Travis W. Banks

Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family

In hexaploid wheat, leaf rust resistance gene Lr1 is located at the distal end of the long arm of chromosome 5D. To clone this gene, an F1-derived doubled haploid population and a recombinant inbred line population from a cross between the susceptible cultivar AC Karma and the resistant line 87E03-S2B1 were phenotyped for resistance to Puccinia triticina race 1-1 BBB that carries the avirulence gene Avr1. A high-resolution genetic map of the Lr1 locus was constructed using microsatellite, resistance gene analog (RGA), BAC end (BE), and low pass (LP) markers. A physical map of the locus was constructed by screening a hexaploid wheat BAC library from cultivar Glenlea that is known to have Lr1. The locus comprised three RGAs from a gene family related to RFLP marker Xpsr567. Markers specific to each paralog were developed. Lr1 segregated with RGA567-5 while recombinants were observed for the other two RGAs. Transformation of the susceptible cultivar Fielder with RGA567-5 demonstrated that it corresponds to the Lr1 resistance gene. In addition, the candidate gene was also confirmed by virus-induced gene silencing. Twenty T1 lines from resistant transgenic line T0-938 segregated for resistance, partial resistance and susceptibility to Avr1 corresponding to a 1:2:1 ratio for a single hemizygous insertion. Transgene presence and expression correlated with the phenotype. The resistance phenotype expressed by Lr1 seemed therefore to be dependant on the zygosity status. T3-938 sister lines with and without the transgene were further tested with 16 virulent and avirulent rust isolates. Rust reactions were all as expected for Lr1 thereby providing additional evidence toward the Lr1 identity of RGA567-5. Sequence analysis of Lr1 indicated that it is not related to the previously isolated Lr10 and Lr21 genes and unlike these genes, it is part of a large gene family.

SNP Discovery through Next-Generation Sequencing and Its Applications

The decreasing cost along with rapid progress in next-generation sequencing and related bioinformatics computing resources has facilitated large-scale discovery of SNPs in various model and nonmodel plant species. Large numbers and genome-wide availability of SNPs make them the marker of choice in partially or completely sequenced genomes. Although excellent reviews have been published on next-generation sequencing, its associated bioinformatics challenges, and the applications of SNPs in genetic studies, a comprehensive review connecting these three intertwined research areas is needed. This paper touches upon various aspects of SNP discovery, highlighting key points in availability and selection of appropriate sequencing platforms, bioinformatics pipelines, SNP filtering criteria, and applications of SNPs in genetic analyses. The use of next-generation sequencing methodologies in many non-model crops leading to discovery and implementation of SNPs in various genetic studies is discussed. Development and improvement of bioinformatics software that are open source and freely available have accelerated the SNP discovery while reducing the associated cost. Key considerations for SNP filtering and associated pipelines are discussed in specific topics. A list of commonly used software and their sources is compiled for easy access and reference.

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control†

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox‐sensitive proteome upon wheat seed dormancy release. Using thiol‐specific fluorescent labelling, solubility‐based protein fractionation, 2‐D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non‐dormant, abscisic, or gibberellic acid‐treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox‐, stress‐, and pathogen‐responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non‐dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria

Puccinia triticina (Pt) is a representative of several cereal‐infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2‐D PAGE MS/MS and gel‐based LC‐MS (GeLC‐MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome‐predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT‐PCR, all were shown to be strongly upregulated and four expressed solely upon infection.

Generation of a wheat leaf rust, Puccinia triticina, EST database from stage‐specific cDNA libraries

SUMMARY Thirteen cDNA libraries constructed from small amounts of leaf rust mRNA using optimized methods served as the source for the generation of 25 558 high-quality DNA sequence reads. Five life-cycle stages were sampled: resting urediniospores, urediniospores germinated over water or plant extract, compatible, interactive stages during appressorium or haustorium formation just before sporulation, and an incompatible interaction. mRNA populations were subjected to treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification. Pathogen and host sequences from interactive libraries were differentiated in silico using cereal and fungal sequences, codon usage analyses, and by means of a partial prototype cDNA microarray hybridized with genomic DNAs. This yielded a non-redundant unigene set of 9760 putative fungal sequences consisting of 6616 singlets and 3144 contigs, representing 4.7 Mbp. At an E-value 10(-5), 3670 unigenes (38%) matched sequences in various databases and collections but only 694 unigenes (7%) were similar to genes with known functions. In total, 296 unigenes were identified as most probably wheat and ten as rRNA sequences. Annotation rates were low for germinated urediniospores (4%) and appressoria (2%). Gene sets obtained from the various life-cycle stages appear to be remarkably different, suggesting drastic reprogramming of the transcriptome during these major differentiation processes. Redundancy within contigs yielded information about possible expression levels of certain genes among stages. Many sequences were similar to genes from other rusts such as Uromyces and Melampsora species; some of these genes have been implicated in pathogenicity and virulence.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

BackgroundRust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.ResultsTo support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.ConclusionsThe current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Temporal Gene Expression Profiling of the Wheat Leaf Rust Pathosystem Using cDNA Microarray Reveals Differences in Compatible and Incompatible Defence Pathways

In this study, we detail the construction of a custom cDNA spotted microarray containing 7728 wheat ESTs and the use of the array to identify host genes that are differentially expressed upon challenges with leaf rust fungal pathogens. Wheat cultivar RL6003 (Thatcher Lr1) was inoculated with Puccinia triticina virulence phenotypes BBB (incompatible) or TJB (7-2) (compatible) and sampled at four different time points (3, 6, 12, and 24 hours) after inoculation. Transcript expression levels relative to a mock treatment were measured. One hundred ninety two genes were found to have significantly altered expression between the compatible and incompatible reactions. Among those were genes involved in photosynthesis, the production of reactive oxygen species, ubiquitination, signal transduction, as well as in the shikimate/phenylpropanoid pathway. These data indicate that various metabolic pathways are affected, some of which might be used by RL6003 to mount a coordinated defense against an incompatible fungal pathogen.

Single-Feature Polymorphism Mapping in Bread Wheat

Probe hybridization data from the Affymetrix GeneChip platform can be used to identify genetic polymorphisms. Developed from gene expression data, these single‐feature polymorphism (SFP) markers are located in or are tightly associated with gene sequences. Using two different methods to identify SFPs, 1035 and 875 SFPs were mapped in a segregating population of 64 doubled haploid lines from the Triticum aestivum L. cross RL4452 × ‘AC Domain’. Statistical associations between the SFP maps and the rice (Oryza sativa L. ssp. japonica) genome were in agreement with known cereal syntenic blocks, and the mapping data were corroborated by previously established physical locations for wheat expressed sequence tags. This approach allowed the rapid identification of markers from the genic regions of the complex hexaploid wheat genome.

Mapping the sensory perception of apple using descriptive sensory evaluation in a genome wide association study

Breeding apples is a long-term endeavour and it is imperative that new cultivars are selected to have outstanding consumer appeal. This study has taken the approach of merging sensory science with genome wide association analyses in order to map the human perception of apple flavour and texture onto the apple genome. The goal was to identify genomic associations that could be used in breeding apples for improved fruit quality. A collection of 85 apple cultivars was examined over two years through descriptive sensory evaluation by a trained sensory panel. The trained sensory panel scored randomized sliced samples of each apple cultivar for seventeen taste, flavour and texture attributes using controlled sensory evaluation practices. In addition, the apple collection was subjected to genotyping by sequencing for marker discovery. A genome wide association analysis suggested significant genomic associations for several sensory traits including juiciness, crispness, mealiness and fresh green apple flavour. The findings include previously unreported genomic regions that could be used in apple breeding and suggest that similar sensory association mapping methods could be applied in other plants.

In silico physical mapping software for the Triticum aestivum genome

The large size of the Triticum aestivum genome makes it unlikely that a complete genome sequence for wheat will be available in the near future. Exploiting the conserved genome organization between wheat and rice and existing genomic resources, we have constructed in silico physical mapping software for wheat, assigning a gross physical location(s) into chromosome bins to 22,626 representative wheat gene sequences. To validate the predictions from the software we compared the predicted locations of ten ESTs to their positions experimentally determined by SNP marker analysis. Six of the sequences were correctly positioned on the map including four that demonstrated a high level of colinearity with their orthologous rice genomic region. This tool will facilitate the development of molecular markers for regions of interest and the creation of map-based cloning strategies in areas demonstrating high levels of sequence conservation and organization between wheat and rice.