Two alternative recessive quantitative trait loci influence resistance to spring black stem and leaf spot in Medicago truncatula

Background Knowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. In this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by Phoma medicaginis, an economically important necrotrophic pathogen of Medicago spp., was examined in the model legume M. truncatula. Results Macroscopically, the resistant response of accession SA27063 was characterised by small, hypersensitive-like spots following inoculation while the susceptible interaction with accessions A17 and SA3054 showed necrotic lesions and spreading chlorosis. No unique cytological differences were observed during early infection (<48 h) between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in SA27063. In both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. Two F2 populations segregating for resistance to spring black stem and leaf spot were established between SA27063 and the two susceptible accessions, A17 and SA3054. The cross between SA27063 and A17 represented a wider cross than between SA27063 and SA3054, as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of F2 progeny. In the SA27063 × A17 F2 population a highly significant quantitative trait locus (QTL, LOD = 7.37; P < 0.00001) named resistance to the necrotroph P homa m edicaginis one (rnpm1) genetically mapped to the top arm of linkage group 4 (LG4). rnpm1 explained 33.6% of the phenotypic variance in the population's response to infection depicted on a 1–5 scale and was tightly linked to marker AW256637. A second highly significant QTL (LOD = 6.77; P < 0.00001), rnpm2, was located on the lower arm of LG8 in the SA27063 × SA3054 map. rnpm2 explained 29.6% of the phenotypic variance and was fine mapped to a 0.8 cM interval between markers h2_16a6a and h2_21h11d. rnpm1 is tightly linked to a cluster of Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) genes and disease resistance protein-like genes, while no resistance gene analogues (RGAs) are apparent in the genomic sequence of the reference accession A17 at the rnpm2 locus. Conclusion The induction of defence responses and cell death in the susceptible interaction following infection by P. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. A QTL for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions. Both loci are recessive in nature, and the simplest explanation for the existence of two separate QTLs is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined P. medicaginis virulence factors

Identification of QTLs associated with resistance to Phomopsis pod blight (Diaporthe toxica) in Lupinus albus

Phomopsis blight in Lupinus albus is caused by a fungal pathogen, Diaporthe toxica. It can invade all plant parts, leading to plant material becoming toxic to grazing animals, and potentially resulting in lupinosis. Identifying sources of resistance and breeding for resistance remains the best strategy for controlling Phomopsis and reducing lupinosis risks. However, loci associated with resistance to Phomopsis blight have not yet been identified. In this study, quantitative trait locus (QTL) analysis identified genomic regions associated with resistance to Phomopsis pod blight (PPB) using a linkage map of L. albus constructed previously from an F8 recombinant inbred line population derived from a cross between Kiev-Mutant (susceptible to PPB) and P27174 (resistant to PPB). Phenotyping was undertaken using a detached pod assay. In total, we identified eight QTLs for resistance to PPB on linkage group (LG) 3, LG6, LG10, LG12, LG17 and LG27 from different phenotyping environments. However, at least one QTL, QTL-5 on LG10 was consistently detected in both phenotyping environments and accounted for up to 28.2% of the total phenotypic variance. The results of this study showed that the QTL-2 on LG3 interacts epistatically with QTL-5 and QTL-6, which map on LG10 and LG12, respectively

Construction of a comparative genetic map in faba bean (Vicia faba L.); conservation of genome structure with Lens culinaris

Background The development of genetic markers is complex and costly in species with little pre-existing genomic information. Faba bean possesses one of the largest and least studied genomes among cultivated crop plants and no gene-based genetic maps exist. Gene-based orthologous markers allow chromosomal regions and levels of synteny to be characterised between species, reveal phylogenetic relationships and chromosomal evolution, and enable targeted identification of markers for crop breeding. In this study orthologous codominant cross-species markers have been deployed to produce the first exclusively gene-based genetic linkage map of faba bean (Vicia faba), using an F6 population developed from a cross between the lines Vf6 (equina type) and Vf27 (paucijuga type). Results Of 796 intron-targeted amplified polymorphic (ITAP) markers screened, 151 markers could be used to construct a comparative genetic map. Linkage analysis revealed seven major and five small linkage groups (LGs), one pair and 12 unlinked markers. Each LG was comprised of three to 30 markers and varied in length from 23.6 cM to 324.8 cM. The map spanned a total length of 1685.8 cM. A simple and direct macrosyntenic relationship between faba bean and Medicago truncatula was evident, while faba bean and lentil shared a common rearrangement relative to M. truncatula. One hundred and four of the 127 mapped markers in the 12 LGs, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the faba bean and M. truncatula genomes. However chromosomal rearrangements were observed that could explain the difference in chromosome numbers between these three legume species. These rearrangements suggested high conservation of M. truncatula chromosomes 1, 5 and 8; moderate conservation of chromosomes 2, 3, 4 and 7 and no conservation with M. truncatula chromosome 6. Multiple PCR amplicons and comparative mapping were suggestive of small-scale duplication events in faba bean. This study also provides a preliminary indication for finer scale macrosynteny between M. truncatula, lentil and faba bean. Markers originally designed from genes on the same M. truncatula BACs were found to be grouped together in corresponding syntenic areas in lentil and faba bean. Conclusion Despite the large size of the faba bean genome, comparative mapping did not reveal evidence for polyploidisation, segmental duplication, or significant rearrangements compared to M. truncatula, although a bias in the use of single locus markers may have limited the detection of duplications. Non-coding repetitive DNA or transposable element content provides a possible explanation for the difference in genome sizes. Similar patterns of rearrangements in faba bean and lentil compared to M. truncatula support phylogenetic studies dividing these species into the tribes Viceae and Trifoliae. However, substantial macrosynteny was apparent between faba bean and M. truncatula, with the exception of chromosome 6 where no orthologous markers were found, confirming previous investigations suggesting chromosome 6 is atypical. The composite map, anchored with orthologous markers mapped in M. truncatula, provides a central reference map for future use of genomic and genetic information in faba bean genetic analysis and breeding

Transposable element genomic fissuring in Pyrenophora teres is associated with genome expansion and dynamics of host-pathogen genetic interactions

Syme, Martin, Wyatt, Lawrence, Muria-Gonzalez, Friesen and Ellwood. Pyrenophora teres, P. teres f. teres (PTT) and P. teres f. maculata (PTM) cause significant diseases in barley, but little is known about the large-scale genomic differences that may distinguish the two forms. Comprehensive genome assemblies were constructed from long DNA reads, optical and genetic maps. As repeat masking in fungal genomes influences the final gene annotations, an accurate and reproducible pipeline was developed to ensure comparability between isolates. The genomes of the two forms are highly collinear, each composed of 12 chromosomes. Genome evolution in P. teres is characterized by genome fissuring through the insertion and expansion of transposable elements (TEs), a process that isolates blocks of genic sequence. The phenomenon is particularly pronounced in PTT, which has a larger, more repetitive genome than PTM and more recent transposon activity measured by the frequency and size of genome fissures. PTT has a longer cultivated host association and, notably, a greater range of host-pathogen genetic interactions compared to other Pyrenophora spp., a property which associates better with genome size than pathogen lifestyle. The two forms possess similar complements of TE families with Tc1/Mariner and LINE-like Tad-1 elements more abundant in PTT. Tad-1 was only detectable as vestigial fragments in PTM and, within the forms, differences in genome sizes and the presence and absence of several TE families indicated recent lineage invasions. Gene differences between P. teres forms are mainly associated with gene-sparse regions near or within TE-rich regions, with many genes possessing characteristics of fungal effectors. Instances of gene interruption by transposons resulting in pseudogenization were detected in PTT. In addition, both forms have a large complement of secondary metabolite gene clusters indicating significant capacity to produce an array of different molecules. This study provides genomic resources for functional genetics to help dissect factors underlying the host-pathogen interactions

A first genome assembly of the barley fungal pathogen Pyrenophora teres f. teres

Background: Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the cause of one of barley’s most important diseases, net form of net blotch. Here we report the first genome assembly for this species based solely on short Solexa sequencing reads of isolate 0-1. The assembly was validated by comparison to BAC sequences, ESTs, orthologous genes and by PCR, and complemented by cytogenetic karyotyping and the first genome-wide genetic map for P. teres f. teres. Results: The total assembly was 41.95 Mbp and contains 11,799 gene models of 50 amino acids or more. Comparison against two sequenced BACs showed that complex regions with a high GC content assembled effectively. Electrophoretic karyotyping showed distinct chromosomal polymorphisms between isolates 0-1 and 15A, and cytological karyotyping confirmed the presence of at least nine chromosomes. The genetic map spans 2477.7 cM and is composed of 243 markers in 25 linkage groups, and incorporates SSR markers developed from the assembly. Among predicted genes, non-ribosomal peptide synthetases and efflux pumps in particular appear to have undergone a P. teres f. teres-specific expansion of non-orthologous gene families. Conclusions: This study demonstrates that paired-end Solexa sequencing can successfully capture coding regions of a filamentous fungal genome. The assembly contains a plethora of predicted genes that have been implicated in a necrotrophic lifestyle and pathogenicity and presents a significant resource for examining the bases for P. teres f. teres pathogenicity

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.Centro de Investigaciones AgropecuariasFil: Sotiropoulos, Alexandros G. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Arango-Isaza, Epifanía. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Ban, Tomohiro. Yokohama City University. Kihara Institute for Biological Research; JapónFil: Barbieri, Chiara. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Barbieri, Chiara. Max Planck Institute for Evolutionary Anthropology. Department of Linguistic and Cultural Evolution; AlemaniaFil: Bourras, Salim. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Bourras, Salim. University of Agricultural Sciences. Department of Forest Mycology and Plant Pathology; SueciaFil: Cowger, Christina. North Carolina State University; Estados Unidos. USDA-ARS Department of Plant Pathology; Estados UnidosFil: Czembor, Paweł C. National Research Institute. Plant Breeding and Acclimatization Institute; PoloniaFil: Ben-David, Roi. ARO-Volcani Center. Institute of Plant Sciences. Department of Vegetables and Field Crops; IsraelFil: Dinoor, Amos. University of Jerusalem. The Robert H. Smith Faculty of Agriculture, Food & Environment. Department of Plant Pathology and Microbiology; IsraelFil: Ellwood, Simon R. Curtin University. School of Molecular and Life Sciences. Centre for Crop and Disease Management; AustraliaFil: Graf, Johannes. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Hatta, Koichi. National Agricultural Research Organization. Hokkaido Agricultural Research Center Field Crop Research and Development; JapónFil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigaciones Agropecuarias; ArgentinaFil: Wicker, Thomas. University of Zurich. Department of Plant and Microbial Biology; Suiz

Genetic diversity and population structure of Ascochyta rabiei from the western Iranian Ilam and Kermanshah provinces using MAT and SSR markers

Knowledge of genetic diversity in A. rabiei provides different levels of information that are important in the management of crop germplasm resources. Gene flow on a regional level indicates a significant potential risk for the regional spread of novel alleles that might contribute to fungicide resistance or the breakdown of resistance genes. Simple sequence repeat (SSR) and mating type (MAT) markers were used to determine the genetic structure, and estimate genetic diversity and the prevalence of mating types in 103 Ascochyta rabiei isolates from seven counties in the Ilam and Kermanshah provinces of western Iran (Ilam, Aseman abad, Holaylan, Chardavol, Dareh shahr, Gilangharb, and Sarpul). A set of 3 microsatellite primer pairs revealed a total of 75 alleles; the number of alleles varied from 15 to 34 for each marker. A high level of genetic variability was observed among A. rabiei isolates in the region. Genetic diversity was high (He = 0.788) within populations with corresponding high average gene flow and low genetic distances between populations. The smallest genetic distance was observed between isolates from Ilam and Chardavol. Both mating types were present in all populations, with the majority of the isolates belonging to Mat1-1 (64%), but within populations the proportions of each mating type were not significantly different from 50%. Results from this study will be useful in breeding for Ascochyta blight-resistant cultivars and developing necessary control measures

The First Genetic and Comparative Map of White Lupin (Lupinus albus L.): Identification of QTLs for Anthracnose Resistance and Flowering Time, and a Locus for Alkaloid Content

We report the first genetic linkage map of white lupin (Lupinus albus L.). An F8 recombinant inbred line population developed from Kiev mutant × P27174 was mapped with 220 amplified fragment length polymorphism and 105 gene-based markers. The genetic map consists of 28 main linkage groups (LGs) that varied in length from 22.7 cM to 246.5 cM and spanned a total length of 2951 cM. There were seven additional pairs and 15 unlinked markers, and 12.8% of markers showed segregation distortion at P < 0.05. Syntenic relationships between Medicago truncatula and L. albus were complex. Forty-five orthologous markers that mapped between M. truncatula and L. albus identified 17 small syntenic blocks, and each M. truncatula chromosome aligned to between one and six syntenic blocks in L. albus. Genetic mapping of three important traits: anthracnose resistance, flowering time, and alkaloid content allowed loci governing these traits to be defined. Two quantitative trait loci (QTLs) with significant effects were identified for anthracnose resistance on LG4 and LG17, and two QTLs were detected for flowering time on the top of LG1 and LG3. Alkaloid content was mapped as a Mendelian trait to LG11

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, Sotiropoulos et al. analyze a global sample of 172 mildew genomes, providing evidence that humans drove global spread of the pathogen throughout history and that mildew rapidly evolved through hybridization with local fungal strains.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

CodingQuarry: Highly accurate hidden Markov model gene prediction in fungal genomes using RNA-seq transcripts

Background: The impact of gene annotation quality on functional and comparative genomics makes gene prediction an important process, particularly in non-model species, including many fungi. Sets of homologous protein sequences are rarely complete with respect to the fungal species of interest and are often small or unreliable, especially when closely related species have not been sequenced or annotated in detail. In these cases, protein homology-based evidence fails to correctly annotate many genes, or significantly improve ab initio predictions. Generalised hidden Markov models (GHMM) have proven to be invaluable tools in gene annotation and, recently, RNA-seq has emerged as a cost-effective means to significantly improve the quality of automated gene annotation. As these methods do not require sets of homologous proteins, improving gene prediction from these resources is of benefit to fungal researchers. While many pipelines now incorporate RNA-seq data in training GHMMs, there has been relatively little investigation into additionally combining RNA-seq data at the point of prediction, and room for improvement in this area motivates this study. Results: CodingQuarry is a highly accurate, self-training GHMM fungal gene predictor designed to work with assembled, aligned RNA-seq transcripts. RNA-seq data informs annotations both during gene-model training and in prediction. Our approach capitalises on the high quality of fungal transcript assemblies by incorporating predictions made directly from transcript sequences. Correct predictions are made despite transcript assembly problems, including those caused by overlap between the transcripts of adjacent gene loci. Stringent benchmarking against high-confidence annotation subsets showed CodingQuarry predicted 91.3% of Schizosaccharomyces pombe genes and 90.4% of Saccharomyces cerevisiae genes perfectly. These results are 4-5% better than those of AUGUSTUS, the next best performing RNA-seq driven gene predictor tested. Comparisons against whole genome Sc. pombe and S. cerevisiae annotations further substantiate a 4-5% improvement in the number of correctly predicted genes. Conclusions: We demonstrate the success of a novel method of incorporating RNA-seq data into GHMM fungal gene prediction. This shows that a high quality annotation can be achieved without relying on protein homology or a training set of genes. CodingQuarry is freely available (https://sourceforge.net/projects/codingquarry/), and suitable for incorporation into genome annotation pipelines