Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array

Background A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNPbased linkage map of an apple rootstock progeny. Results Of the 7,867 Malus SNP markers on the array, 1,823 (23.2 %) were heterozygous in one of the two parents of the progeny, 1,007 (12.8 %) were heterozygous in both parental genotypes, whilst just 2.8 % of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the ‘Golden Delicious’ genome sequence. A total of 311 markers (13.7 % of all mapped markers) mapped to positions that conflicted with their predicted positions on the ‘Golden Delicious’ pseudo-chromosomes, indicating the presence of paralogous genomic regions or misassignments of genome sequence contigs during the assembly and anchoring of the genome sequence. Conclusions We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the ‘Golden Delicious’ reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts

When analysing the secretome of the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we identified hemolysin co-regulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the Pst DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of hcp genes and tested the fitness of hcp knock-out mutants in host plant colonization and in inter-microbial competition. We found that the hcp2 gene is expressed, most actively at the stationary growth phase, and that the Hcp2 protein is secreted via T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and it does not contribute to virulence or colonisation in tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition against yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive in conditions where it has to compete with other micro-organisms for resources.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S. enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.

Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ~200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.

Association mapping of partitioning loci in barley

Background: Association mapping, initially developed in human disease genetics, is now being applied to plant species. The model species Arabidopsis provided some of the first examples of association mapping in plants, identifying previously cloned flowering time genes, despite high population sub-structure. More recently, association genetics has been applied to barley, where breeding activity has resulted in a high degree of population sub-structure. A major genotypic division within barley is that between winter- and spring-sown varieties, which differ in their requirement for vernalization to promote subsequent flowering. To date, all attempts to validate association genetics in barley by identifying major flowering time loci that control vernalization requirement (VRN-H1 and VRN-H2) have failed. Here, we validate the use of association genetics in barley by identifying VRN-H1 and VRN-H2, despite their prominent role in determining population sub-structure. Results: By taking barley as a typical inbreeding crop, and seasonal growth habit as a major partitioning phenotype, we develop an association mapping approach which successfully identifies VRN-H1 and VRN-H2, the underlying loci largely responsible for this agronomic division. We find a combination of Structured Association followed by Genomic Control to correct for population structure and inflation of the test statistic, resolved significant associations only with VRN-H1 and the VRN-H2 candidate genes, as well as two genes closely linked to VRN-H1 (HvCSFs1 and HvPHYC). Conclusion: We show that, after employing appropriate statistical methods to correct for population sub-structure, the genome-wide partitioning effect of allelic status at VRN-H1 and VRN-H2 does not result in the high levels of spurious association expected to occur in highly structured samples. Furthermore, we demonstrate that both VRN-H1 and the candidate VRN-H2 genes can be identified using association mapping. Discrimination between intragenic VRN-H1 markers was achieved, indicating that candidate causative polymorphisms may be discerned and prioritised within a larger set of positive associations. This proof of concept study demonstrates the feasibility of association mapping in barley, even within highly structured populations. A major advantage of this method is that it does not require large numbers of genome-wide markers, and is therefore suitable for fine mapping and candidate gene evaluation, especially in species for which large numbers of genetic markers are either unavailable or too costly.

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Shoot calcium and magnesium concentrations differ between subtaxa, are highly heritable, and associate with potentially pleiotropic loci in Brassica oleracea

Calcium (Ca) and magnesium (Mg) are the most abundant group II elements in both plants and animals. Genetic variation in shoot Ca and shoot Mg concentration (shoot Ca and Mg) in plants can be exploited to biofortify food crops and thereby increase dietary Ca and Mg intake for humans and livestock. We present a comprehensive analysis of within-species genetic variation for shoot Ca and Mg, demonstrating that shoot mineral concentration differs significantly between subtaxa (varietas). We established a structured diversity foundation set of 376 accessions to capture a high proportion of species-wide allelic diversity within domesticated Brassica oleracea, including representation of wild relatives (C genome, 1n = 9) from natural populations. These accessions and 74 modern F-1 hybrid cultivars were grown in glasshouse and field environments. Shoot Ca and Mg varied 2- and 2.3-fold, respectively, and was typically not inversely correlated with shoot biomass, within most subtaxa. The closely related capitata (cabbage) and sabauda (Savoy cabbage) subtaxa consistently had the highest mean shoot Ca and Mg. Shoot Ca and Mg in glasshouse-grown plants was highly correlated with data from the field. To understand and dissect the genetic basis of variation in shoot Ca and Mg, we studied homozygous lines from a segregating B. oleracea mapping population. Shoot Ca and Mg was highly heritable (up to 40). Quantitative trait loci (QTL) for shoot Ca and Mg were detected on chromosomes C2, C6, C7, C8, and, in particular, C9, where QTL accounted for 14 to 55 of the total genetic variance. The presence of QTL on C9 was substantiated by scoring recurrent backcross substitution lines, derived from the same parents. This also greatly increased the map resolution, with strong evidence that a 4-cM region on C9 influences shoot Ca. This region corresponds to a 0.41-Mb region on Arabidopsis (Arabidopsis thaliana) chromosome 5 that includes 106 genes. There is also evidence that pleiotropic loci on C8 and C9 affect shoot Ca and Mg. Map-based cloning of these loci will reveal how shoot-level phenotypes relate to Ca 21 and Mg 21 uptake and homeostasis at the molecular level.

Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.