Martin L. Carson

Century-old mystery of Puccinia striiformis life history solved with the identification of Berberis as an alternate host.

The life history of Puccinia striiformis remains a mystery because the alternate host has never been identified. Inoculation of grasses using aeciospores from naturally infected Berberis chinensis and B. koreana resulted in infection on Poa pratensis, producing uredinia typical of stripe rust caused by P. striiformis. Analyses using real-time polymerase chain reaction and DNA sequence confirmed the rust fungus as P. striiformis. Pycnia and aecia were produced on B. chinensis, B. holstii, B. koreana, and B. vulgaris after inoculation using germinating telia of P. striiformis f. sp. tritici. Wheat inoculated with aeciospores from B. chinensis resulted in uredinia, which demonstrated that Berberis spp. also serve as alternate hosts for the wheat stripe rust pathogen. The elucidation of the complete life history for P. striiformis f. sp. tritici will provide a powerful tool to rapidly advance our knowledge of the genetics of this rust fungus, and will lead to the development of improved strategies for a better control of stripe rust.

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Population Genomics Related to Adaptation in Elite Oat Germplasm

An oat association‐mapping panel contributed by active breeding programs worldwide. Characterized population structure and found subdivisions related to adaptation Characterized genome‐wide and chromosome‐specific linkage disequilibrium Performed association‐mapping and post hoc modeling of heading date Found several consistently associated QTL

Pathogen and host genotype differently affect pathogen fitness through their effects on different life-history stages

BackgroundAdaptation of pathogens to their hosts depends critically on factors affecting pathogen reproductive rate. While pathogen reproduction is the end result of an intricate interaction between host and pathogen, the relative contributions of host and pathogen genotype to variation in pathogen life history within the host are not well understood. Untangling these contributions allows us to identify traits with sufficient genetic variation for selection to act and to identify mechanisms of coevolution between pathogens and their hosts. We investigated the effects of pathogen and host genotype on three life-history components of pathogen fitness; infection efficiency, latent period, and sporulation capacity, in the oat crown rust fungus, Puccinia coronata f.sp. avenae, as it infects oats (Avena sativa).ResultsWe show that both pathogen and host genotype significantly affect total spore production but do so through their effects on different life-history stages. Pathogen genotype has the strongest effect on the early stage of infection efficiency, while host genotype most strongly affects the later life-history stages of latent period and sporulation capacity. In addition, host genotype affected the relationship between pathogen density and the later life-history traits of latent period and sporulation capacity. We did not find evidence of pathogen-by-host genotypic (GxG) interactions.ConclusionOur results illustrate mechanisms by which variation in host populations will affect the evolution of pathogen life history. Results show that different pathogen life-history stages have the potential to respond differently to selection by host or pathogen genotype and suggest mechanisms of antagonistic coevolution. Pathogen populations may adapt to host genotypes through increased infection efficiency while their plant hosts may adapt by limiting the later stages of pathogen growth and spore production within the host.

THE JACK OF ALL TRADES IS MASTER OF NONE: A PATHOGEN'S ABILITY TO INFECT A GREATER NUMBER OF HOST GENOTYPES COMES AT A COST OF DELAYED REPRODUCTION

A trade‐off between a pathogens ability to infect many hosts and its reproductive capacity on each host genotype is predicted to limit the evolution of an expanded host range, yet few empirical results provide evidence for the magnitude of such trade‐offs. Here, we test the hypothesis for a trade‐off between the number of host genotypes that a fungal pathogen can infect (host genotype range) and its reproductive capacity on susceptible plant hosts. We used strains of the oat crown rust fungus that carried widely varying numbers of virulence (avr) alleles known to determine host genotype range. We quantified total spore production and the expression of four pathogen life‐history stages: infection efficiency, time until reproduction, pustule size, and spore production per pustule. In support of the trade‐off hypothesis, we found that virulence level, the number of avr alleles per pathogen strain, was correlated with significant delays in the onset of reproduction and with smaller pustule sizes. Modeling from our results, we conclude that trade‐offs have the capacity to constrain the evolution of host genotype range in local populations. In contrast, long‐term trends in virulence level suggest that the continued deployment of resistant host lines over wide regions of the United States has generated selection for increased host genotype range.

Quantitative trait loci in the Ogle/TAM O-301 oat mapping population controlling resistance to Puccinia coronata in the field.

Crown rust is the most damaging disease of cultivated oat (Avena sativa) and genetic resistance is the primary means of controlling the disease. Quantitative trait loci (QTL) with major and minor effects have been identified in Ogle1040 and TAM O-301 (most notably, Pc58 and PcNQMG/LGCG from TAM O-301 and OT-27 from Ogle1040) through single-isolate greenhouse and field tests. To map loci and determine the effectiveness of previously identified QTL against naturally occurring pathogen populations in highly disease-conducive environments, the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population was grown in Texas and Louisiana over 2 years and in Manitoba, Canada. The genetic region characterized by the Pc58 resistance gene complex, particularly Pc58a, accounted for most of the diseased leaf area (DLA) and infection type (IT) variance in all five experiments. Additionally, the genetic region characterized by PcNQMG/LGCG accounted for a portion of the IT variance in three experiments. Although no QTL was detected on OT-27 in this study, all the markers on this linkage group were associated (P < 0.0001) with reducing both IT and DLA using single-marker analysis. Screening with 25 Puccinia coronata isolates from six different states indicated that Pc58abc and Pc58a were highly effective, while characterization using F(2) populations derived from OT RILs containing the two main genetic regions responsible for crown rust resistance in TAM O-301 (Pc58 and PcNQMG/LGCG) and a minor QTL in Ogle (OT-27) indicated that Pc58a, in combination with a locus in Ogle1040, provided high levels of resistance to natural races in Texas. This study provides new information and key loci in OT mapping population and may be useful for effective control of crown rust in North America.

Development and characterization of novel, polymorphic microsatellite markers for oat crown rust, Puccinia coronata

We report the development of 37 novel and polymorphic microsatellite markers for oat crown rust, Puccinia coronata f.sp. avenae. The allelic diversity ranged from two to 16 alleles per locus. Observed heterozygosity ranged from 0.000 to 0.971, and expected heterozygosity from 0.057 to 0.848. Thirteen of the loci were not in Hardy‐Weinberg equilibrium, due to either the presence of null alleles, small sample size, or the effects of population subdivision (Wahlunds effect). All 37 primer pairs were tested with P. graminis and P. triticina showing that they are specific to P. coronata.

Quantitative Trait Loci from Two Genotypes of Oat (Avena sativa) Conditioning Resistance to Puccinia coronata

Developing oat cultivars with partial resistance to crown rust would be beneficial and cost-effective for disease management. Two recombinant inbred-line populations were generated by crossing the susceptible cultivar Provena with two partially resistant sources, CDC Boyer and breeding line 94197A1-9-2-2-2-5. A third mapping population was generated by crossing the partially resistant sources to validate the quantitative trait locus (QTL) results. The three populations were evaluated for crown rust severity in the field at Louisiana State University (LSU) in 2009 and 2010 and at the Cereal Disease Laboratory (CDL) in St. Paul, MN, in 2009, 2010, and 2011. An iSelect platform assay containing 5,744 oat single nucleotide polymorphisms was used to genotype the populations. From the 2009 CDL test, linkage analyses revealed two QTLs for partial resistance in the Provena/CDC Boyer population on chromosome 19A. One of the 19A QTLs was also detected in the 2009 LSU test. Another QTL was detected on chromosome 12D in the CDL 2009 test. In the Provena/94197A1-9-2-2-2-5 population, only one QTL was detected, on chromosome 13A, in the CDL 2011 test. The 13A QTL from the Provena/94197A1-9-2-2-2-5 population was validated in the CDC Boyer/94197A1-9-2-2-2-5 population in the CDL 2010 and 2011 tests. Comparative analysis of the significant marker sequences with the rice genome database revealed 15 candidate genes for disease resistance on chromosomes 4 and 6 of rice. These genes could be potential targets for cloning from the two resistant parents.

Genome-Wide Association Mapping of Crown Rust Resistance in Oat Elite Germplasm

Multienvironment genome‐wide association study of reaction to crown rust in elite oat Oat response to inoculation with 10 well‐characterized Puccinia coronata isolates evaluated Adult plant response to crown rust assessed in 10 location–years Patterns of association compared against genotypes of differential gene stocks QTL placed in the context of current literature