Haplotype-based association mapping of genomic regions associated with Zymoseptoria tritici resistance using 217 diverse wheat genotypes.

BACKGROUND: Septoria tritici blotch (STB) is considered to be one of the most destructive foliar wheat diseases and is caused by Zymoseptoria tritici. The yield losses are severe and in Northwestern Europe can reach up to 50%. The efficacy of fungicides is diminishing due to changes in the genetic structure of the pathogen. Therefore, resistance breeding is the most effective strategy of disease management. Recently, genome-wide association studies (GWAS) have become more popular due to their robustness in dissecting complex traits, including STB resistance in wheat. This was made possible by the use of large mapping populations and new sequencing technologies. High-resolution mapping benefits from historical recombination and greater allele numbers in GWAS. RESULTS: In our study, 217 wheat genotypes of diverse origin were phenotyped against five Z. tritici isolates (IPO323, IPO88004, IPO92004, IPO86036 and St1-03) and genotyped on the DArTseq platform. In polytunnel tests two disease parameters were evaluated: the percentage of leaf area covered by necrotic lesions (NEC) and the percentage of leaf area covered by lesions bearing pycnidia (PYC). The disease escape parameters heading date (Hd) and plant height (Ht) were also measured. Pearson's correlation showed a positive effect between disease parameters, providing additional information. The Structure analysis indicated four subpopulations which included from 28 (subpopulation 2) to 79 genotypes (subpopulation 3). All of the subpopulations showed a relatively high degree of admixture, which ranged from 60% of genotypes with less than 80% of proportions of the genome attributed to assigned subpopulation for group 2 to 85% for group 4. Haplotype-based GWAS analysis allowed us to identify 27 haploblocks (HBs) significantly associated with analysed traits with a p-value above the genome-wide significance threshold (5%, which was -log10(p) > 3.64) and spread across the wheat genome. The explained phenotypic variation of identified significant HBs ranged from 0.2% to 21.5%. The results of the analysis showed that four haplotypes (HTs) associated with disease parameters cause a reduction in the level of leaf coverage by necrosis and pycnidia, namely: Chr3A_HB98_HT2, Chr5B_HB47_HT1, Chr7B_HB36_HT1 and Chr5D_HB10_HT3. CONCLUSIONS: GWAS analysis enabled us to identify four significant chromosomal regions associated with a reduction in STB disease parameters. The list of valuable HBs and wheat varieties possessing them provides promising material for further molecular analysis of resistance loci and development of breeding programmes.

The use of weighted multiple linear regression to estimate QTL × QTL × QTL interaction effects of winter wheat (Triticum aestivum L.) doubled-haploid lines.

Knowledge of the magnitude of gene effects and their interactions, their nature, and contribution to determining quantitative traits is very important in conducting an effective breeding program. In traditional breeding, information on the parameter related to additive gene effect and additive-additive interaction (epistasis) and higher-order additive interactions would be useful. Although commonly overlooked in studies, higher-order interactions have a significant impact on phenotypic traits. Failure to account for the effect of triplet interactions in quantitative genetics can significantly underestimate additive QTL effects. Understanding the genetic architecture of quantitative traits is a major challenge in the post-genomic era, especially for quantitative trait locus (QTL) effects, QTL-QTL interactions, and QTL-QTL-QTL interactions. This paper proposes using weighted multiple linear regression to estimate the effects of triple interaction (additive-additive-additive) quantitative trait loci (QTL-QTL-QTL). The material for the study consisted of 126 doubled haploid lines of winter wheat (Mandub × Begra cross). The lines were analyzed for 18 traits, including percentage of necrosis leaf area, percentage of leaf area covered by pycnidia, heading data, and height. The number of genes (the number of effective factors) was lower than the number of QTLs for nine traits, higher for four traits and equal for five traits. The number of triples for unweighted regression ranged from 0 to 9, while for weighted regression, it ranged from 0 to 13. The total aaagu effect ranged from - 14.74 to 15.61, while aaagw ranged from - 23.39 to 21.65. The number of detected threes using weighted regression was higher for two traits and lower for four traits. Forty-nine statistically significant threes of the additive-by-additive-by-additive interaction effects were observed. The QTL most frequently occurring in threes was 4407404 (9 times). The use of weighted regression improved (in absolute value) the assessment of QTL-QTL-QTL interaction effects compared to the assessment based on unweighted regression. The coefficients of determination for the weighted regression model were higher, ranging from 0.8 to 15.5%, than for the unweighted regression. Based on the results, it can be concluded that the QTL-QTL-QTL triple interaction had a significant effect on the expression of quantitative traits. The use of weighted multiple linear regression proved to be a useful statistical tool for estimating additive-additive-additive (aaa) interaction effects. The weighted regression also provided results closer to phenotypic evaluations than estimator values obtained using unweighted regression, which is closer to the true values.

Evaluation of Fusarium Head Blight Resistance Effects by Haplotype-Based Genome-Wide Association Study in Winter Wheat Lines Derived by Marker Backcrossing Approach.

Fusarium head blight (FHB) of wheat caused by Fusarium species is a destructive disease, causing grain yield and quality losses. Developing FHB-resistant cultivars is crucial to minimize the extent of the disease. The first objective of this study was incorporation of Fhb1 from a resistant donor into five Polish wheat breeding lines with good agronomical traits and different origins. We also performed a haplotype-based GWAS to identify chromosome regions in derived wheat families associated with Fusarium head blight resistance. As a result of marker-assisted backcrossing (MABC), five wheat combinations were obtained. Fungal inoculation and disease assessment were conducted for two years, 2019 and 2020. In 2019 the average phenotypic response of type II resistance was 2.2, whereas in 2020 it was 2.1. A haploblock-based GWAS performed on 10 phenotypic traits (related to type of resistance, year of experiment and FHB index) revealed nine marker-trait associations (MTA), among which six belong to chromosome 2D, two to 3B and one to 7D. Phenotypic variation (R2) explained by the identified haplotypes in haploblocks ranged from 6% to 49%. Additionally, an association weight matrix (AWM) was created, giving the partial correlation-information theory (PCIT) pipeline of 171 edges and 19 nodes. The resultant data and high level of explained phenotypic variance of MTA create the opportunity for data utilization in MAS.

Stem rust on barberry species in Europe: Host specificities and genetic diversity.

The increased emergence of cereal stem rust in southern and western Europe, caused by the pathogen Puccinia graminis, and the prevalence of alternate (sexual) host, Berberis species, have regained attention as the sexual host may serve as source of novel pathogen variability that may pose a threat to cereal supply. The main objective of the present study was to investigate the functional role of Berberis species in the current epidemiological situation of cereal stem rust in Europe. Surveys in 11 European countries were carried out from 2018 to 2020, where aecial infections from five barberry species were collected. Phylogenetic analysis of 121 single aecial clusters of diverse origin using the elongation factor 1-α gene indicated the presence of different special forms (aka formae speciales) of P. graminis adapted to different cereal and grass species. Inoculation studies using aecial clusters from Spain, United Kingdom, and Switzerland resulted in 533 stem rust isolates sampled from wheat, barley, rye, and oat, which confirmed the presence of multiple special forms of P. graminis. Microsatellite marker analysis of a subset of 192 sexually-derived isolates recovered on wheat, barley and rye from the three populations confirmed the generation of novel genetic diversity revealed by the detection of 135 multilocus genotypes. Discriminant analysis of principal components resulted in four genetic clusters, which grouped at both local and country level. Here, we demonstrated that a variety of Berberis species may serve as functional alternate hosts for cereal stem rust fungi and highlights the increased risks that the sexual cycle may pose to cereal production in Europe, which calls for new initiatives within rust surveillance, epidemiological research and resistance breeding.

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade.

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.

Wheat Stem Rust Back in Europe: Diversity, Prevalence and Impact on Host Resistance.

The objective of this study was to investigate the re-emergence of a previously important crop pathogen in Europe, Puccinia graminis f.sp. tritici, causing wheat stem rust. The pathogen has been insignificant in Europe for more than 60 years, but since 2016 it has caused epidemics on both durum wheat and bread wheat in local areas in southern Europe, and additional outbreaks in Central- and West Europe. The prevalence of three distinct genotypes/races in many areas, Clade III-B (TTRTF), Clade IV-B (TKTTF) and Clade IV-F (TKKTF), suggested clonal reproduction and evolution by mutation within these. None of these genetic groups and races, which likely originated from exotic incursions, were detected in Europe prior to 2016. A fourth genetic group, Clade VIII, detected in Germany (2013), was observed in several years in Central- and East Europe. Tests of representative European wheat varieties with prevalent races revealed high level of susceptibility. In contrast, high diversity with respect to virulence and Simple Sequence Repeat (SSR) markers were detected in local populations on cereals and grasses in proximity to Berberis species in Spain and Sweden, indicating that the alternate host may return as functional component of the epidemiology of wheat stem rust in Europe. A geographically distant population from Omsk and Novosibirsk in western Siberia (Russia) also revealed high genetic diversity, but clearly different from current European populations. The presence of Sr31-virulence in multiple and highly diverse races in local populations in Spain and Siberia stress that virulence may emerge independently when large geographical areas and time spans are considered and that Sr31-virulence is not unique to Ug99. All isolates of the Spanish populations, collected from wheat, rye and grass species, were succesfully recovered on wheat, which underline the plasticity of host barriers within P. graminis. The study demonstrated successful alignment of two genotyping approaches and race phenotyping methodologies employed by different laboratories, which also allowed us to line up with previous European and international studies of wheat stem rust. Our results suggest new initiatives within disease surveillance, epidemiological research and resistance breeding to meet current and future challenges by wheat stem rust in Europe and beyond.

Identifying a novel powdery mildew resistance gene in a barley landrace from Morocco.

Powdery mildew is a barley foliar disease that causes great loss in yield. Because of the limited number of effective resistance genes, efforts to identify new sources of resistance are frequently focused on genetically diversified landraces. The goal of this study was to characterise the powdery mildew resistance gene in barley line 2553-3 selected from the Moroccan landrace. Phytopathological testing against a set of differential pathogen isolates revealed different pattern responses of this gene from those of other known resistance genes. F2 and F2:3 (2553-3 × Manchuria) mapping populations were employed to investigate resistance inheritance. Two approaches were applied for the linkage analysis: in the first approach, 22 resistant and 21 susceptible homozygous F2 plants genotyped by the DArTseq platform (Diversity Arrays Technology, Pty. Ltd.) were used; in the second, 94 F2 plants were genotyped by converted DArTseq markers and SSRs. Both analyses delineated a new resistance gene on the short arm of chromosome 2H. The authors propose MlMor as a gene symbol for newly characterized powdery mildew resistance genes in barley line 255-3-3. The results presented herein provide a good foundation for the development of closer linkage markers and MAS breeding.

Resistance of (Aegilops tauschii × Secale cereale) × Triticosecale Hybrids to Leaf Rust (Puccinia triticina) Determined on the Macroscopic and Microscopic Level.

Leaf rust caused by Puccinia triticina Eriks belongs to the most important fungal pathogens of wheat (Triticum aestivum L.) and triticale (× Triticosecale). Effective resistance to leaf rust is both, cost-effective and environmentally safe. Many wild Aegilops species carry unknown resistances against fungal diseases and are characterized by a high genetic variability. The main goal of this work was to examine the resistance of (Aegilops tauschii × Secale cereale) × Triticosecale hybrids to leaf rust in inoculation tests with different races of P. triticina. Hybrid plants were selected for the presence of 2D chromosome/s in the triticale background using fluorescence and genomic in situ hybridization. The presence of leaf rust resistance genes was confirmed with closely linked molecular markers, i.e., Xgdm35 and Xgwm296. 14 genotypes of BC2F4 - BC2F6 hybrid plants with the monosomic addition of chromosome 2D (M2DA) were analyzed together with nine control lines. Resistance was determined at the macroscopic and microscopic level at the seedling and adult plant stage (flag leaf). In general, results revealed limited resistance of hybrid plants at the seedling stage, followed by an increase of the resistance level at later stages of plant development. This indicates that respective hybrid plants may exhibit APR resistance conferred by Lr22a introgressed from Ae. tauschii. On the basis of the macroscopic and microscopic analysis, this kind of resistance turned out to be additive and race-specific. We selected four monosomic 2D addition triticale genotypes highly resistant to P. triticina infection at the two main stages of plant development. From the selected genotypes, we obtained 26 doubled haploid lines among which two lines with doubled additional chromosomes 2D of Ae. tauschii can be used for further breeding to increase leaf rust resistance of cultivated triticale.

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei).

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.

QTL mapping of partial resistance in winter wheat to Stagonospora nodorum blotch.

Stagonospora nodorum blotch is an important foliar and glume disease in cereals. Inheritance of resistance in wheat appears to be quantitative. To date, breeding of partially resistant cultivars has been the only effective way to combat this pathogen. The partial resistance components, namely length of incubation period, disease severity, and length of latent period, were evaluated on a population of doubled haploids derived from a cross between the partially resistant Triticum aestivum 'Liwilla' and susceptible Triticum aestivum 'Begra'. Experiments were conducted in a controlled environment and the fifth leaf was examined. Molecular analyses were based on bulked segregant analyses using 240 microsatellite markers. Four QTLs were significantly associated with partial resistance components and were located on chromosomes 2B, 3B, 5B, and 5D. The percentage of phenotypic variance explained by a single QTL ranged from 14 to 21% for incubation period, from 16 to 37% for disease severity, and from 13 to 28% for latent period,

Sequence diversity of mating-type genes in Phaeosphaeria avenaria.

Phaeosphaeria avenaria, one of the causal agents of stagonospora leaf blotch diseases in cereals, is composed of two subspecies, P. avenaria f. sp. triticea (Pat) and P. avenaria f. sp. avenaria (Paa). The Pat subspecies was grouped into Pat1-Pat3, based on restriction fragment length polymorphism (RFLP) and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences in previous studies. Mating-type genes and their potential use in phylogeny and molecular classification were studied by DNA hybridization and PCR amplification. The majority of Pat1 isolates reported to be homothallic and producing sexual reproduction structures on cultural media had only the MAT1-1 gene. Minor sequence variations were found in the conserved region of MAT1-1 gene in Pat1 isolates. However, both mating-type genes, MAT1-1 and MAT1-2, were identified in P. avenaria isolates represented by ATCC12277 from oats (Paa) and the Pat2 isolates from foxtail barley ( Hordeum jubatum L.). Cluster analyses based on mating-type gene conserved regions revealed that cereal Phaeosphaeria is not phylogenetically closely related to other ascomycetes, including Mycosphaerella graminicola (anamorph Septoria tritici). The sequence diversity of mating-type genes in Pat and Paa supports our previous phylogenetic relationship and molecular classification based on RFLP fingerprinting and rDNA ITS sequences.