Asparagus cultivars grown worldwide are thought to have originated from a limited genetic base, however, selection has resulted in variation for climatic adaptation and other traits. Understanding genetic diversity of the crop is important to guide breeding decisions. The objectives of this research were to identify SNPs among 64 cultivated lines from different geographic areas and assess genetic variation, population structure and linkage disequilibrium. More than 55,000 SNPs were identified by GBS and subjected to filtration for minor allele frequency and missing data, resulting in 12,886 markers for all subsequent analysis. Markers exhibited a wide range of Expected Heterozygosity (He), Polymorphic Information Content (PIC) and Observed Heterozygosity (Ho) with mean values of 0.370, 0.310, and 0.450 respectively. Population STRUCTURE analysis indicated that the 64 lines were grouped into two, four, seven, and nine subpopulations. For K = 4, four distinct groups were defined: (1) New Zealand, New Jersey, France, and California; (2) Canada; (3) China, The Netherlands, and Germany; and (4) England, Denmark, Spain, Turkey, and India. The results were further confirmed by PCA, and a phylogenetic tree. LD declined rapidly with an increase in physical distance. A considerable amount of genetic diversity was observed, despite previous suggestions that asparagus cultivars may have originated from one open-pollinated population.

Field winterhardiness is a critical trait in rose cultivars ( Rosa × hybrida ) grown in northern climates. Although the molecular basis of cold hardiness has been well documented in model organisms such as Arabidopsis thaliana , little is known about the genetics and mechanisms underlying winterhardiness in roses. This research aims to explore the genetic control of winterhardiness for application in breeding programs using quantitative trail loci (QTL) analysis in two biparental rose populations derived from cold-hardy roses of the Canadian Explorer Series Collection. Field winterhardiness was assessed as a complex trait with winter damage and regrowth recorded in multiyear and multilocation trials in Ontario and Saskatchewan, Canada. In addition, this research explored the relationship between field measurements and electrolyte leakage recorded under artificial conditions. Electrolyte leakage had limited utility for application in rose breeding programs as a substitute for field evaluation, but did enable identification of QTL associated with potential cold hardiness candidate genes. A QTL for electrolyte leakage mapped to a genomic region that harbors a CBF1 -like transcription factor. A total of 14 QTLs associated with field winter damage and regrowth were discovered, and they explained between 11% and 37% of the observed phenotypic variance. Two QTL associated with winter damage and regrowth overlapped with a known QTL for black spot ( Diplocarpon rosae ) disease resistance, Rdr1 , in an environment under high disease pressure. Due to the complexity of field winterhardiness and its direct reliance on intertwined factors, such as overall plant health, moisture status, snow cover, and period of prolonged sub-zero temperatures, field trials are the ultimate measurement of field winterhardiness. Transgressive segregation was observed for all traits, and it was most likely due to complementary gene action. Field winter damage and regrowth were highly heritable in single environments, but they were subject to genotype × environment interaction resulting from pest pressure and severe climatic conditions.

Additional file of 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

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.

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.

The wheat gene Lr1 imparts resistance to certain races of the leaf rust pathogen Puccinia triticina. However, the biological basis underlying the complex resistance mechanisms remains largely unknown. Gene expression profiling analyses using the Affymetrix GeneChip® Wheat Genome Array were performed with six independent Lr1 transgenic lines sampled before inoculation (0 h) and at 6 and 24 h post-inoculation (HPI) with the avirulent P. triticina race CCDS. For the 6- and 24-HPI time points, 3,384 probe sets corresponding to 2,976 transcripts were expressed differentially compared with the plants before inoculation. Genes involved in defence signalling are triggered as early as 6 HPI and include COI1, the primary component of jasmonic acid signalling. By 24 HPI, a shift in plant cellular metabolism, namely carbon conservation, is notable. The enzymes of glycolysis and gluconeogenesis are down-regulated; however, enzymes linked to alternative carbon sequestration processes such as the glyoxylate cycle are up-regulated. Also at 24 HPI, secondary signalling molecules belonging to the various hormone signalling pathways were triggered. These microarray data suggest that the Lr1-mediated leaf rust resistance is achieved by early onset of defence signalling followed by a coordinated interplay of metabolism alteration and elicitation of defence responses.

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.

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.

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.

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.

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 ~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.

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.

Leaf rust resistance gene Lr1 was cloned and demonstrated by transformation of the susceptible cultivar Fielder. Transgenic line T0-938 had a single hemizygote Lr1 insertion and segregated in subsequent generations. Unlike the majority of R-genes, Lr1 has a co-dominant gene action, i.e., the heterozygotes exhibit an intermediate level of resistance. A gene expression experiment using T0-938 derived lines was performed to study the resistance, intermediate and susceptible pathways triggered in the presence of Lr1 in its homozygous or heterozygous states and in the absence of Lr1. The 27-Affymetrix chip experiment comprised 3 independent biological replicates, 3 genotypes (T4-938- homozygous for Lr1 (R), T4-938-sister line without Lr1 (S) and F1-938-heterozygous for Lr1 (I)), and 3 time points (before inoculation, 6h and 24h after inoculation). These lines genetically differed almost exclusively by their Lr1 gene dosage and provided near perfect isogenic lines for profiling the expression pathways triggered during the compatible and incompatible Lr1 wheat leaf rust pathosystem. Gene expression was measured by comparison to the expression levels before inoculation. A total of 584 of the 61,127 probe sets were differentially regulated in at least one of the 6 possible genotype × time point comparisons. Only 4 and 10 of these differentially regulated genes showed the same trends in all three genotypes at 6 and 24 hours after inoculation, respectively. The majority of genes differentially expressed in the three genotypes and at the two time points differed, indicating that pathways to resistance and susceptibility were largely not shared. Interestingly, the strict number of differentially regulated genes was higher in the intermediate and compatible reactions as it was in the incompatible reaction. Expression profiling analysis across genotypes and time points will be presented.

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 R2values > 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 R2values 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.

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.

The development of wheat genetic maps based on microsatellite (SSR) markers is giving researchers an unprecedented view into genome organization and breeding population structure. When this DNA-based technology is coupled with phenotypic data, QTL analysis and trait mapping are facilitated. This ultimately enables design of experiments to assemble complex genotypes that pyramid multiple traits