification of virus species by a multiplex RT-PCR with BaYMV- and BaMMV-specific primer sets M: 2000bp DNA marker (Takara Bio, Beijing, China); 1: Yangzhou Supi 1; 2: Yangzhou Dan 2; 3: Yancheng Supi 1; 4: Yancheng Dan 2; 5: amplifying with H₂O. (B) The VPg coding sequences of different virus strains amplified from two susceptible barley accessions. M: 2000bp DNA marker (Takara Bio, Beijing, China) 1: amplifying BaYMV VPg from Yangzhou Supi 1; 2: amplifying BaMMV VPg from Yangzhou Dan 2; 3: amplifying BaYMV VPg from Yancheng Supi 1; 4: amplifying BaMMV VPg from Yancheng Dan 2; 5: Negative control, amplifying BaYMV VPg primer with H₂O; 6: Negative control, amplifying BaMMV VPg primer with H₂O. (C) Differences of BaYMV-VPg coding sequence between the Yangzhou testing site and the Yancheng testing site. The blue SNP is the synonymous mutation and the red SNP is the non-synonymous mutation.

Figure 5. BaYMV disease phenotype of 181 barley accessions.

(A) Standardized area under the disease progress steps (sAUDPS) index at 3 years (2015, 2016, and 2020) in the Yangzhou testing site and 2 years (2019 and 2020) in the Yancheng testing site. (B) AMMI biplot for the sAUDPS indexes in different environments. Blue numbers represent accessions. Brown numbers represent the stability of the environment.

Figure 6. Association analysis for BaYMV disease resistance in the Yangzhou testing site and the Yancheng testing site by FarmCPU model.

(A/C) Manhattan maps show marker-trait associations in the Yangzhou and Yancheng testing sites, respectively. The abscissa shows the physical location of the SNPs, and the SNPs on different chromosomes were distinguished by color. SNP density was expressed in a manner corresponding to the abscissa. (B/D) Quantile–Quantile Plots of genome-wide association analysis in the Yangzhou and Yancheng testing sites, respectively.