Genome-Wide Association Analyses Track Genomic Regions for Resistance to Ascochyta rabiei in Australian Chickpea Breeding Germplasm.

Ascochyta blight (AB), caused by a necrotrophic fungus, Ascochyta rabiei (syn. Phoma rabiei) has the potential to destroy the chickpea industry worldwide, due to limited sources of genetic resistance in the cultivated gene pool, high evolutionary potential of the pathogen and challenges with integrated disease management. Therefore, the deployment of stable genetic resistance in new cultivars could provide an effective disease control strategy. To investigate the genetic basis of AB resistance, genotyping-by-sequencing based DArTseq-single nucleotide polymorphism (SNP) marker data along with phenotypic data of 251 advanced breeding lines and chickpea cultivars were used to perform genome-wide association (GWAS) analysis. Host resistance was evaluated seven weeks after sowing using two highly aggressive single spore isolates (F17191-1 and TR9571) of A. rabiei. GWAS analyses based on single-locus and multi-locus mixed models and haplotyping trend regression identified twenty-six genomic regions on Ca1, Ca4, and Ca6 that showed significant association with resistance to AB. Two haplotype blocks (HB) on chromosome Ca1; HB5 (992178-1108145 bp), and HB8 (1886221-1976301 bp) were associated with resistance against both isolates. Nine HB on the chromosome, Ca4, spanning a large genomic region (14.9-56.6 Mbp) were also associated with resistance, confirming the role of this chromosome in providing resistance to AB. Furthermore, trait-marker associations in two F3 derived populations for resistance to TR9571 isolate at the seedling stage under glasshouse conditions were also validated. Eighty-nine significantly associated SNPs were located within candidate genes, including genes encoding for serine/threonine-protein kinase, Myb protein, quinone oxidoreductase, and calmodulin-binding protein all of which are implicated in disease resistance. Taken together, this study identifies valuable sources of genetic resistance, SNP markers and candidate genes underlying genomic regions associated with AB resistance which may enable chickpea breeding programs to make genetic gains via marker-assisted/genomic selection strategies.

Current population structure and pathogenicity patterns of Ascochyta rabiei in Australia.

Ascochyta blight disease, caused by the necrotrophic fungus Ascochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agroecologically distinct regions. Thanks to continuous monitoring efforts over the past 6 years, a comprehensive collection of A. rabiei isolates was collated from the major Australian chickpea production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterized using a DArTseq genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred. Overall, the Australian A. rabiei population displayed a far lower genetic diversity (average Nei's gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency. Among these, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population. The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the Australian A. rabiei population. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.

Evidence and Consequence of a Highly Adapted Clonal Haplotype within the Australian Ascochyta rabiei Population.

The Australian Ascochyta rabiei (Pass.) Labr. (syn. Phoma rabiei) population has low genotypic diversity with only one mating type detected to date, potentially precluding substantial evolution through recombination. However, a large diversity in aggressiveness exists. In an effort to better understand the risk from selective adaptation to currently used resistance sources and chemical control strategies, the population was examined in detail. For this, a total of 598 isolates were quasi-hierarchically sampled between 2013 and 2015 across all major Australian chickpea growing regions and commonly grown host genotypes. Although a large number of haplotypes were identified (66) through short sequence repeat (SSR) genotyping, overall low gene diversity (Hexp = 0.066) and genotypic diversity (D = 0.57) was detected. Almost 70% of the isolates assessed were of a single dominant haplotype (ARH01). Disease screening on a differential host set, including three commonly deployed resistance sources, revealed distinct aggressiveness among the isolates, with 17% of all isolates identified as highly aggressive. Almost 75% of these were of the ARH01 haplotype. A similar pattern was observed at the host level, with 46% of all isolates collected from the commonly grown host genotype Genesis090 (classified as "resistant" during the term of collection) identified as highly aggressive. Of these, 63% belonged to the ARH01 haplotype. In conclusion, the ARH01 haplotype represents a significant risk to the Australian chickpea industry, being not only widely adapted to the diverse agro-geographical environments of the Australian chickpea growing regions, but also containing a disproportionately large number of aggressive isolates, indicating fitness to survive and replicate on the best resistance sources in the Australian germplasm.

Genome Analysis Identified Novel Candidate Genes for Ascochyta Blight Resistance in Chickpea Using Whole Genome Re-sequencing Data.

Ascochyta blight (AB) is a fungal disease that can significantly reduce chickpea production in Australia and other regions of the world. In this study, 69 chickpea genotypes were sequenced using whole genome re-sequencing (WGRS) methods. They included 48 Australian varieties differing in their resistance ranking to AB, 16 advanced breeding lines from the Australian chickpea breeding program, four landraces, and one accession representing the wild chickpea species Cicer reticulatum. More than 800,000 single nucleotide polymorphisms (SNPs) were identified. Population structure analysis revealed relatively narrow genetic diversity amongst recently released Australian varieties and two groups of varieties separated by the level of AB resistance. Several regions of the chickpea genome were under positive selection based on Tajima's D test. Both Fst genome- scan and genome-wide association studies (GWAS) identified a 100 kb region (AB4.1) on chromosome 4 that was significantly associated with AB resistance. The AB4.1 region co-located to a large QTL interval of 7 Mb∼30 Mb identified previously in three different mapping populations which were genotyped at relatively low density with SSR or SNP markers. The AB4.1 region was validated by GWAS in an additional collection of 132 advanced breeding lines from the Australian chickpea breeding program, genotyped with approximately 144,000 SNPs. The reduced level of nucleotide diversity and long extent of linkage disequilibrium also suggested the AB4.1 region may have gone through selective sweeps probably caused by selection of the AB resistance trait in breeding. In total, 12 predicted genes were located in the AB4.1 QTL region, including those annotated as: NBS-LRR receptor-like kinase, wall-associated kinase, zinc finger protein, and serine/threonine protein kinases. One significant SNP located in the conserved catalytic domain of a NBS-LRR receptor-like kinase led to amino acid substitution. Transcriptional analysis using qPCR showed that some predicted genes were significantly induced in resistant lines after inoculation compared to non-inoculated plants. This study demonstrates the power of combining WGRS data with relatively simple traits to rapidly develop "functional makers" for marker-assisted selection and genomic selection.

SNP-Based Linkage Mapping for Validation of QTLs for Resistance to Ascochyta Blight in Lentil.

Lentil (Lens culinaris Medik.) is a self-pollinating, diploid, annual, cool-season, food legume crop that is cultivated throughout the world. Ascochyta blight (AB), caused by Ascochyta lentis Vassilievsky, is an economically important and widespread disease of lentil. Development of cultivars with high levels of durable resistance provides an environmentally acceptable and economically feasible method for AB control. A detailed understanding of the genetic basis of AB resistance is hence highly desirable, in order to obtain insight into the number and influence of resistance genes. Genetic linkage maps based on single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers have been developed from three recombinant inbred line (RIL) populations. The IH × NF map contained 460 loci across 1461.6 cM, while the IH × DIG map contained 329 loci across 1302.5 cM and the third map, NF × DIG contained 330 loci across 1914.1 cM. Data from these maps were combined with a map from a previously published study through use of bridging markers to generate a consensus linkage map containing 689 loci distributed across seven linkage groups (LGs), with a cumulative length of 2429.61 cM at an average density of one marker per 3.5 cM. Trait dissection of AB resistance was performed for the RIL populations, identifying totals of two and three quantitative trait loci (QTLs) explaining 52 and 69% of phenotypic variation for resistance to infection in the IH × DIG and IH × NF populations, respectively. Presence of common markers in the vicinity of the AB_IH1- and AB_IH2.1/AB_IH2.2-containing regions on both maps supports the inference that a common genomic region is responsible for conferring resistance and is associated with the resistant parent, Indianhead. The third QTL was derived from Northfield. Evaluation of markers associated with AB resistance across a diverse lentil germplasm panel revealed that the identity of alleles associated with AB_IH1 predicted the phenotypic responses with high levels of accuracy (~86%), and therefore have the potential to be widely adopted in lentil breeding programs. The availability of RIL-based maps, a consensus map, and validated markers linked to AB resistance provide important resources for lentil improvement.

Use of metabolomics for the chemotaxonomy of legume-associated Ascochyta and allied genera.

Chemotaxonomy and the comparative analysis of metabolic features of fungi have the potential to provide valuable information relating to ecology and evolution, but have not been fully explored in fungal biology. Here, we investigated the chemical diversity of legume-associated Ascochyta and Phoma species and the possible use of a metabolomics approach using liquid chromatography-mass spectrometry for their classification. The metabolic features of 45 strains including 11 known species isolated from various legumes were extracted, and the datasets were analyzed using chemometrics methods such as principal component and hierarchical clustering analyses. We found a high degree of intra-species consistency in metabolic profiles, but inter-species diversity was high. Molecular phylogenies of the legume-associated Ascochyta/Phoma species were estimated using sequence data from three protein-coding genes and the five major chemical groups that were detected in the hierarchical clustering analysis were mapped to the phylogeny. Clusters based on similarity of metabolic features were largely congruent with the species phylogeny. These results indicated that evolutionarily distinct fungal lineages have diversified their metabolic capacities as they have evolved independently. This whole metabolomics approach may be an effective tool for chemotaxonomy of fungal taxa lacking information on their metabolic content.