Mapping QTL associated with resistance to Pseudomonas syringae pv. actinidiae in kiwifruit (Actinidia chinensis var. chinensis).

Pseudomonas syringae pv. actinidiae (Psa) is a bacterial pathogen of kiwifruit. This pathogen causes leaf-spotting, cane dieback, wilting, cankers (lesions), and in severe cases, plant death. Families of diploid A. chinensis seedlings grown in the field show a range of susceptibilities to the disease with up to 100% of seedlings in some families succumbing to Psa. But the effect of selection for field resistance to Psa on the alleles that remain in surviving seedlings has not been assessed. The objective of this work was to analyse, the effect of plant removal from Psa on the allele frequency of an incomplete-factorial-cross population. This population was founded using a range of genotypically distinct diploid A. chinensis var. chinensis parents to make 28 F1 families. However, because of the diversity of these families, low numbers of surviving individuals, and a lack of samples from dead individuals, standard QTL mapping approaches were unlikely to yield good results. Instead, a modified bulk segregant analysis (BSA) overcame these drawbacks while reducing the costs of sampling and sample processing, and the complexity of data analysis. Because the method was modified, part one of this work was used to determine the signal strength required for a QTL to be detected with BSA. Once QTL detection accuracy was known, part two of this work analysed the 28 families from the incomplete-factorial-cross population that had multiple individuals removed due to Psa infection. Each family was assigned to one of eight bulks based on a single parent that contributed to the families. DNA was extracted in bulk by grinding sampled leaf discs together before DNA extraction. Each sample bulk was compared against a bulk made up of WGS data from the parents contributing to the sample bulk. The deviation in allele frequency from the expected allele frequency within surviving populations using the modified BSA method was able to identify 11 QTLs for Psa that were present in at least two analyses. The identification of these Psa resistance QTL will enable marker development to selectively breed for resistance to Psa in future kiwifruit breeding programs.

The Relative Power of Structural Genomic Variation versus SNPs in Explaining the Quantitative Trait Growth in the Marine Teleost Chrysophrys auratus.

BACKGROUND: Genetic diversity provides the basic substrate for evolution. Genetic variation consists of changes ranging from single base pairs (single-nucleotide polymorphisms, or SNPs) to larger-scale structural variants, such as inversions, deletions, and duplications. SNPs have long been used as the general currency for investigations into how genetic diversity fuels evolution. However, structural variants can affect more base pairs in the genome than SNPs and can be responsible for adaptive phenotypes due to their impact on linkage and recombination. In this study, we investigate the first steps needed to explore the genetic basis of an economically important growth trait in the marine teleost finfish Chrysophrys auratus using both SNP and structural variant data. Specifically, we use feature selection methods in machine learning to explore the relative predictive power of both types of genetic variants in explaining growth and discuss the feature selection results of the evaluated methods. METHODS: SNP and structural variant callers were used to generate catalogues of variant data from 32 individual fish at ages 1 and 3 years. Three feature selection algorithms (ReliefF, Chi-square, and a mutual-information-based method) were used to reduce the dataset by selecting the most informative features. Following this selection process, the subset of variants was used as features to classify fish into small, medium, or large size categories using KNN, naïve Bayes, random forest, and logistic regression. The top-scoring features in each feature selection method were subsequently mapped to annotated genomic regions in the zebrafish genome, and a permutation test was conducted to see if the number of mapped regions was greater than when random sampling was applied. RESULTS: Without feature selection, the prediction accuracies ranged from 0 to 0.5 for both structural variants and SNPs. Following feature selection, the prediction accuracy increased only slightly to between 0 and 0.65 for structural variants and between 0 and 0.75 for SNPs. The highest prediction accuracy for the logistic regression was achieved for age 3 fish using SNPs, although generally predictions for age 1 and 3 fish were very similar (ranging from 0-0.65 for both SNPs and structural variants). The Chi-square feature selection of SNP data was the only method that had a significantly higher number of matches to annotated genomic regions of zebrafish than would be explained by chance alone. CONCLUSIONS: Predicting a complex polygenic trait such as growth using data collected from a low number of individuals remains challenging. While we demonstrate that both SNPs and structural variants provide important information to help understand the genetic basis of phenotypic traits such as fish growth, the full complexities that exist within a genome cannot be easily captured by classical machine learning techniques. When using high-dimensional data, feature selection shows some increase in the prediction accuracy of classification models and provides the potential to identify unknown genomic correlates with growth. Our results show that both SNPs and structural variants significantly impact growth, and we therefore recommend that researchers interested in the genotype-phenotype map should strive to go beyond SNPs and incorporate structural variants in their studies as well. We discuss how our machine learning models can be further expanded to serve as a test bed to inform evolutionary studies and the applied management of species.

Differential expression analyses reveal extensive transcriptional plasticity induced by temperature in New Zealand silver trevally (Pseudocaranx georgianus).

Ectotherm species, such as marine fishes, depend on environmental temperature to regulate their vital functions. In finfish aquaculture production, being able to predict physiological responses in growth and other economic traits to temperature is crucial to address challenges inherent in the selection of grow-out locations. This will become an even more significant issue under the various predicted future climate change scenarios. In this study, we used the marine teleost silver trevally (Pseudocaranx georgianus), a species currently being explored as a candidate for aquaculture in New Zealand, as a model to study plasticity in gene expression patterns and growth in response to different temperatures. Using a captive study population, temperature conditions were experimentally manipulated for 1 month to mimic seasonal extremes. Phenotypic differences in growth were measured in 400 individuals, and gene expression patterns of pituitary gland and liver were determined in a subset of 100 individuals. Results showed that growth increased 50% in the warmer compared with the colder condition, suggesting that temperature has a large impact on metabolic activities associated with growth. A total of 265,116,678 single-end RNA sequence reads were aligned to the trevally genome, and 28,416 transcript models were developed (27,887 of these had GenBank accessions, and 17,980 unique gene symbols). Further filtering reduced this set to 8597 gene models. 39 and 238 differentially expressed genes (DEGs) were found in the pituitary gland and the liver, respectively (|log2FC| > 0.26, p-value < 0.05). Of these, 6 DEGs showed a common expression pattern between both tissues, all involved in housekeeping functions. Temperature-modulated growth responses were linked to major pathways affecting metabolism, cell regulation and signalling, previously shown to be important for temperature tolerance in other fish species. An interesting finding of this study was that genes linked to the reproductive system were up-regulated in both tissues in the high treatment, indicating the onset of sexual maturation. Few studies have investigated the thermal plasticity of the gene expression in the main organs of the somatotropic axis simultaneously. Our findings indicate that trevally exhibit substantial growth differences and predictable plastic regulatory responses to different temperature conditions. We identified a set of genes that provide a list of candidates for further investigations for selective breeding objectives and how populations may adapt to increasing temperatures.

The genome of New Zealand trevally (Carangidae: Pseudocaranx georgianus) uncovers a XY sex determination locus.

BACKGROUND: The genetic control of sex determination in teleost species is poorly understood. This is partly because of the diversity of mechanisms that determine sex in this large group of vertebrates, including constitutive genes linked to sex chromosomes, polygenic constitutive mechanisms, environmental factors, hermaphroditism, and unisexuality. Here we use a de novo genome assembly of New Zealand silver trevally (Pseudocaranx georgianus) together with sex-specific whole genome sequencing data to detect sexually divergent genomic regions, identify candidate genes and develop molecular makers. RESULTS: The de novo assembly of an unsexed trevally (Trevally_v1) resulted in a final assembly of 579.4 Mb in length, with a N50 of 25.2 Mb. Of the assembled scaffolds, 24 were of chromosome scale, ranging from 11 to 31 Mb in length. A total of 28,416 genes were annotated after 12.8 % of the assembly was masked with repetitive elements. Whole genome re-sequencing of 13 wild sexed trevally (seven males and six females) identified two sexually divergent regions located on two scaffolds, including a 6 kb region at the proximal end of chromosome 21. Blast analyses revealed similarity between one region and the aromatase genes cyp19 (a1a/b) (E-value < 1.00E-25, identity > 78.8 %). Males contained higher numbers of heterozygous variants in both regions, while females showed regions of very low read-depth, indicative of male-specificity of this genomic region. Molecular markers were developed and subsequently tested on 96 histologically-sexed fish (42 males and 54 females). Three markers amplified in absolute correspondence with sex (positive in males, negative in females). CONCLUSIONS: The higher number of heterozygous variants in males combined with the absence of these regions in females support a XY sex-determination model, indicating that the trevally_v1 genome assembly was developed from a male specimen. This sex system contrasts with the ZW sex-determination model documented in closely related carangid species. Our results indicate a sex-determining function of a cyp19a1a-like gene, suggesting the molecular pathway of sex determination is somewhat conserved in this family. The genomic resources developed here will facilitate future comparative work, and enable improved insights into the varied sex determination pathways in teleosts. The sex marker developed in this study will be a valuable resource for aquaculture selective breeding programmes, and for determining sex ratios in wild populations.

Construction of a high-density genetic map for hexaploid kiwifruit (Actinidia chinensis var. deliciosa) using genotyping by sequencing.

Commercially grown kiwifruit (genus Actinidia) are generally of two sub-species which have a base haploid genome of 29 chromosomes. The yellow-fleshed Actinidia chinensis var. chinensis, is either diploid (2n = 2x = 58) or tetraploid (2n = 4x = 116) and the green-fleshed cultivar A. chinensis var. deliciosa "Hayward," is hexaploid (2n = 6x = 174). Advances in breeding green kiwifruit could be greatly sped up by the use of molecular resources for more efficient and faster selection, for example using marker-assisted selection (MAS). The key genetic marker that has been implemented for MAS in hexaploid kiwifruit is for gender testing. The limited marker-trait association has been reported for other polyploid kiwifruit for fruit and production traits. We have constructed a high-density linkage map for hexaploid green kiwifruit using genotyping-by-sequence (GBS). The linkage map obtained consists of 3686 and 3940 markers organized in 183 and 176 linkage groups for the female and male parents, respectively. Both parental linkage maps are co-linear with the A. chinensis "Red5" reference genome of kiwifruit. The linkage map was then used for quantitative trait locus (QTL) mapping, and successfully identified QTLs for king flower number, fruit number and weight, dry matter accumulation, and storage firmness. These are the first QTLs to be reported and discovered for complex traits in hexaploid kiwifruit.

CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants.

BACKGROUND: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. RESULTS: We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologiesCleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. CONCLUSIONS: We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

Comparison of two different host plant genera responding to grapevine leafroll-associated virus 3 infection.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Microarray analysis of kiwifruit (Actinidia chinensis) bark following challenge by the sucking insect Hemiberlesia lataniae (Hemiptera: Diaspididae).

Both commercial and experimental genotypes of kiwifruit (Actinidia spp.) exhibit large differences in response to insect pests. An understanding of the vine's physiological response to insect feeding and its genetic basis will be important in assisting the development of varieties with acceptable levels of pest resistance. This experiment describes transcriptome changes observed in the bark of kiwifruit 2 and 7 days after the commencement of feeding by the armored scale insect pest, Hemiberlesia lataniae. Using a cDNA microarray consisting of 17,512 unigenes, we measured transcriptome changes and analyzed these into functional ontology categories using MapMan. Results are available in the GEO database GSE73922 and are described fully in Ref. Hill et al. (2015) [1]. After 7 days, transcripts associated with photosynthesis were down-regulated and secondary metabolism was up-regulated. Differential expression of transcripts associated with stress response was consistent with a defense response involving both effector and herbivore-triggered immunities, with predominant involvement of the salicylic acid phytohormonal pathway. This hypothesis was supported by the results of two laboratory experiments. The methods described here could be further adapted and applied to the study of plant responses to a wide range of sessile sucking pests.

Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

UNLABELLED: Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. METHOD: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS.

Transcriptome Analysis of Kiwifruit (Actinidia chinensis) Bark in Response to Armoured Scale Insect (Hemiberlesia lataniae) Feeding.

The kiwifruit cultivar Actinidia chinensis 'Hort16A' is resistant to the polyphagous armoured scale insect pest Hemiberlesia lataniae (Hemiptera: Diaspididae). A cDNA microarray consisting of 17,512 unigenes selected from over 132,000 expressed sequence tags (ESTs) was used to measure the transcriptomic profile of the A. chinensis 'Hort16A' canes in response to a controlled infestation of H. lataniae. After 2 days, 272 transcripts were differentially expressed. After 7 days, 5,284 (30%) transcripts were differentially expressed. The transcripts were grouped into 22 major functional categories using MapMan software. After 7 days, transcripts associated with photosynthesis (photosystem II) were significantly down-regulated, while those associated with secondary metabolism were significantly up-regulated. A total of 643 transcripts associated with response to stress were differentially expressed. This included biotic stress-related transcripts orthologous with pathogenesis related proteins, the phenylpropanoid pathway, NBS-LRR (R) genes, and receptor-like kinase-leucine rich repeat signalling proteins. While transcriptional studies are not conclusive in their own right, results were suggestive of a defence response involving both ETI and PTI, with predominance of the SA signalling pathway. Exogenous application of an SA-mimic decreased H. lataniae growth on A. chinensis 'Hort16A' plants in two laboratory experiments.

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity.

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.

Effects of kiwifruit extracts on colonic gene and protein expression levels in IL-10 gene-deficient mice.

Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity using in vitro models of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effects in vivo against inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10(-/-)) mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While all Il10(-/-) mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated from Il10(-/-) and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriate in vivo models before making dietary recommendations, as a food that looks promising in vitro may not be effective in vivo.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species.

BACKGROUND: Inappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohn's Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/- mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF.CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/- mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/- mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD. RESULTS: At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44 k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups. Genes differentially expressed in Il10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). CONCLUSIONS: Our results suggest that colonic inflammation in Il10-/- mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF.CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds.

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Conversion of MapMan to allow the analysis of transcript data from Solanaceous species: effects of genetic and environmental alterations in energy metabolism in the leaf.

The tomato microarray TOM1 offers the possibility to monitor the levels of several thousand transcripts in parallel. The microelements represented on this tomato microarray have been putatively assigned to unigenes, and organised in functional classes using the MapMan ontology (Thimm et al., 2004. Plant J. 37: 914-939). This ontology was initially developed for use with the Arabidopsis ATH1 array, has a low level of redundancy, and can be combined with the MapMan software to provide a biologically structured overview of changes of transcripts, metabolites and enzyme activities. Use of this application is illustrated using three case studies with published or novel TOM1 array data sets for Solanaceous species. Comparison of previously reported data on transcript levels in potato leaves in the middle of the day and the middle of the night identified coordinated changes in the levels of transcripts of genes involved in various metabolic pathways and cellular events. Comparison with diurnal changes of gene expression in Arabidopsis revealed common features, illustrating how MapMan can be used to compare responses in different organisms. Comparison of transcript levels in new experiments performed on the leaves of the cultivated tomato S. lycopersicum and the wild relative S. pennellii revealed a general decrease of levels of transcripts of genes involved in terpene and, phenylpropanoid metabolism as well as chorismate biosynthesis in the crop compared to the wild relative. This matches the recently reported decrease of the levels of secondary metabolites in the latter. In the third case study, new expression array data for two genotypes deficient in TCA cycle enzymes is analysed to show that these genotypes have elevated levels of transcripts associated with photosynthesis. This in part explains the previously documented enhanced rates of photosynthesis in these genotypes. Since the Solanaceous MapMan is intended to be a community resource it will be regularly updated on improvements in tomato gene annotation and transcript profiling resources.