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Early induced genes ranked by the ratio of expression at three hours post infection (HPI) compared with in vitro (cutoff 5-fold). (DOCX 20 kb)

Reads per kilobase per million (RPKM) values of genes encoding proteins predicted to be secreted via T2SS. (DOCX 25 kb)

Genes most highly upregulated in the mid phase of the infection time course (3â 24Â h post infection, (HPI)). Genes are ranked on the ratio of maximum Reads Per Kilobase per Million over that time period compared with in vitro expression (cutoff 5-fold). (DOCX 24 kb)

Comparison of predicted paired end distance to genome.Heatmaps of alignment distance scores for the alignment of the read pairs from the 9Kb long-insert mate-paired-end (LIMP) library to each of the 29 chromosomes within the Red5 whole genome assembly and. Individual chromosome plots were prepared using hagfish_blockplot from the software program â hagfishâ ( https://github.com/mfiers/hagfish/ ). Individual images were cropped for height (not length) then cut and pasted into a table format for easier viewing. Each image depicted the entire length of the chromosome but all images are of standard length irrespective of chromosome length. Green regions indicate mate pairs aligning to the whole genome sequence within the expected distance of the library. Black indicates regions without mate pair alignment. Pinkish-red indicates regions where the distance between mated paired end reads is shorter (assembly compression relative to physical genome) or longer (assembly expansion relative to physical genome). (PPTX 432Â kb)

The manual annotation process.Flow diagram of manual annotation process. A. Timeline showing the manual annotation process. *see materials and methods. B. Annotation followed a 5 step process. The annotator training was completed in the form of both workshops and YouTube training videos. ** https://www.youtube.com/playlist?list=PLcBe8nhQVgUg1zqOsdeRuVq9QVsLfj_Y9 . (PPTX 47Â kb)

Expression levels of secondary metabolite gene clusters. Means of reads per kilobase per million (RPKM) for each gene across all time points with standard deviations. (DOCX 13 kb)

Genes expressed in the mid phase of infection that do not encode the T3SS or T3SEs. Effectors were ranked by the highest level of expression between 3 and 24 HPI compared with in vitro (cutoff 5-fold). (DOCX 21 kb)

Expression levels of individual Psa effectors over the infection time course. Effectors are ranked by the highest level of expression between 3 and 12Â h post infection (HPI) in reads per kilobase per million (RPKM). Effectors likely to be disrupted, duplicated or pseudogenes were not included. (DOCX 22 kb)

Expression of non-effector genes with upstream HrpL boxes. Genes are ranked by the ration of expression at 12Â h post infection (HPI) compared with in vitro expression. (DOCX 18 kb)

Genes upregulated late in the time course . Genes were ranked based on the ratio of expression at 120Â h post infection (HPI) compared with 1.5 HPI. (DOCX 22 kb)

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

Negli ultimi anni si è intensificata la ricerca di nuove varietà precoci di actinidia a polpa verde principalmente per motivi colturali ed economici. Una raccolta anticipata, infatti, riduce il rischio di danni causati da gelate precoci mentre la collocazione del prodotto sul mercato in un periodo antecedente ad Hayward (compreso tra Settembre e la fine dell’anno) puo` trovare riscontri positivi a livello di prezzo (primizia). La cultivar Summer 3373 è al momento considerata tra le più promettenti nel futuro prossimo poiché può essere raccolta 40-45 giorni prima di Hayward e non richiede un lungo periodo di frigo-conservazione. Il seguente studio, iniziato con la messa a dimora delle piante nel Marzo 2002, ha lo scopo di caratterizzare la varietà. A partire dal 2003 sono stati rilevati i principali cambiamenti fenologici delle piante: epoca e percentuale di germogliamento, epoca di fioritura, sviluppo e crescita dei frutti e loro epoca di maturazione. Tramite le tradizionali metodologie distruttive ed analisi non distruttive (tecnologia NIRs) è stata, inoltre, seguita l’evoluzione delle principali caratteristiche chimico-fisiche dei frutti a partire da un mese circa prima della prevista data di raccolta (prima decade di Settembre). Lo studio è continuato negli anni successivi per verificare l’effettivo anticipo di maturazione rispetto alla cultivar Hayward e l’effetto della frigo-conservazione sui frutti.

L’attuale panorama varietale dell’actinidia si sta diversificando sempre più rispetto alla situazione stagnante di qualche anno fa data la disponibilità di potenziali parentali appartenenti a specie diverse per il miglioramento genetico. In particolare, nei prossimi anni è lecito aspettarsi l’introduzione di nuove selezioni a polpa gialla che affiancheranno quelle già esistenti. A tale proposito, da alcuni anni, è in corso un programma di miglioramento genetico a partire da linee parentali di A. chinensis. Tale programma è svolto presso l’azienda sperimentale dell’Università di Bologna in collaborazione con il Dipartimento di Scienze agrarie e ambientali dell’Università di Udine. Nel presente lavoro si riportano i risultati ottenuti dall’analisi di 303 individui appartenenti a 15 diverse famiglie di incrocio e messi a dimora nel 2003. Per ogni individuo sono stati rilevati l’epoca di germogliamento e di fioritura per tutti gli individui presenti; l’accrescimento e la maturazione dei frutti per le selezioni femminili. Al termine della stagione 2006, sono state individuate alcune selezioni che presentano caratteristiche di interesse, quali una buona pezzatura e buone caratteristiche organolettiche dei frutti. Tali selezioni saranno oggetto di uno studio più approfondito nel corso dei prossimi anni.

Il CPPU è stato recentemente registrato in Italia per l’uso sulla coltura di actinidia. Peraltro, nonostante i numerosi studi condotti, il meccanismo di azione non è ancora stato completamento chiarito. L’applicazione di CPPU ha come principale effetto l’aumento della pezzatura dei frutti, sebbene non sempre ciò sia accompagnato da un accumulo di sostanza secca (%SS). La %SS è correlata con la concentrazione di carboidrati e con la qualità finale dei frutti. Il presente lavoro ha affrontato lo studio della relazione tra l’applicazione del formulato e l’accumulo di carboidrati. Il CPPU alla concentrazione di 10ppm è stato applicato per immersione su frutticini 28 giorni dopo la fioritura. I frutti erano portati da germogli anulati dove erano stati imposti diversi rapporti foglie /frutti pari a 3:1 (alta carica) o 6:1 (bassa carica). Gli effetti su pezzatura e peso secco dei frutti sono stati rilevati a partire da 7 giorni dal trattamento. I frutti trattati con CPPU hanno presentato un peso fresco e secco maggiore del controllo mentre la %SS diminuiva a partire da 3 settimane dopo il trattamento. La bassa carica di frutti combinata con l’applicazione di CPPU ha determinato un aumento in %SS rispetto all’alta carica sebbene i valori siano sempre risultati inferiori ai rispettivi controlli non trattati. Il contenuto di amido nei frutti trattati è risultato inferiore rispetto al controllo mentre il contenuto in zuccheri solubili è invece risultato superiore, suggerendo una modifica nell’equilibrio con l’accumulo di amido. La bassa carica di frutti e l’applicazione di CPPU hanno accelerato il processo di maturazione, come per l’appunto indicato dalla più alta concentrazione di zuccheri solubili alla data di raccolta.