Identification of a polymorphism within the Rosa multiflora muRdr1A gene linked to resistance to multiple races of Diplocarpon rosae W. in tetraploid garden roses (Rosa × hybrida).

KEY MESSAGE: A QTL for resistance to several races of black spot co-located with the known Rrd1 locus in Rosa. A polymorphism in muRdr1A linked to black spot resistance was identified and molecular markers were designed. Black spot, caused by Diplocarpon rosae, is one of the most serious foliar diseases of landscape roses that reduces the marketability and weakens the plants against winter survival. Genetic resistance to black spot (BS) exists and race-specific resistance is a good target to implement marker-assisted selection. High-density single nucleotide polymorphism-based genetic maps were created for the female parent of a tetraploid cross between 'CA60' and 'Singing in the Rain' using genotyping-by-sequencing following a two-way pseudo-testcross strategy. The female linkage map was generated based on 227 individuals and included 31 linkage groups, 1055 markers, with a length of 1980 cM. Race-specific resistance to four D. rosae races (5, 7, 10, 14) was evaluated using a detached leaf assay. BS resistance was also evaluated under natural infection in the field. Resistance to races 5, 10 and 14 of D. rosae and field resistance co-located on chromosome 1. A unique sequence of 32 bp in exon 4 of the muRdr1A gene was identified in 'CA60' that co-segregates with D. rosae resistance. Two diagnostic markers, a presence/absence marker and an INDEL marker, specific to this sequence were designed and validated in the mapping population and a backcross population derived from 'CA60.' Resistance to D. rosae race 7 mapped to a different location on chromosome 1.

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.

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.

BACKGROUND: Rust 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. RESULTS: To 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. CONCLUSIONS: The 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.

Molecular characterization of the Sasanda LTR copia retrotransposon family uncovers their recent amplification in Triticum aestivum (L.) genome.

Retrotransposons constitute a major proportion of the Triticeae genomes. Genome-scale studies have revealed their role in evolution affecting both genome structure and function and their potential for the development of novel markers. In this study, family members of an LTR copia retrotransposon which mediated the duplication of the gene encoding the high molecular weight glutenin subunit Bx7 in cultivar Glenlea were characterized. This novel element was named Sasanda_EU157184-1 (TREP3516). High density filters of the Glenlea hexaploid wheat BAC library were screened with a Sasanda long terminal repeat (LTR)-specific probe and approximately 1,075 positive clones representing an estimated copy number of 347 elements per haploid genome were identified. The 242 BAC clones with the strongest hybridization signal were selected. To maximize isolation of complete elements, this subset of clones was screened with a reverse transcriptase (RT) domain probe and DNA was isolated from the 133 clones that produced a strong hybridization signal. Left (5') and right (3') LTRs as well as the RT domains were PCR amplified and sequencing was carried out on the final subset of 121 clones. Evolutionary relationships were inferred from a data set consisting of 100 RT, 102 5' LTR and 100 3' LTR sequences representing 233, 451 and 495 informative sites for comparison, respectively. Neighbour-joining tree indicated that the element is at least 1.8 million years old and has evolved into a minimum of five sub-families. The insertion times of the 89 complete elements were estimated based on the divergence between their LTRs. Corroborating the inference from the RT domain, analysis of the LTR domains also indicated bursts of amplification from 2.6 million years ago (MYA) to now, except for one member dated to 4.6 +/- 0.7 MYA, which corresponds to the interval of divergence of Triticum and Aegilops (3 MYA) and divergence of Triticum and Rye (7 MYA). In 44 elements, the 5' and 3' LTRs were identical indicating recent transposition activity. The element can be used to develop retrotransposon-based markers such as sequence-specific amplified polymorphism, retrotransposon microsatellite amplified polymorphism and inter-retrotransposon amplified polymorphism, all of which are well suited for genotyping studies.

Identifying regions of the wheat genome controlling seed development by mapping expression quantitative trait loci.

Statistical methods established for the genetic analysis of quantitative traits can be applied to gene expression data. Quantitative trait locus (QTL) analysis can associate the expression of genes or groups of genes with particular genomic regions, and thereby identify regions regulating gene expression. A segregating population of 41 doubled haploid (DH) lines from the hard red spring wheat cross RL4452 x 'AC Domain' was used to map expression level polymorphisms. This population had previously been mapped with microsatellites, and includes a full QTL analysis for agronomic and seed quality traits. Expression analysis on mRNA from developing seed grown in two field locations was conducted on 39 of the 41 DH lines using the Affymetrix GeneChip Wheat Genome Array. Analysis of the hybridization intensity identified 1484 Affymetrix probe sets in the first location and 10,280 probe sets in the second location, where the hybridization intensity varied significantly between genotypes of the population. A common set of 1455 probe sets differing in intensity between genotypes in both locations was used for mapping, and 542 QTLs were identified that each mapped to a single chromosome interval, illustrating that major gene expression QTLs could be found in wheat. Genomic regions corresponding to multiple gene expression QTLs were identified. Comparison of expression mapping data with physical mapping of wheat expressed sequence tag (EST) sequences using rice synteny, as well as logarithm of odds (LOD) score analysis, showed that both cis- and trans-acting expression QTLs were present. Chromosomes 1D and 4B may contain significant trans-regulatory regions in this population.

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.

Complete Genome Sequence of Erwinia amylovora Bacteriophage vB_EamM_Ea35-70.

The complete genome of an Erwinia amylovora bacteriophage, vB_EamM_Ea35-70 (Ea35-70), is 271,084 bp, encodes 318 putative proteins, and contains one tRNA. Comparative analysis with other Myoviridae genomes suggests that Ea35-70 is related to the Phikzlikevirus genus within the family Myoviridae, since 26% of Ea35-70 proteins share homology to proteins in Pseudomonas phage φKZ.

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.

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.

Genome-wide linkage disequilibrium analysis in bread wheat and durum wheat.

Bread wheat and durum wheat were examined for linkage disequilibrium (LD) using microsatellite markers distributed across the genome. The allele database consisted of 189 bread wheat accessions genotyped at 370 loci and 93 durum wheat accessions genotyped at 245 loci. A significance level of p < 0.001 was set for all comparisons. The bread and durum wheat collections showed that 47.9% and 14.0% of all locus pairs were in LD, respectively. LD was more prevalent between loci on the same chromosome compared with loci on independent chromosomes and was highest between adjacent loci. Only a small fraction (bread wheat, 0.9%; durum wheat, 3.2%) of the locus pairs in LD showed R2 values > 0.2. The LD between adjacent locus pairs extended (R2 > 0.2) approximately 2-3 cM, on average, but some regions of the bread and durum wheat genomes showed high levels of LD (R2 = 0.7 and 1.0, respectively) extending 41.2 and 25.5 cM, respectively. The wheat collections were clustered by similarity into subpopulations using unlinked microsatellite data and the software Structure. Analysis within subpopulations showed 14- to 16-fold fewer locus pairs in LD, higher R2 values for those pairs in LD, and LD extending further along the chromosome. The data suggest that LD mapping of wheat can be performed with simple sequence repeats to a resolution of <5 cM.

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 F(1)-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 T (1) lines from resistant transgenic line T (0)-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. T (3)-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.

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.

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.

Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis.

Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.