Дисертації з теми "Apple (Malus x domestica Borkh.)"

The domesticated apple (Malus × domestica) is one of the most cultivated plant over the world and is one of the most genetically polymorphic agricultural species. Studying the genetic diversity of the apple germplasm could provide important hints about the domestication process as giving a valuable resource for high reso- lution genetic mapping, QTL analysis and breeding programs. Advances in next generation sequencing technologies have driven the costs of DNA sequencing down to the point that whole genome re-sequencing (WGS) is now feasible for high di- versity, large genome species. The aim of this work is to gain information on genome-wide genetic variability patterns in apple and to identify regions of the genome that may have been selected during the process of plant domestication. SNPs were called from Illumina short reads for 63 apple cultivars represen- tative of European germplasm diversity. The identified SNPs (over 15 millions) were filtered for quality and to avoid repeated and paralogous regions. Additional filters (minor allele frequency and Hardy-Weinberg equilibrium) were applied to discard variants derived from genotyping errors resulting in a final number of 426,321 SNPs . The SNPs kept after the quality filters were used to study the population structure and the genetic diversity. A weak stratification of the ana- lyzed population emerged both from the principal component analysis (PCA) and a model based clustering approach performed using fastStructure. This analysis showed the presence of three subpopulations with a high level of admixture. FST between each couple of sub-groups was 0.055, 0.083 and 0.096 that indicate a moderate differentiation. Two different approaches were used to identify selective sweeps. The first is based on allelic frequencies and the site frequency spectrum (SFS) and it is implemented in the software SweeD. The second is based on linkage disequilibrium patterns and the omega statistic and it is implemented in the software OmegaPlus. Regions that were identified by both softwares were merged and used as candidate regions for positive selection resulting in 1,194 sweeps on the whole genome. A total of 153 gene predictions were extracted from these candidate regions and annotated using Gene Ontology terms and mapping on the KEGG pathway database. Similarity searches were also performed against plant databases to find gene orthologs and to better understand the function of candidates. The annotation revealed that genes under positive selection are involved in pathways like photosynthesis, protein ubiquitination, plant hormone signal transduction and starch and sucrose metabolism. In particular for the plant hormone signal transduction, were identified the auxin influx carrier and a SAUR family protein that lead to cell enlargement and plant growth and the ethylene insensitive protein 2 that leads to fruit ripening and senescence. The genes identified in regions under positive selection that were functionally annotated are consistent with the domestication traits for a better fruit: bigger, tastier and sweeter

The domesticated apple (Malus × domestica) is one of the most cultivated plant over the world and is one of the most genetically polymorphic agricultural species. Studying the genetic diversity of the apple germplasm could provide important hints about the domestication process as giving a valuable resource for high resolution genetic mapping, QTL analysis and breeding programs. Advances in next generation sequencing technologies have driven the costs of DNA sequencing down to the point that whole genome re-sequencing (WGS) is now feasible for high diversity, large genome species. The aim of this work is to gain information on genome-wide genetic variability patterns in apple and to identify regions of the genome that may have been selected during the process of plant domestication. SNPs were called from Illumina short reads for 63 apple cultivars representative of European germplasm diversity. The identified SNPs (over 15 millions) were filtered for quality and to avoid repeated and paralogous regions. Additional filters (minor allele frequency and Hardy-Weinberg equilibrium) were applied to discard variants derived from genotyping errors resulting in a final number of 426,321 SNPs . The SNPs kept after the quality filters were used to study the population structure and the genetic diversity. A weak stratification of the analyzed population emerged both from the principal component analysis (PCA) and a model based clustering approach performed using fastStructure. This analysis showed the presence of three subpopulations with a high level of admixture. FST between each couple of sub-groups was 0.055, 0.083 and 0.096 that indicate a moderate differentiation. Two different approaches were used to identify selective sweeps. The first is based on allelic frequencies and the site frequency spectrum (SFS) and it is implemented in the software SweeD. The second is based on linkage disequilibrium patterns and the omega statistic and it is implemented in the software OmegaPlus. Regions that were identified by both softwares were merged and used as candidate regions for positive selection resulting in 1,194 sweeps on the whole genome. A total of 153 gene predictions were extracted from these candidate regions and annotated using Gene Ontology terms and mapping on the KEGG pathway database. Similarity searches were also performed against plant databases to find gene orthologs and to better understand the function of candidates. The annotation revealed that genes under positive selection are involved in pathways like photosynthesis, protein ubiquitination, plant hormone signal transduction and starch and sucrose metabolism. In particular for the plant hormone signal transduction, were identified the auxin influx carrier and a SAUR family protein that lead to cell enlargement and plant growth and the ethylene insensitive protein 2 that leads to fruit ripening and senescence. The genes identified in regions under positive selection that were functionally annotated are consistent with the domestication traits for a better fruit: bigger, tastier and sweeter
Il melo domestico (Malus × domestica) è una delle piante più coltivate al mondo ed è tra le specie agricole geneticamente più polimorfiche. Studiare la diversità genetica in melo può dare importanti suggerimenti sul processo di domesticazione e valide risorse per creare mappe genetiche ad alta risoluzione, per analisi di QTL e nei programmi di breeding. I miglioramenti nelle tecnologie di sequenziamento del DNA, dette NGS, hanno ridotto di molto i costi del sequenziamento al punto che i risequenziamenti completi di genomi sono ora fattibili anche per specie ad alta diversità genetica e dal genoma molto grande. Lo scopo di questo lavoro è l'analisi della variabilità genetica dell’intero genoma di melo e l'identificazione di regioni genomiche sottoposte a selezione durante il processo di domesticazione. A tale scopo 63 cultivar di melo, rappresentanti l’intera diversità del germoplasma europeo, sono state sequenziate con teconolgia Illumina. Dalle sequenze sono stati predetti oltre 15 milioni di SNP che sono stati filtrati eliminare le predizioni scadenti o legate a regioni ripetute e paraloghe. Ulteriori filtri (minor allele frequency e Hardy-Weinberg equilibrium) sono stati applicati per eliminare gli SNP derivati da errori di genotipizzazione. Il numero finale degli SNP filtrati è risultato di 426'321. Gli SNP rimasti dopo i filtri di qualità sono stati usati per studiare la struttura di popolazione e la diversità genetica. Dall'analisi delle componenti principali e da un metodo di clusterizzazione implementato in fastStructure, è emersa una debole stratificazione della popolazione analizzata. Questa analisi ha mostrato la presenza di tre sottopopolazioni con un alto livello di admixture. L’FST tra ogni coppia di sottopopolazioni è risultato di 0,055, 0,083 and 0,096 indicando un livello di differenziazione moderato. Due diversi approcci sono stati usati per identificare 'selective sweep'. Il primo è basato sulle frequenze alleliche e sul 'site frequency spectrum' (SFS) ed è implementato nel software SweeD. Il secondo è basato sui pattern di 'linkage disequilibrium' e la statistica ω ed è implementato nel software OmegaPlus. Le regioni del genoma che sono state identificate da entrambi i software sono state usate come regioni candidate sotto selezione positiva. In tutto il genoma le regioni sotto selezione sono risultate 1'194. In totale 153 predizioni geniche sono state estratte dalle regioni candidate e annotate usando i termini della Gene Ontology e con i pathway metabolici descritti nel database KEGG. Ricerche di similarità in database di piante sono state fatte per trovare geni ortologhi e per capire meglio la funzione dei geni candidati. L'annotazione ha rivelato che i geni sotto selezione positiva sono coinvolti in vari processi quali la fotosintesi, l'ubiquitinazione di proteine, la trasduzione del segnale ormonale delle piante o il metobolismo di amidi e zuccheri. In particolare, per la trasduzione del segnale, sono stati identificati l'importatore dell'auxina e una proteina della famiglia SAUR che agiscono sull'aumento della dimensione cellulare e sulla crescita della pianta e la proteina 2 insensibile all'etilene che porta alla maturazione del frutto e alla senescenza. Le annotazioni funzionali disponibili ascrivono i geni identificati a ruoli fisiologici coerenti con i tratti fenotipici attesi per un processo di domesticazione. Per esempio i tratti legati al miglioramento delle caratterisitche del frutto come la dimensione, il gusto e la dolcezza

Philosophiae Doctor - PhD
Apple fruit quality is of utmost importance to apple farmers and breeders in the selection and commercialization of new cultivars. Fruit size, colour, texture, firmness and taste are all traits that affect the quality of fruit. In this study the genetic contribution of these traits, and others were evaluated in order to generate the genetic markers required for the application of marker assisted selection in fruit quality breeding. Three mapping populations, ‘Prima’ x ‘Anna’, ‘Golden Delicious’ x ‘Priscilla’ and ‘Golden Delicious’ x ‘Anna’, consisting of 87, 87 and 141 respectively, were used in the study. Fruit samples were analysed, using a range of visual, physical and sensory measurements, over a period of three years, and the data was then correlated using statistical analysis. Traits analysed included stripe-ness, fruit colour, fruit size, fruit form, ground colour, russet, texture, fruit firmness, juiciness, sugar content, acidity, taste, skin toughness, %TSS, fruit mass and diameter. ANOVA detected significant levels of variation between the three families for all traits except taste and russet; while highly significant ‘within family’ variation was also observed for all traits in pre- and post-storage analyses, except for sugar content (sweetness) and fruit form. Within family variation also contributed the largest percentage towards the variance components of all traits. Heritability estimates found stripe-ness to be the most heritable trait, from subjective analyses, while heritability values ranged from 0.41 to 0.84 for instrumentally measured traits. The genetic maps for the three populations were generated using both published microsatellites and new EST-SSR and DART markers, using JoinMap 4.0". The integrated genetic linkage maps of ‘Prima’ x ‘Anna’, ‘Golden Delicious’ x ‘Priscilla’, ‘Golden Delicious’ x ‘Anna’ consisted of 398 (133 SSR and 265 DArT), 353 (80 SSR and 273 DArT) and 213 (87 SSR and 126 DArT) markers respectively. The maps were 1021.6cM, 1079cM and 1302.7cM in length, respectively. Location of quantitative trait loci (QTL) for 14 fruit quality traits was detected using MapQTL 5.0" and a total of 79 pre-storage and 60 poststorage QTLs were identified on the three mapping populations. Comparative genome analysis and the role of various genes on the outcome of fruit quality can now be investigated. Using the integrated genetic maps, and the QTLs identified, candidate markers associated with these QTL can be used for marker-assisted selection, to increase the speed and efficiency of the apple breeding program.

Amongst the many problems facing the apple industry, apple scab is one of the most challenging experienced by producers. This disease is caused by Venturia inaequalis, which causes lesions to develop on both the fruit and leaves. The fungus is usually controlled by extensive use of sprays, but molecular genetics have made more environmentally friendly techniques available. This study was aimed at constructing a genetic linkage map from apple, which would be used in marker-assisted selection (MAS).

 

The domesticated apple (Malus x domestica Borkh.), belonging to the Malus genus of the Rosaceae family, is one of the edible pomaceous fruits. Since it is one of the important commercial fruit crops worldwide, the quality of the fruit is crucial to breeders and farmers as it ultimately determines acceptance of a cultivar for consumption. Fruit quality is also a critical determinant factor that is used to estimate the potential of apples to have a long shelf life. The introduction of marker-assisted selection (MAS) has allowed hastening of traditional breeding and selection of high-quality apple cultivars. The availability of genetic linkage maps, constructed by positioning molecular markers throughout the apple genome, enables the detection and analysis of major genes and quantitative trait loci (QTLs) contributing to the quality traits of a given genotype. 
herefore, the primary aim of this study was to construct a genetic linkage map of the &lsquo
Golden Delicious&rsquo
x &lsquo
Dietrich&rsquo
population for the identification of QTLs associated with fruit quality traits and then to examine the apple fruit pulp proteome with a specific focus on fruit firmness. In this regard, genomic DNA was extracted from leaves of the &lsquo
Golden Delicious&rsquo
x Dietrich&rsquo
population and used in megaplex PCR reactions. The PCR products were analysed prior to scoring of alleles. Polymorphic markers were then used to construct genetic linkage maps. The genetic linkage maps constructed in this study comprise of 167 simple sequence repeats (SSR) markers, 33 of these were newly developed markers. The 17 linkage groups of apple were constructed and aligned to existing apple genetic maps. The maps span 1,437.8 cM and 1,491.5 cM for &lsquo
Golden Delicious&rsquo
and &lsquo
Dietrich&rsquo
, respectively.

Fruit thinning is critical to the success of apple industry because most apple trees are prone to biennial bearing characterized by heavy bloom and over-cropping in the â onâ year and low or no cropping in the â offâ year. Fruit thinning can improve fruit size, increase return bloom, and reduce alternate bearing habit of apple trees. Chemical thinning has been widely used as a horticultural practice by growers for years in apple and other fruit production. However, its thinning results are often variable and very difficult to predict since parameters as well as their interactions (e.g. concentration, environmental condition, cultivar response) that affect thinning effectiveness have been poorly defined and the regulatory mechanisms at molecular level remain to be illustrated. Therefore, the purpose of this study is to characterize the physiological and molecular responses of the apple trees to the thinning treatments, and then use key genes as molecular markers for screening potential thinning agents. The long-term goal is to understand how the applied chemicals and environmental factors interact and regulate key regulatory genes as well as the thinning effectiveness during thinning process, and establish a predictable model for the improvement of fruit thinning consistency and effectiveness in apple and other fruit trees. Effects of naphthaleneacetic acid (NAA), shading, amimoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP) on young apple fruit abscission, leaf and fruit ethylene production, and the expression of genes related to ethylene biosynthesis, perception and cell wall degradation were examined in â Golden Deliciousâ apples (Malus x domestica borkh.). NAA at 15 mgâ ¢L-1 and shading increased fruit abscission and ethylene production of leaves and fruit when applied at a 12-mm stage of fruit development, whereas AVG, an inhibitor of ethylene biosynthesis, at 250 mgâ ¢L-1 reduced NAA-induced fruit abscission and ethylene production of leaves and fruit. 1-MCP at 160 mgâ ¢L-1 had no effect on fruit abscission but induced ethylene production by both leaves and fruit. Changes in the gene expression pattern responding to each treatment were analyzed by real-time quantitative PCR. NAA treatment was found to enhance the expression of genes related to ethylene biosynthesis (MdACS5A, MdACS5B and MdACO1) and perception (MdETR1, MdETR1b, MdETR2, MdERS1 and MdERS2). AVG reduced NAA-induced expression of these genes except for MdERS2 in the fruit abscission zone (FAZ). NAA increased the expression of a polygalacturonase gene (MdPG2) in the FAZ but not in the fruit cortex (FC), whereas AVG reduced NAA-enhanced expression of MdPG2 in the FAZ. These results suggest that ethylene biosynthesis, ethylene perception, and MdPG2 gene are involved in young fruit abscission caused by NAA. On the other hand, 1-MCP did not affect the expression of MdACS5A and MdACS5B in the FAZ, although it enhanced the expression of these two genes in the FC from 6 to 24 hours post-treatment. The expression of MdACO1 in both tissues was increased by 1-MCP after 3 or more days post-treatment. 1-MCP had only a small influence on the expression of most ethylene receptor genes, with the exception of MdETR1, which was up-regulated in the FC to a level similar to that observed for NAA treatment. In response to 1-MCP, in the FAZ, the expression of MdCel1 and MdPG2 was up-regulated at the beginning and the end, respectively, of the experiment, but otherwise remained at or below control levels. 1-MCP did not inhibit NAA-induced abscission of young apple fruit, suggesting that abscission does not solely depend on ethylene signal transduction, or that the periods of effectiveness for 1-MCP and ethylene were asynchronous in this study. Gene expression analysis also revealed that both NAA and shading enhanced the expression of gene related to ABA biosynthesis in the FAZ, which suggested the involvement of ABA in young fruit abscission. Global gene expression profile during young fruit abscission was analyzed using an apple oligonucleotide microarray. More than 700 genes were identified with reproducible changes in transcript abundance in the FAZ after NAA treatment. Genes associated with abscission, ethylene, ABA, cell wall degradation, mitochondrial activity, glycolysis, lipid catabolism, secondary metabolism, abiotic stress, and apoptosis were upregulated, while genes involved in regulation of cell cycle, cell wall biosynthesis, photosynthesis, carbon fixation, chromatin assembly, auxin transport/efflux, cytoskeleton function, and flower development were generally downregulated. Comparison of changes in the gene expression patterns during NAA-induced fruit abscission with shading-induced fruit abscission revealed similarities, but also considerable differences. The data suggested that young fruit were shutting down growth and energy production and undergoing a certain kind of programmed cell death (PCD) induced by these different stimuli. Gene expression analysis in abscising young fruit revealed that photosynthesis, carbon utilization, and ABA/ethylene pathways appeared to operate in both NAA- and shading-induced fruit abscission. However, more sugar signaling genes were regulated in shading-induced fruit abscission, compared to NAA-induced fruit abscission.
Ph. D.

Thesis (PhD)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Various experiments were undertaken over a period of five years to investigate the feasibility of initiating a large-scale programme of controlled apple breeding and selection for the improvement of climatic adaptation, using budbreak number (NB) as a practical criterion of selection. NB is preferred to time of budbreak as sole criterion on the grounds that early budbreak is associated with low NB under local conditions. Variation within and between adult and juvenile seedling families was investigated and the genetic control of the traits involved was assessed, as well as direct and correlated responses to selection. In initial experiments different rating criteria for NB as measure of chilling requirement were tested in association with vegetative and reproductive budbreak time and flowering duration, viz, a classification index based on number and distribution of budbreak (pDS grade), an index where shoot length with increased budbreak was included in the index calculation (pDS index) and bud break number expressed as number per 100 cm of shoot length (NB index). Variance analysis (ANOVA and Variance component analyses) detected significant variation within seedling families for budbreak time and NB, but estimates of genetic components of variance between families were generally low. High genetic variance among seedlings within families is most likely due to the high level of heterozygosity in the parental cultivars as is characteristic of vegetatively propagated crops. Intra-class correlation coefficients for clones within and between families indicate moderate genetic determination for NB with broad sense heritabilities around 30 percent. Realized heritabilities calculated from response to two-way truncation selection were between 40 and 60 percent. For budbreak time (reproductive and vegetative), the broad sense heritability averaged around 75 and 69 percent, respectively, indicating a high degree of genetic determination. Significant response to selection for NB of one-year-old shoots of young seedlings and from seedlings grown into adult trees showed that pre-selection for increased budbreak successfully identified seedlings genetically inclined to more and better distribution of budbreak within a set time of 21 days after initial budbreak. Correlated responses indicated additional advantages of practical and horticultural value, viz, uniformity and position of bud break, and the number and length of side shoots. In general, it is concluded from responses to two-way selection that utilizable genetic variance in NB is present within seedling families and thus that selection may successfully be applied as an early screening method for increased budbreak in adult trees. The NB index of intact one-year-old shoots under prevailing sub-optimal winter conditions is therefore proposed as criterion of selection for improvement of climatic adaptation, and combined selection utilizing genetic variation between and within crosses as the selection method.
AFRIKAANSE OPSOMMING: Verskeie proewe is oor 'n periode van vyf jaar uitgevoer om die toepaslikheid van 'n grootskaalse appelteel- en seleksieprogram vir die verbetering van klimaatsaanpasbaarheid te ondersoek met 'aantal knopbreke' (NB) as praktiese seleksiekriterium. NB word verkies bo tyd van knopbreek op grond daarvan dat vroeë knopbreek onder plaaslike toestande met lae NB gepaard gaan. Variasie binne en tussen volwasse en jong saailingfamilies en die genetiese beheer van die betrokke eienskappe is ondersoek, asook direkte en gekoreleerde seleksieresponsie. In die aanvangs-eksperimente is verskillende kriteria vir die kwantifisering van aantal knopbreke getoets as potensiële maatstawwe van die inherente kouebehoefte in appelsaailinge. Die tyd van vegetatiewe en reproduktiewe knopbreek en blomperiode is ook getoets. Die volgende indekse is gebruik: 'n klassifikasie-indeks om die aantal en verspreiding van knopbreke te beskryf (pDS graad), 'n indeks waar die lootlengte, met verhoogde aantal knopbreke, ingesluit is in die berekening van die indekswaarde (PDS indeks), en knopbreke uitgedruk as die aantal per 100 cm lootlengte (NB indeks). Variansie analise (ANOVA en variansie komponent analise) het betekenisvolle variasie binne saailingfamilies aangetoon vir tyd van, en aantal knopbreke. Ramings van genetiese komponente van variansie tussen families was relatief klein. Hoë genetiese variansie tussen saailinge binne families is waarskynlik te wyte aan die hoë vlak van heterosigositeit in die ouergenotipes, wat kenmerkend is van gewasse wat vegetatief voortgeplant word. Intraklas korrelasie koëffisiënte vir klone tussen en binne families het gedui op 'n middelmatige oorerflikheid in die breë sin (ongeveer 30 persent) vir aantal knopbreke. Verhaalde oorerflikhede wat bereken is vanaf responsie op twee-rigting atknottingsseleksie was tussen 40 en 60 persent. Vir tyd van knopbreek (vegetatief en reproduktief) was die breësin oorerflikhede ongeveer 75 en 69 persent, onderskeidelik, wat aanduidend is van 'n hoë graad van genetiese bepaling. Betekenisvolle responsie op seleksie vir NB van jong saailinge en saailingbome wat volwassenheid bereik het toon dat pre-seleksie vir knopbreke saailinge kan identifiseer wat geneties meer knopbreke en 'n beter verspreiding van knoppe binne 'n periode van 21 dae na die eerste knopbreek lewer. Gekorreleerde responsie op seleksie toon 'n addisionele voordeel van praktiese en tuinboukundige belang, naamlik, meer en langer sylote. In opsomming kan dit gestel word dat responsie op twee-rigting seleksie bruikbare genetiese variasie vir NB binne saailingfamilies ontgin het en dat seleksie vir verhoogde aantal knopbreke suksesvol toegepas kan word. Die NB indeks op een-jaar-oue hout word dus voorgestel as seleksiekriterium vir verbetering van klimaatsaanpasbaarheid onder plaaslike sub-optimale wintertoestande, en gekombineerde seleksie "combined selection" wat genetiese variasie binne en tussen kruisings benut as seleksiemetode.

The unveiling of small RNA (sRNA)-mediated gene regulatory pathways has profoundly shaped our understanding of the complexity of gene regulation. In eukaryotes, sRNAs have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. To gain insight into the roles of small RNAs in apple and peach, we conducted sRNA-seq, computational analysis and molecular experiments to genome-widely characterize their microRNAs (miRNAs) and trans-acting siRNA (tasiRNA) pathways.
We identified totally 75 miRNAs or families, including 23 conserved, 10 less-conserved and 42 apple-specific ones, and 118 miRNA target genes in apple. Two classical trans-acting siRNA (tasiRNA) pathways, miR390-TAS3 and miR828-TAS4, were characterized with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. In contrast to the location of the miR159 target site in a sequence-divergent region, the target sites of miR828 and miR858 are located in the region encoding the conserved R3 repeat domain of MYB proteins. 10 out of the 19 miR828-targeted MYBs undergo the biogenesis of various phased siRNA (phasiRNA), which potentially regulate diverse genes outside the MYB family. In peach, totally 94 miRNAs or families and 80 target genes were identified. Similar pathways of tasiRNA (miR828-TAS4 and miR390-TAS3) or phasiRNA (miR828-MYB-siRNA) processing were also characterized in peach.
Taking advantage of reverse computation and public available deep-sequencing data, we demonstrated that the miRNA-TAS-PPR-siRNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to instigate phasiRNA production from PPR �genes, which are triggered by different 22-nt miRNAs, including miR7122, miR1509, and fve-PPRtri1/2 and through distinct mechanistic strategies, like miRNA direct-targeting or indirect-targeting through TAS-like genes, one-hit or two-hit, or even two layers of tasiRNA-TAS interactions. We found that the MIRNA genes of these miRNA triggers show great identity with the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Combined results from phylogenetic analyses and conservation extent profiling revealed that the super-miR7122 was potentially evolved from another miRNA superfamily (super-miR4376), which probably originated from the miR390. Additionally, the miR482/2118-NB-LRR-siRNA pathway was found to be conserved, but evolved with distinct features, in apple and peach.
Taken together, widespread and complex miRNA and tasiRNA regulatory networks have been adapted in apple and peach. They add another crucial layer of regulation on gene activity and stability, and must exert essential functions in all aspects of plant life.

Ph. D.

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malus x domestica Borkh.), is converted to fructose by SORBITOL DEHYDROGENASE (SDH; EC 1.1.1.14) which is active in apple fruit throughout fruit development. Apple fruit set and early development is very sensitive to carbohydrate availability, but details on carbohydrate metabolism during this phase are limited. The first objective of this work was to determine if SORBITOL DEHYDROGENASE, the primary enzyme responsible for metabolism of the major phloem-transported carbohydrate sorbitol, is present and active during apple fruit set and early development. The second objective of this work was to determine if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Nine different genes encoding SDH were determined from analysis of a cDNA library and genomic-clones. Northern, Western and ELISA analyses showed that SDH transcripts and SDH protein were present in the fruit during the first 5 weeks after bloom and comprised 7 to 8 % of the total extractable protein. Whole fruit SDH activity was highest at 2 to 3 weeks after bloom in each of three cultivars, Lodi, Redchief Delicious and Fuji. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g FW, and seed SDH activity contributed significantly to whole fruit SDH activity during the first five weeks of development after bloom. Five of the nine SDH genes present in apple genome were expressed in apple fruit (SDH1, SDH2, SDH3, SDH6, SDH9). Expression of SDH6 and SDH9 was seed-specific and expression of SDH2 was cortex-specific. Using 2D SDS-PAGE and Western analyses, SDH isomers with pI values 4.2, 4.8, 5.5 and 6.3 were found in seeds, and SDH isomers with pI values 5.5, 6.3, 7.3 and 8.3 were found in cortex. The present work is the first to show that SDH is differentially expressed and highly active in seed and cortex during early development. Thus, SDH during apple fruit set and early development may play a primary role in defining fruit sink activity.

Philosophiae Doctor - PhD
Apple scab, powdery mildew and woolly apple aphid are a major concern for apple breeders and producers. Control of these diseases is a significant economic and marketing priority for the South African apple industry. Application of chemicals and orchard management practices are the main methods for controlling these diseases. These diseases require an average of 15 chemical sprays per season, which leads to increased production costs for the farmer. The increased cost of chemical based control programs and demand from consumers for ‘organic apples’ grown with very little to no chemical sprays makes it important to breed for commercial apple cultivars with endogenous disease resistance genes (R-genes). The use of genetic tools (apple genetic linkage maps and the apple genome sequence) to track and introgress endogenous R-genes in breeding and to confer durable disease resistance in commercial apple cultivars will lead to a more cost effective means of disease control for apple producers. Historically, most breeding programmes rely on recurrent conventional breeding systems. This involves the crossing of apple selections showing resistance to a given disease with a susceptible elite variety. This is followed by phenotyping the progeny to identify trees exhibiting segregating field resistance. Several crosses and backcrossing are required to produce resistant varieties and to fix the resistance trait using this breeding strategy. This breeding technique is time consuming, especially in perennial tree species such as apples, which have a long juvenile period. Molecular markers have enabled the building of genetic maps, which has allowed for tracking of the inheritance of genes contributing towards the observed resistances. This has given breeders the opportunity to start the implementation of marker-assisted-breeding (MAB) and marker-assistedselection (MAS). MAB and MAS greatly reduce the time required to select for favourable genotypes, given that MAB facilitates efficient selection for inherited traits at the seedling stage. With the publication of the apple genome sequence, the identification of the genes involved in disease resistances has been made possible and this will allow researchers to venture into cisgenics for apples, which will further reduce the time required for the introgression of desirable genes into commercial cultivars. The main thrust of this research was to generate dense genetic linkage maps for three mapping populations segregating for apple scab, woolly apple aphid and powdery mildew resistance. The three mapping populations are ‘Mildew Resistant’ x ‘Golden Delicious’, ‘Russian Seedling’ x ‘Golden Delicious’ and Malus platycarpa x ‘Mildew Resistant’ and are Malus full-sib outbreed mapping populations. The generation of the genetic maps was for use in the subsequent identification candidate disease resistance QTLs/genes that can be implemented in apple cisgenics. Integrated genetic maps using SSRs, DArTs and SNP marker data were generated for all the three crosses. The integrated map of ‘Mildew Resistance’ x ‘Golden Delicious’ consists of 1, 563 markers with a total map length of 1, 298.8 cM. The ‘Russian Seedling’ x ‘Golden Delicious’ genetic map is composed of 979 markers with a total map length of 1, 729.9 cM. The Malus platycarpa x ‘Mildew Resistant’ integrated map has 616 markers and a total map length of 1,324.3 cM. Due to the fragmentation of some of the linkage groups in the ‘Russian Seedling’ x ‘Golden Delicious’ and in the Malus platycarpa x ‘Mildew Resistant’ genetic maps, a phylogenetic analysis was performed to evaluate the genetic distances between the parents of the crosses in order to understand the cause of the fragmentation of these two integrated genetic maps. QTLs were detected through the statistical correlation of the phenotypic and map data using restricted Multiple QTL Mapping (rMQM) from MapQTL® 6.0. The genome-wide LOD score minimum QTL detection thresholds were determined using 10 000 permutations for each population. The minimum QTL detection threshold for accepting a putative QTL was then determined to be 4.5 for ‘Mildew Resistant’ x ‘Golden Delicious’ and 4.6 for both the ‘Malus platycarpa’ x ‘Mildew Resistant’ and ‘Russian Seedling’ x ‘Golden Delicious’ mapping populations. A total of 17 putative QTLs were detected for the ‘Mildew Resistant’ x ‘Golden Delicious’ population, 10 putative QTLs for the Malus platycarpa x ‘Mildew Resistant’ population and nine putative QTLs for the ‘Russian Seedling’ x ‘Golden Delicious’ population were detected for the three diseases under study. The two putative QTLs for apple scab resistance detected on LG 02 of the ‘Russian Seedling’ x ‘Golden Delicious’ map coincided with the loci previously identified as encoding two apple scab resistance genes Vh2 and Vh4 on ‘Russian apple’. The QTL for apple scab resistance identified on the proximal QTL of LG 02 co-localized with SNP marker R_8936738_Lg2 on the loci where Vh4 was previously identified. The distal QTL on LG 02 shown to encode the Vh2 R-gene was linked with the SNP marker R_32981524_Lg2. With ‘Russian apple’ being known to carry a natural pyramid of R-genes for apple scab on LG 02, therefore, the ‘Russian Seedling’ used in this study was screened by a set of 14 SSR markers to determine if it was related to ‘Russian apple. The 14 SSRs produced identical alleles to those amplified by ‘Russian apple’, which means “Russian Seedling’ and ‘Russian apple’ are closely related or identical. The LG 02 pseudo-chromosome sequence was extracted from the NCBI database housing the apple genome sequence and was then used to mine for the putative R-genes within the two QTL regions. The region corresponding to the Vh2 loci, which was roughly a 600 kb region, had two clusters of ABC (PDR) disease resistance related genes. These were predicted using a full Pfam domain search and were only detected on the negative strand. The 60 kb region corresponding to the Vh4 loci comprised a cluster of LRR domains that were also detected on the negative strand using a full Pfam domain search. This 60 kb region was further analysed using Phytozome and Genome Database for Rosaceae (GDR) leading to two candidate disease resistance genes being identified. Ten consensus gene sequences were present within the 60 kb region, with only two transcripts MDP0000657246 and MDP0000128458 identified as being disease resistance related genes. The MDP0000657246 was identified on the contig MDC000294 of the Malus x domestica reference genome as being a Leucine Rich Repeat protein kinase family, which is one of the most abundant disease resistance family mainly involved in the gene-for-gene resistance mechanism. The MDP0000128458 locus was identified on contig MDC015161 as being a Ser/Thr phosphatase 7. The Ser/Thr phosphatase genes have been associated with the regulation of MAP kinase cascades that have been shown to induce the hypersensitive response (HR) in tobacco. Therefore these two genes are likely to be the loci associated with the hypersensitive response associated with the infection of apples with race 4 of apple scab, carrying the Vh4 apple scab resistance gene. Recurrent putative QTLs were detected that still need to be validated in order to be used for MAB. The ‘Russian Seedling’ x ‘Golden Delicious’ cross produced a single powdery mildew resistance QTL located on LG08 and conferring a 1:1 resistance to susceptible phenotypic segregation ratio. These results indicate that the source of the resistance thus was a single dominant resistance gene. The ‘Mildew Resistant’ x ‘Golden Delicious’ mapping population also showed two stable QTLs one for powdery mildew on LG 03, which co-segregated with SNP GD_LG03snp00866 and in addition SNP R_13071892_Lg10 was also identified to be co-segregating with the QTL for apple scab resistance on LG10. However, none of these recurrent QTLs co-localized with known genes or QTLs. For the phylogenetic analysis, re-sequenced data using the Illumina® sequencing technologies and the apple SNP chip data for ‘Russian Seedling’, ‘Mildew Resistant’, Malus platycarpa, a Chinese accession of Malus sieversii and ‘Anna’ where used to infer relatedness of the five genotypes. The Chinese accession of Malus sieversii was included in the analysis since ‘Russian Seedling’ was thought to be relatively close genetically. Whilst ‘Anna’ is known to be a low chilling cultivar of Malus x domestica (Borkh) and therefore would add in the phylogenetic placement of ‘Mildew Resistant’ and Malus platycarpa. These were sequenced to coverage of approximately 60X for ‘Russian Seedling’ and 6X for the other four genotypes. The sequence data was aligned to the reference Malus x domestica cv Golden Delicious mitochondrial genome sequence. Phylogenetic analysis was then performed using both the data from the apple SNP-chip and the aligned mitochondrial genomes. The results from both sets of data supported the putative evolutionary distances between the five genotypes. ‘Russian Seedling’ and M. sieversii were closely related, while both were genetically divergent from the closely related ‘Anna’ and ‘Golden Delicious’ commercial cultivars. This analysis however indicated that ‘Mildew Resistant’ was relatively closely related to ‘Golden Delicious’ and hence the low number of markers showing segregation distortions for the ‘Mildew Resistant’ x ‘Golden Delicious’ population in the 17 LGs of the integrated map. However, the other two mapping population exhibited a high number of markers with segregation distortions. Markers which are closely associated with disease resistance to apple scab powdery mildew and woolly apple aphid resistance will play a major role in the identification of the genes responsible for the resistances being observed. The identification of the two candidate genes for the Vh4 gene associated with apple scab resistance will be the platform from which a cisgenic programme can be implemented in the South African apple breeding program.

Philosophiae Doctor - PhD
Apple (Malus x domestica) production in the Western Cape, South Africa, is one of the major contributors to the gross domestic product (GDP) of the region. The production of apples is affected by a number of diseases. One of the economically important diseases is apple scab that is caused by the pathogenic fungus, Venturia inaequalis. Research to introduce disease resistance ranges from traditional plant breeding through to genetic manipulation. Parallel disease management regimes are also implemented to combat the disease, however, such strategies are increasingly becoming more ineffective since some fungal strains have become resistant to fungicides. The recently sequenced apple genome has opened the door to study the plant pathogen interaction at a molecular level. This study reports on proteomic and transcriptomic analyses of apple seedlings infected with Venturia inaequalis. In the proteomic analysis, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify apple leaf proteins extracted from infected and uninfected apple seedlings. Using MelanieTM 2-DE Gel Analysis Software version 7.0 (Genebio, Geneva, Switzerland), a comparative analysis of leaf proteome expression patterns between the uninfected and infected apple leaves were conducted. The results indicated proteins with similar expression profiles as well as qualitative and quantitative differences between the two leaf proteomes. Thirty proteins from the apple leaf proteome were identified as differentially expressed. These were selected for analysis using a combination of MALDI-TOF and MALDI-TOF-TOF MS, followed by database searching. Of these spots, 28 were positively identified with known functions in photosynthesis and carbon metabolism (61%), protein destination and storage (11%), as well as those involved in redox/response to stress, followed by proteins involved in protein synthesis and disease/defence (7%), nucleotide and transport (3%). RNA-Seq was used to identify differentially expressed genes in response to the fungal infection over five time points namely Day 0, 2, 4, 8 and 12. cDNA libraries were constructed, sequenced using Illumina HiScan SQTM and MiSeqTM instruments. Nucleotide reads were analysed by aligning it to the apple genome using TopHat spliceaware aligner software, followed by analysis with limma/voom and edgeR, R statistical packages for finding differentially expressed genes. These results showed that 398 genes were differentially expressed in response to fungal infection over the five time points. These mapped to 1164 transcripts in the apple transcripts database, which were submitted to BLAST2GO. Eighty-six percent of the genes obtained a BLAST hit to which 77% of the BLAST hits were assigned GO terms. These were classed into three ontology categories i.e. biological processes, molecular function and cellular components. By focussing on the host responsive genes, modulation of genes involved in signal perception, transcription, stress/detoxification, defence related proteins, transport and secondary metabolites have been observed. A comparative analysis was performed between the Day 4 proteomic and Day 4 transcriptomic data. In the infected and uninfected apple leaf proteome of Day 4, we found 9 proteins responsive to fungal infection were up-regulated. From the transcriptome data of Day 4, 162 genes were extracted, which mapped to 395 transcripts in the apple transcripts. These were submitted to BLAST2GO for functional annotation. Proteins encoded by the up-regulated transcripts were functionally categorised. Pathways affected by the up-regulated genes are carbon metabolism, protein synthesis, defence, redox/response to stress. Up-regulated genes were involved in signal perception, transcription factors, stress/detoxification, defence related proteins, disease resistance proteins, transport and secondary metabolites. We found that the same pathways including energy, disease/defence and redox/response to stress were affected for the comparative analysis. The results of this study can be used as a starting point for targeting host responsive genes in genetic manipulation of apple cultivars.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Limited information is available on the apple preferences of the South African consumer market, which is characterised by diverse consumers from different age and ethnic groups with different food preferences. White, coloured and black consumers from different age groups were selected from the Stellenbosch area, Western Cape, South Africa. Consumer preference analysis for apple eating quality and appearance, and descriptive sensory analysis (DSA) were performed on nine commercial apple cultivars. Analysis of variance (ANOVA) conducted on mean preference scores for each age and ethnic group showed that preference generally differed between these groups. However, Ward’s statistical clustering that was applied to the same data set showed that the sociodemographic composition of consumer groups with similar apple preferences is not homogenous. Three consumer clusters were identified with similar preferences for apple eating quality (E1-3) and appearance (A1-3): E1 liked firmness and therefore tolerated sour taste and disliked mealiness. Although E1 liked sweet fruit, they indicated lower preference for sweet fruit compared to E2 and E3. E2 liked sour taste and apple flavour more compared to the other clusters while E3 disliked sour taste and had the highest preference for sweetness. Although coloured and black consumers generally disliked sour taste and E3 constituted a larger proportion of these consumers, the coloured and black consumers who liked or tolerated sour taste constituted approximately 41% of the total consumer population in the Western Cape. White and younger (<26 years) consumers were mostly in cluster E1 liking firm fruit. Peel colour preferred by the appearance preference clusters were: Green and pink bi-colour (A1), green/yellow and red-striped (A2); and red peel colour (A3). Consumers preferred the appearance of cultivars that associated with the eating quality attributes that they liked. When consumers’ preference for the eating quality of five cultivars were analysed during presentation with different levels of visual pictorial information (no, correct and incorrect photograph), mismatches between expected and actual eating quality preference resulted in lower preference scores. Apple breeding is time-consuming and expensive. Comprehensive knowledge of fruit quality parameters that drive consumer preference is required to streamline the breeding process. Eating quality and appearance attributes of four apple breeding families were subjected to instrumental and individual assessment by a trained assessor and DSA by a trained panel. Instrumental measurements could not predict the sensory attributes analysed by the individual assessor. Sensory textural attributes, apple flavour and sweet taste as quantified by DSA and instrumental measurement of titratable acidity (TA) and total soluble solids (TSS)/TA, but not TSS, could predict consumer preference. The assessor responsible for individual assessment could not predict the preference of the total consumer group. A quantitative genetic analysis of the data was carried out to quantify within- and between-family variation using ANOVA, variance components and heritability estimates. Variation between families was shown for attributes relating to colour and acidity, but not for sweet taste, TSS and apple flavour. Strong genetic control that was generally shown for colour attributes predicts a rapid selection response. Most attributes were inherited quantitatively, but TA showed complicated inheritance mechanisms.
AFRIKAANSE OPSOMMING: Min inligting is beskikbaar oor die appelvoorkeure van die diverse Suid-Afrikaanse verbruikersmark wat bestaan uit verbruikers van verskillende ouderdomme en etnisiteite met verskillende voedselvoorkeure. Wit, bruin en swart verbruikers van verskillende ouderdomsgroepe is geselekteer in Stellenbosch in die Wes-Kaap, Suid-Afrika. Verbruikersvoorkeuranalise vir die eetkwaliteit en voorkoms van appels en beskrywende sensoriese analise (BSA) is uitgevoer op nege kommersiële appelkultivars. Analise van variansie (ANOVA), uitgevoer op gemiddelde voorkeurdata per ouderdom en etniese groep, het getoon dat die voorkeure van hierdie verskillende groepe oor die algemeen verskil het. Volgens Ward statistiese groepering op dieselfde datastel was die sosiodemografiese samestelling van verbruikersgroepe met soortgelyke voorkeure egter nie homogeen nie. Drie verbruikersgroepe is geïdentifiseer met soortgelyke voorkeure vir appel eetkwaliteit (E1-3) en voorkoms (V1-3). E1 het ‘n voorkeur vir fermheid, ‘n afkeur vir melerigheid en verdra suurheid. Alhoewel E1 van soet vrugte gehou het, het hulle ‘n laer voorkeur vir soetheid as E2 en E3. E2 het ‘n voorkeur vir suurheid en appelgeur terwyl E3 ‘n afkeur vir suur smaak en die hoogste voorkeur vir soetheid getoon het. Alhoewel swart en bruin verbruikers meestal ‘n renons in suur smaak getoon het en meer van hierdie verbruikers tot E3 behoort, maak swart en bruin verbruikers wat suur smaak aanvaar ongeveer 41% van die totale verbruikersgroep in die Wes-Kaap uit. Wit en jonger (<26 jaar) verbruikers was meestal in E1 en het ‘n voorkeur vir fermheid getoon. Vrugkleurvoorkeure was vir groen en pienk (tweekleurig) (V1), groen/geel en rooi gestreep (V2) en rooi (V3). Verbruikers het ‘n voorkeur gehad vir die voorkoms van kultivars waarvan hulle die eetkwaliteit verkies het. Die effek van gevestigde kleur en smaak assosiasies is getoets deur verbruikers te versoek om die eetkwaliteit van vyf kultivars te evalueer tydens aanbieding daarvan met drie vlakke van visuele inligting, naamlik geen, korrekte en verkeerde foto. Verwarring tussen verwagte en werklike eetkwaliteit het gelei tot ‘n laer voorkeur. Teling van appels is tydrowend en duur. Uitgebreide kennis van die vrugkwaliteit parameters wat verbruikersvoorkeur dryf, is noodsaaklik vir effektiewe teling. Eienskappe wat verband hou met die eetkwaliteit en voorkoms van saailinge is in vier appelfamilies geanaliseer. Die eienskappe is geassesseer deur middel van instrumentele en individuele evaluasie deur ‘n opgeleide assessor asook deur BSA deur ‘n opgeleide paneel. Instrumentele analise kon nie die vlakke van sensoriese eienskappe voorspel soos waargeneem deur die individuele assessor nie. BSA van sensoriese tekstuureienskappe, appelgeur en soetheid, en instrumentele meting van titreerbare suur (TS) en totale oplosbare vastestowwe (TOV)/TS, maar nie TOV nie, kon verbruikersvoorkeur voorspel. Die assessor wat individuele evaluasies uitgevoer het, kon nie die voorkeur van ‘n groot verbruikersgroep akkuraat voorspel nie. Kwantitatiewe genetiese analise van die data is uitgevoer en binne- en tussen-familie variasie is gekwantifiseer deur middel van ANOVA, variansie komponente en oorerflikheidsskattings. Variasie tussen families is gevind vir kleureienskappe en suurheid, maar nie vir soet smaak, TOV en appelgeur nie. Resultate het getoon dat kleureienskappe meestal aan sterk genetiese beheer onderworpe is en dit dui op vinnige vordering met seleksie vir vrugkleur. Vrugeienskappe is meestal kwantitatief oorgeërf. Oorerwing van TS blyk ingewikkeld te wees.

To date five classes of resistance proteins have been identified in plants and these include the intracellular protein kinases, receptor-like protein kinases with extracellular leucinerich repeat (LRR) domain, LRR proteins that encode membrane bound extracellular proteins, toxin reductase and intracellular LRR proteins with a nucleotide-binding site (NBS). These proteins recognise &ldquo
invading pathogen&rdquo
and in turn trigger defence response systems that act to protect plants from invading pathogens. The NBS-LRR genes which constitutes the major class encode a family of resistance proteins that are made up of a centrally located nucleotide binding site domain and a C-terminal leucine rich repeat receptor. This class of genes constitute the largest family of resistance genes identified in plants to date. They make up the majority of proteins involved in the plant basal and inducible defence systems against pathogen infection.

Leaves and fruit of nine apple (Malus x domestica Borkh.) genotypes were evaluated for insect injury in 1998 and 1999. Foliar and fruit injury from 12 insect species was inconsistently affected by genotype. Spraying trees with oil affected neither fruit insect injury nor fruit phytotoxicity. In choice feeding assays, incidence of Japanese beetle (JB) feeding and leaf area consumed was greater for 'Liberty' than for 'York.' Genotypes did not differ in no-choice feeding assays. Choice and no-choice feeding assays between apple and oak indicated that JB could distinguish host plants in an artificial environment. Trichome density appeared different among three genotypes. 'York', the non-preferred genotype, had highest specific leaf weight and concentration of phloridzin, a feeding repellent. 'Liberty' the preferred genotype, had the lowest specific leaf weight, and had the highest concentration of quercitrin, a feeding stimulant. Olfactory stimuli of JB was evaluated with a Y-tube olfactometer. Beetles preferred the side of the Y-tube containing leaf tissue of apple or Virginia creeper over the side with no leaf. Beetles did not choose one plant species over the other. Bias test of beetle orientation in the Y-tube olfactometer indicated that in the morning, but not the afternoon, beetles preferentially moved into the left side of the Y-tube. Humidity did not affect beetle orientation. In darkness JB preferred a leaf disc over a paper disc and beetles tended to remain on the leaf.
Ph. D.

>Magister Scientiae - MSc
The Rosaceae family contains major temperate crops such as the domesticated apple(Malus x domestica Borkh.), peach (Prunus persica L. Batsch) and European pear (Pyrus communis L.). However, despite its evident economic importance, it is generally poorly studied in genomic terms, relative to the other major crop groups. Microsatellite and Diversity Array Technology (DArT) genetic markers have been exploited in this work and are essential tools in genetic map construction and marker-assisted selection (MAS) of high quality apples and other rosaceous crops. Microsatellites are advantageous in that they are co-dominant, highly polymorphic, abundant, transferable and reliably reproducible; hence their use in this study. In order for budbreak to take place in a timely and homogenous fashion, apple trees need a period of exposure to low temperatures.Within orchards the application of chemicals that induce budbreak in unsuitable environments is required to produce apples from cultivars that require high chilling levels. However, this and other practices using chemicals in orchards tend to pollute the environment. One of the solutions to this problem is to breed low chill apples such as ‘Anna’ cultivar, which was used as one of the parents in this study.This work was aimed at understanding the underlying genetic factors that determine chilling requirements for the time of initial vegetative budbreak trait in the apple cross ‘Anna’ x ‘Austin’. This was achieved through linkage map construction using SSR and DArT molecular markers followed by QTL analysis. This thesis has therefore exploited the large number of Expressed Sequence Tags (ESTs) and genome sequence data for the apple, using Tandem Repeats Finder, to design a total of 98 new SSR primers pairs. The other 369 SSR markers used in this work were from published work. JoinMap! 4.1 software was used to create an integrated genetic map with 17 linkage groups, for the domesticated apple cultivar, ‘Austin’ x ‘Anna’ mapping population with 80 individuals.The result of this process was a genetic map 1 212cM in length, and a total of 429 markers (314 DArT and 115 SSR), at an average density of a marker every 4 cM. This map was used identify the Quantitative Trait Loci (QTLs) determining chilling requirements for time of vegetative budbreak (IVB). In this process, putative IVB QTLs were identified in the ‘Anna’ x ‘Austin’ mapping population using the rMQM analysis function of MapQTL! 6.0, for both adult and seedling data collected over 3 growing seasons from 1996 to 1998. These QTLs were detected on linkage groups 2, 9 and 14,and explained 0.3 to 12.8 % of the observed phenotypic variation for the adult population,and 5.3 - 21 % for the seedling population. Seedling (LG 14) and adult (LGs 5, 7, 10) specific QTLs were also detected for the ‘Anna’ x ‘Austin’ cross. These QTLs will provide the basis for marker validation on related mapping populations in the apple breeding programme, and for the future identification of candidate genes controlling the process of budbreak.

L’objectif de cette thèse était de comprendre comment les caractéristiques structurales des pommes peuvent être liées aux facteurs structurels des purées après cuisson et fragmentation tissulaire. Les caractéristiques structurales du fruit ont été modulées par les cultivars, les pratiques culturales et la maturation, et les conditions du procédé (thermique : 50–95 °C et mécanique : 100–3000 tr/min) ont été modulées grâce à un cuiseur-broyeur. La structure de la purée (volume occupé par les particules, taille des particules, viscosité du sérum) et la texture (viscosité, seuil d’écoulement, G’ et G’’) ont ensuite été analysées et comparées entre les matières premières et les conditions du procédé. Les pectines ont été extraites et leur composition chimique ainsi que leur structure ont été corrélées à la structure de la purée. La taille des particules s´est montré être le déterminant majeur de la texture des purées en absence de dilution ou de concentration. Le degré d’adhésion cellulaire (défini par la structure et la composition des pectines) a eu un impact plus important sur la taille des particules que la taille des cellules individuelles (définie par les cultivars ou les pratiques culturales). D’autres facteurs structuraux, tels que la viscosité du sérum ou la quantité de pulpe, n’ont contribué à la texture des purées qu’à taille de particules constante. La fragmentation tissulaire, déterminant la taille des particules pendant le procédé, a été principalement affectée par l’intensité du cisaillement. Le stockage post-récolte des pommes et des températures élevées (95 °C) ont induit une dégradation et une solubilisation des pectines, en particulier par l'hydrolyse des chaînes latérales des rhamnogalacturonanes I. Cela a réduit l’adhésion cellulaire et la fragmentation tissulaire a ainsi été favorisée. Ces résultats ont permis d´approfondir la compréhension de la fragmentation tissulaire et des changements de texture au cours du procédé ce qui permettra de fournir des directives à l’industrie pour mieux gérer la diversité et l’hétérogénéité des fruits pendant le procédé de transformation des fruits en purée
The objective of this thesis was to understand how structural characteristics in raw apples can be linked to structural factors in purees after cooking and tissue fragmentation. Structural characteristics of the fruit were modulated by cultivars, agricultural practices and maturation, and process conditions (thermal: 50–95 °C and mechanical: 100–3000 rpm) were modulated in a cooker-cutter during processing. Puree’s structure (volume occupied by particles, particle size, serum viscosity) and texture (viscosity, yield stress, G’ and G’’) were then analysed and compared between raw materials and process conditions. Pectins were extracted and their chemical composition and structure were correlated to puree’s structure. Particle size appeared to be the most important determinant of puree’s texture when there is no dilution or concentration of the fruit tissue. The extent of cell adhesion (defined by pectin structure and composition) determined particle size more than individual cell size (defined by varietal effects or agricultural practices). Other structural factors only contributed to puree’s texture once particle size was constant. Tissue fragmentation, determining particle size during processing, was principally affected by shear intensity. Post-harvest maturity of the raw apples and high temperatures (95 °C) induced pectin degradation, especially rhamnogalacturonan I side chain hydrolysis, and solubilisation. This led to reduced cell adhesion and tissue fragmentation was additionally favoured. The results deepened the understanding of tissue fragmentation and textural changes during processing and provided guidelines for industry to manage diversity and heterogeneity of raw fruits during processing

Stellenbosch University. Faculty of AgriSciences. Dept. of Horticulture.
Thesis (PhD (Agric))--Stellenbosch University, 2006.
ENGLISH ABSTRACT: Market preferences for larger fruit have forced producers to adopt cultural practices that will ensure bigger fruit even if this comes at the expense of reduced total yields. In order to obtain acceptable fruit size there must be an adequate supply of photosynthetic carbon products especially during the cell division stage of fruit growth. Competition between fruits and between fruit and vegetative growth, as well as adverse climatic conditions, may limit the carbon supply to the fruits at this critical period and thus limit the final fruit size. Growers are showing renewed interest in the use of girdling or scoring in combination with the usual fruit thinning program to achieve growth control and increase fruit size. A new and milder chemical growth retardant, prohexadione-calcium (ProCa), is now also available for vegetative growth control of apple trees. [n this study, the effects of, and potential interaction between scoring, ProCa and fruit thinning were investigated, with respect to shoot and fruit growth, yield and photosynthetic capacity, in 'Royal Gala" 'Fuji' and 'Cripps' Pink' apple trees. ProCa decreased final extension shoot length in all three cultivars. Extension shoots were generally more sensitive than bourse shoots to scoring and ProCa. ProCa seems to be a more effective way of controlling shoot growth than scoring, with 'Fuji' and 'Cripps' Pink' being more sensitive than 'Royal Gala' to the application of ProCa. Scoring led to increased fruit growth rates during the first 40 days after full bloom (DAFB), and culminated in better fruit size at harvest. Scoring improved the total soluble solids concentration (TSS) of 'Fuji' and 'Cripps' Pink' compared to control and ProCa treatments. ProCa inhibited shoot growth effectively, but no evidence was found for improved carbon allocation to fruits and reproductive buds. Yield efficiencies of scored trees were significantly improved in all cultivars during the second season, due to better reproductive bud development after the first year of scoring. On 'Royal Gala' and 'Cripps' Pink', the efficiency of scoring to stimulate reproductive bud development on old and new spurs declined after 4 weeks after full bloom (WAFB). In 'Fuji', scoring later than I WAFB led to a decreasing positive response on old and new spurs. In contrast, reproductive bud development on long shoots increased with later scoring (6 and 8 W AFB) on all cultivars. It seems that the most beneficial time of scoring is 2-4 W AFB, as early as possible during the cell division stage offruit growth, but not before natural drop has occurred. The combination of early-season scoring and application of ProCa seems to hold potential for increasing carbon allocation to the fruit and improving fruit size and quality attributes. Scoring early in the season reduced photosynthetic capacity, and this reduction in carbon availability led to earlier cessation of shoot growth as well as shorter shoots. Later in the season, reduced fruit numbers led to a decrease in photosynthesis. The optimum temperature range for photosynthesis was found to adjust according to seasonal temperature variations. The harvest-induced reduction in sink strength changed stomatal sensitivity to higher temperature. Due to a reduced demand for carbohydrates by the plant, the maximum rate of photosynthesis (Amax) was reduced post-harvest. Following this reduction in sink strength and Am .. , stomata became more sensitive to high leaf temperatures, thus restricting water loss. Pre-harvest there was a strong demand for carbohydrates, therefore stomata were kept open at higher temperatures to ensure a high rate of C02 incorporation, but at a cost with regard to water use efficiency. The sharp increase in dark respiration (Rd) in leaves and fruit with an increase in temperature would mean that significant carbohydrate shortages could occur in trees during source limited periods early in the season, especially under warm weather conditions commonly experienced in the Western Cape region.
AFRIKAANSE OPSOMMING: Die effek van omgewings- en interne faktore op koolstofproduksie en -allokasie in appelbome (Malus domestlca Borkh.). Mark voorkeur vir groter vrugte forseer produsente om tegnieke te gebruik wat groter vrugte sal verseker selfs al beteken dit verlaagde totale opbrengste. Om aanvaarbare vruggrootte te bereik moet daar voldoende voorsiening van fotosinteties geproduseerde koolstofprodukte aan die vrug wees, veral gedurende die selverdelingstadium van vruggroei. Kompetisie tussen vrugte, en tussen vrugte en vegetatiewe groei sal die voorsiening van koolstof gedurende die kritieke stadium aan die vrug beperk en so vruggroote benadeel. Daar is dus hernude belangstelling in die gebruik van ringelering in kombinasie met die gewone vrug uitdunprogramme om groei te beheer en vruggrootte te verbeter. 'n Nuwe, sagter chemiese groeireguleerder, proheksadioon-kalsium (ProCa) is ook nou beskikbaar vir groeibeheer op appelbome. In hierdie studie is die effekte van, en moontlike interaksie tussen ringelering, ProCa en vruguitdunning ondersoek ten opsigte van loot- en vruggroei en opbrengs, sowel as die effekte op gaswisseling op 'Royal Gala', 'Fuji' en 'Cripps' Pink' appelbome. ProCa veroorsaak korter lote in aJ drie kultivars. Verlengingslote is meer sensitief as beurslote vir ringelering en ProCa. ProCa is effektiewer as ringelering om lootgroei te beheer, en 'Fuji' en 'Cripps' Pink' is sensitiewer as 'Royal Gala'. Ringelering lei tot verbeterde vruggroeitempos gedurende die eerste 40 dae na volblom en verbeterde vruggrootte by oes. Ringelering verbeter die totale oplosbare vastestotkonsentrasie (TOVS) van 'Fuji' en 'Cripps' Pink' in vergelyking met kontrole- en ProCa-behandelings, onderskeideHk. ProCa inhibeer lootgroei effektief, maar geen bewyse van verbeterde koolstomllokasie na vrugte en reproduktiewe knoppe is gevind nie. Opbrengseffektiwiteit van geringeleerde borne verhoog gedurende die tweede seisoen van ringelering weens beter reproduktiewe knopontwikkeling. Ringelering later as 4 weke na volblom (WNVB) is minder effektief om reproduktiewe knopontwikkeling op ou en nuwe spore in 'Royal Gala' en 'Cripps' Pink' te stimuleer. Die positiewe effek van ringelering op reproduktiewe knoppe op 'Fuji' verminder sodra dit later as I WNVB gedoen word. Op langlote het 'n laat ringelering (6 en 8 WNVB) die beste effek op reproduktiewe knopontwikkeling. Die mees voordelige tyd om te ringeIeer was 2-4 WNVB, so vroeg as moontlik gedurende die selverdelingstadium van vruggroei, maar nie voor natuurlike vrugval voltooi is nie. Die kombinasie van vroei! ringelering en die toediening van ProCa het potensiaal om koolstofallokasie na die vrug te verbeter, met die gepaardgaande verbetering in vruggrootte en -kwaliteit. RingeJering vroeg in die seisoen inhibeer fotosintese en hierdie verlaging in koolstof beskikbaarheid het tot gevolg dat lootgroei vroei!r gestaak word. Later in die seisoen veroorsaak verlaagde vruggetalle 'n afname in fotosintese. Die optimum temperatuur vir fotosintese verander na gelang van heersende lugtemperature. Die oes-geinduseerde verlaging in sinksterkte verander stomatale sensitiwiteit vir hoo temperature. Weens 'n verJaagde aanvraag vir koolhidrate deur die plant word fotosintese verlaag na-oes. Weens die verlaging is stomata sensitiewer vir hoer temperature en beperk dus waterverlies. V oor oes is daar 'n hoo aanvraag na koolhidrate dus word stomata oop gehou selfs by hoe temperature ten koste van watergebruiksdoeltreffendheid. Die skerp styging in donker respirasie in blare en vrugte met 'n verhoging in temperature sal beteken dat koolhidraat-tekorte kan ontstaan in bome gedurende die bron-beperkte periodes vroeg in die seisoen, veral onder warm toestande 500S wat algemeen ervaar word in die Wes Kaap.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Esse estudo teve como objetivos estudar os aspectos da biologia floral e possÃveis fatores limitantes à polinizaÃÃo e investigar a utilizaÃÃo da abelha Apis mellifera e o papel de outros insetos visitantes na polinizaÃÃo de macieira (Malus domestica Borkh.) em cultivos na regiÃo do semiÃrido nordestino brasileiro. O estudo foi realizado em uma Ãrea experimental que possui 0,5 hectare da fazenda FrutaCorÂ, municÃpio de Russas, CearÃ. Foram estudadas a variedade Julieta, utilizada como doadora de pÃlen e Princesa como receptora. A quebra da dormÃncia do florescimento ocorreu de forma quÃmica e fÃsica para as duas variedades. O estudo foi desenvolvido durante 39 dias, entre o final de outubro e inicio de dezembro de 2013. Flores da var. Julieta duraram menos dias do que as da Princesa (VarJulieta = 2,04  0,197; VarPrincesa 2,93  0,274; Mann-Whitney U = 150, p < 0,0001). A liberaÃÃo do pÃlen na var. Julieta ocorreu a partir das 09:00h do primeiro dia, com pico Ãs 13:00h do segundo dia. A receptividade estigmÃtica na var. Princesa iniciou durante o estÃdio âbalÃoâ mantendo-se atà senescÃncia. Houve uma perda mÃdia de 19,87%  15,79 no nÃmero de anteras da var. Julieta que efetivamente liberaram pÃlen em relaÃÃo ao total produzido por flor. A temperatura mÃdia de 30,21ÂC  4,18, pode ter sido responsÃvel pela reduÃÃo na longevidade, perda de anteras e perda de pÃlen, sendo, portanto, recomendado o aumento no nÃmero de plantas doadora de pÃlen nos cultivos em ambientes semiÃridos. Os visitantes florais mais abundantes foram os insetos e dentre esses, as abelhas representando cerca de 61,86% do total. As flores da macieira apresentaram pico de visitaÃÃo no perÃodo da manhÃ, seguindo atà as 12:00h (81,57%). Por se tratar de uma cultura dependente de polinizaÃÃo cruzada para o vingamento do fruto, foi observada a atratividade das flores para as abelhas em diferentes idades, obtendo 18,66  4,93; 41,3  7,63 e 4,6  1,52, em flores de 1Â, 2 e 3 dia, respectivamente, mostrando que as flores de 2 dia sÃo mais atrativas para as abelhas (p<0,05). Foi observado que apenas uma visita de Apis mellifera foi suficiente para promover o vingamento de frutos, independentemente da idade da flor. A introduÃÃo de colÃnias dessa abelha no pomar se faz necessÃrio, pois A. mellifera foi a espÃcies mais frequentemente observada nas flores da macieira.
This study aimed to investigated aspects of floral biology and possible limiting factors to pollination, investigate the use of bees, Apis mellifera, and the role of other insects visitors in apple pollination (Malus domestica Borkh.) in crops in the Brazilian northeast semiarid region. The study was conducted in an experimental area which has 0.5 hectare on FrutaCor farm, Russas - CearÃ. We used Two appleâs varieties in this study. The Julieta variety was used as pollen donor and the Princessa variety was used as receiving pollen. Theses varieties need chemical and physical treatment to flower break dormancy occurred. The study was conducted for 39 days at the end of October and early December 2013. Julieta Flowers lasted for fewer days than the princessa (VarJulieta = 2.04  0.197, 2.93  0.274 VarPrincesa; Mann-Whitney U = 150, p <0.0001). The pollen release of Julieta started at 09: 00h on the first day and had peaking at 13: 00h in the second day. The stigmatic receptivity of Princessa variety started during the stage "balloon" remaining until senescence. There was an average loss of Julietâ anthers number of 19.87  15.79% that effectively released pollen in relation to the total production per flower. The average of temperature 30,21ÂC  4.18, may have been responsible for the reduction in longevity, loss of anthers and pollen loss, and is therefore recommended to increase the number of pollen donor plants in crops in semi-arid environments. The most abundant flower visitors were insects and among these, the bees representing approximately 61.86% of the total. Apple tree flowers had visitation peak in the morning, 5:00 to 12: 00h (81.57%). Therefore this culture is a cross-pollination-dependent to ripening of fruit, we observed the different ages flowers attractiveness by bees and we obtained 18.66  4.93; 41.3  7.63 and 4.6  1.52, on 1 flowers, 2 and 3 days, respectively, showing that the 2nd day flowers are more attractive to bees (p <0.05). We observed that only a visit of Apis mellifera was enough to promote fruit set, regardless of the age of the flower. The introduction of this bee colonies in the orchard is necessary because A. mellifera was the most frequently observed species in apple tree flowers.

Hábitos de vida saudáveis e uma dieta balanceada aliados ao alto consumo de frutas e vegetais, estão associados à prevenção de doenças e à manutenção da saúde. A maçã possui em sua composição compostos bioativos que podem agir na prevenção e no controle das dislipidemias. Tendo em vista a preocupação da Saúde Pública em encontrar uma fonte alternativa na redução das doenças cardiovasculares, esse trabalho teve como objetivo analisar a composição química da maçã, variedade Gala, e estudar os efeitos do seu consumo no ganho de peso, consumo alimentar, níveis sanguíneos de colesterol total, HDL-C, LDL-C, triglicerídeos, colesterol hepático e colesterol excretado em ratos Wistar hipercolesterolêmicos. Foram utilizados 6 animais em cada grupo de tratamento (controle, 5%, 15% e 25% de maçã na dieta) durante 30 e 60 dias. Foi observado neste estudo, quanto a composição centesimal da maçã, que uma maçã (200g), é capaz de fornecer 14,5% das recomendações de fibras totais e 55% de vitamina C, além de quantidades consideráveis de compostos fenólicos (0,38g/100g em base fresca) e tanino (0,16g/100g em base fresca). No ensaio biológico, os animais de ambos os tempos, apresentaram uma redução não significativa no ganho de peso e no consumo de dieta com o aumento das concentrações de maçã. Ao final de 30 dias, todas as dietas proporcionaram uma redução significativa (p ≤ 0,05) nos níveis de triglicerídeos comparativamente ao grupo controle e não significativa com relação aos níveis de HDL-C. As dietas com 15% e 25% de maçã mostraram reduções significativas nos níveis sanguíneos de colesterol total e LDL-C e um aumento no teor de colesterol excretado em relação ao grupo controle. A dieta com 25% de maçã promoveu uma redução significativa nos níveis de colesterol hepático em comparação ao grupo controle. Aos 60 dias, os níveis sanguíneos de colesterol total, LDL-C, HDL-C e triglicerídeos de ratos alimentados com as dietas 5%, 15% e 25% de maçã foram semelhantes ao grupo controle.A partir desses resultados, pode-se concluir que a maçã Gala é um potente alimento no controle dirigido das dislipidemias em ratos. Uma dieta rica em verduras, legumes e frutas, inclusive a maçã, associado a um estilo de vida saudável, ao longo do tempo, podem ser considerados fundamentais na prevenção e redução do risco de doenças, principalmente as cardiovasculares.
Healthy life habits and an equilibrate diet, associated with a high fruit and vegetable intake, are joined with the prevention of diseases and health maintenance. The apple has in its composition bioactives compounds that can help in the prevention and control of hyperlipidemia. With the worry of Public Health to find an alternative source in the reduction of the cardiovasculars diseases, the objective of this work was to analyse the chemical composition of the Gala apple and to study the effects of its consumption in the gain of weigh, food intake, seric levels of total cholesterol, HDL-C, LDL-C, triglycerides, hepatic cholesterol and feces cholesterol in hypercholesterolemics Wistar rats. Six animals were distributed in each treatments (control, 5%, 15% and 25% apple diet) during 30 and 60 days. This study showed that one apple (200g) can provide 14,5% of the total fiber and 55% of the vitamin C within the recommendation, besides considerable quantities of phenolic compounds (0,38 g/100g wet weigh) and tannins (0,16 g/100g wet weigh). In the biological assay all the animals showed a non significative reduction in the gain of weigh and food intake with the increase of apple concentrations in the diets. At the end of 30 days, all the diets provided a significative reduction in the levels of tryglicerides comparable to the control group and non significative in relation to HDL-C levels. The 15% and 25% apple diets showed significatives reductions in the seric levels of total cholesterol and LDL-C and an increase in the level of feces cholesterol in relation to the control group. The 25% apple diet provided a significative reduction in the hepatic cholesterol levels comparable to the control group. After 60 days, the seric levels of total cholesterol, LDL-C, HDL-C and tryglicerides in rats fed with 5%, 15% and 25% apple diets were alike to the control group. These results confirm the importance of the Gala apple in the rats hyperlipidemia control. A diet rich in vegetables and fruits, including apple, associated with a healthy life habits, along the time, can be considered in the prevention and reduction against the risk of disease, mainly, the cardiovascular ones.

MONTEIRO, Victor Magalhães. Abelhas visitantes florais e potenciais polinzadores da macieira (Malus domestica Borkh.) no semiárido brasileiro. 2014. 75 f. : Dissertação (Mestrado) - Universidade Federal do Ceará, Centro de Ciências, Departamento de Zootecnia, Programa de Pós-Graduação em Zootecnia. Fortaleza-CE, 2014.
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This study aimed to investigated aspects of floral biology and possible limiting factors to pollination, investigate the use of bees, Apis mellifera, and the role of other insects visitors in apple pollination (Malus domestica Borkh.) in crops in the Brazilian northeast semiarid region. The study was conducted in an experimental area which has 0.5 hectare on FrutaCor® farm, Russas - Ceará. We used Two apple’s varieties in this study. The Julieta variety was used as pollen donor and the Princessa variety was used as receiving pollen. Theses varieties need chemical and physical treatment to flower break dormancy occurred. The study was conducted for 39 days at the end of October and early December 2013. Julieta Flowers lasted for fewer days than the princessa (VarJulieta = 2.04 ± 0.197, 2.93 ± 0.274 VarPrincesa; Mann-Whitney U = 150, p <0.0001). The pollen release of Julieta started at 09: 00h on the first day and had peaking at 13: 00h in the second day. The stigmatic receptivity of Princessa variety started during the stage "balloon" remaining until senescence. There was an average loss of Juliet’ anthers number of 19.87 ± 15.79% that effectively released pollen in relation to the total production per flower. The average of temperature 30,21ºC ± 4.18, may have been responsible for the reduction in longevity, loss of anthers and pollen loss, and is therefore recommended to increase the number of pollen donor plants in crops in semi-arid environments. The most abundant flower visitors were insects and among these, the bees representing approximately 61.86% of the total. Apple tree flowers had visitation peak in the morning, 5:00 to 12: 00h (81.57%). Therefore this culture is a cross-pollination-dependent to ripening of fruit, we observed the different ages flowers attractiveness by bees and we obtained 18.66 ± 4.93; 41.3 ± 7.63 and 4.6 ± 1.52, on 1 flowers, 2 and 3 days, respectively, showing that the 2nd day flowers are more attractive to bees (p <0.05). We observed that only a visit of Apis mellifera was enough to promote fruit set, regardless of the age of the flower. The introduction of this bee colonies in the orchard is necessary because A. mellifera was the most frequently observed species in apple tree flowers.
Esse estudo teve como objetivos estudar os aspectos da biologia floral e possíveis fatores limitantes à polinização e investigar a utilização da abelha Apis mellifera e o papel de outros insetos visitantes na polinização de macieira (Malus domestica Borkh.) em cultivos na região do semiárido nordestino brasileiro. O estudo foi realizado em uma área experimental que possui 0,5 hectare da fazenda FrutaCor®, município de Russas, Ceará. Foram estudadas a variedade Julieta, utilizada como doadora de pólen e Princesa como receptora. A quebra da dormência do florescimento ocorreu de forma química e física para as duas variedades. O estudo foi desenvolvido durante 39 dias, entre o final de outubro e inicio de dezembro de 2013. Flores da var. Julieta duraram menos dias do que as da Princesa (VarJulieta = 2,04 ± 0,197; VarPrincesa 2,93 ± 0,274; Mann-Whitney U = 150, p < 0,0001). A liberação do pólen na var. Julieta ocorreu a partir das 09:00h do primeiro dia, com pico às 13:00h do segundo dia. A receptividade estigmática na var. Princesa iniciou durante o estádio “balão” mantendo-se até senescência. Houve uma perda média de 19,87% ± 15,79 no número de anteras da var. Julieta que efetivamente liberaram pólen em relação ao total produzido por flor. A temperatura média de 30,21ºC ± 4,18, pode ter sido responsável pela redução na longevidade, perda de anteras e perda de pólen, sendo, portanto, recomendado o aumento no número de plantas doadora de pólen nos cultivos em ambientes semiáridos. Os visitantes florais mais abundantes foram os insetos e dentre esses, as abelhas representando cerca de 61,86% do total. As flores da macieira apresentaram pico de visitação no período da manhã, seguindo até as 12:00h (81,57%). Por se tratar de uma cultura dependente de polinização cruzada para o vingamento do fruto, foi observada a atratividade das flores para as abelhas em diferentes idades, obtendo 18,66 ± 4,93; 41,3 ± 7,63 e 4,6 ± 1,52, em flores de 1º, 2º e 3º dia, respectivamente, mostrando que as flores de 2º dia são mais atrativas para as abelhas (p<0,05). Foi observado que apenas uma visita de Apis mellifera foi suficiente para promover o vingamento de frutos, independentemente da idade da flor. A introdução de colônias dessa abelha no pomar se faz necessário, pois A. mellifera foi a espécies mais frequentemente observada nas flores da macieira.

The purpose of this study was to determine if different clonal rootstock from apple (Malus domestica Borkh.) formed different vesicular arbuscular mycorrhizal (VAM) associations. Different fertilizers and VAM fungi were tested to determine their effects on apple seedling growth in apple replant diseased (ARD) soils. VAM associations in apple stoolbed nursery were low. Over 80% of all samples had less than 10% mycorrhizal colonization. This reduction in VAM colonization amongst various rootstock clones is a result of detrimental management practices in the stoolbed nursery. Apples grown in a budded nursery had high mycorrhizal colonization, the lowest colonization rate was 30%. Different rootstocks from the budded nursery do not show any significant differences in VAM colonization, whereas clonal rootstocks from the stoolbed nursery do. From the stoolbed nursery, Mailing (M) 2 consistantly showed higher VAM colonization rates, compared to M 4, M7, M9, M 26, Mailing Merton (MM) 106, MM 111, Alnarp 2 and Ottawa 3. Apple replant disease (ARD) is identified as the reason for poor growth of apple seddlings in 5 soils from the Okanagan Valley of British Columbia. Sterilization by autoclaving, pasteurization and formalin increased test seedling height. Air-drying test soil does not affect ARD pot bioassays. However, air-drying the soil and pasteurizing or adding formalin increased plant height significantly more than these treatments in nonair-dried soils. The fertilizer monoammonium phosphate (11-55-0) increased plant height more than ammonium nitrate (34-0-0) while triple superphosphate (0-45-0) did not increase plant height. Root growth was increased by 0-45-0 only. VAM fungi were drastically reduced or eliminated by sterilization and 11-55-0, but not by the other fertilizers. VAM fungi in 2 ARD soils do not overcome ARD. Test seedlings grown in sterilized ARD soils inoculated with 4 species of VAM fungi do not show as great an increase in shoot height compared to the addition of 11-55-0 fertilizer. Root growth shows the inverse response. Glomus intraradices Schenck and Smith, was the best colonizer but inoculation with G. versiforme (Karsten) Berch resulted in the greatest shoot and root growth. Glomus clarum Nicholson and Schenck, and G. monosporum Gerdemann and Trappe, did not result in increases in plant growth in ARD soils. In sterilized ARD soils, VAM fungi do not increase shoot growth as expected, but do increase root growth, suggesting the initial growth of inoculated apple seedlings is root mass. Seedlings given 11-55-0 fertilizers show the reverse pattern of growth. In nonsterilized ARD soils, the growth of seedlings appears to be inversely proportional to VAM colonization.
Land and Food Systems, Faculty of
Graduate

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado que possui grande importância econômica mundialmente, sendo sua produtividade intimamente relacionada à saída do processo de dormência hibernal. Este processo pode ser definido como a incapacidade da planta iniciar o crescimento meristemático mesmo sob condições favoráveis e os mecanismos de controle molecular da dormência em macieiras ainda são pouco compreendidos. O objetivo do presente trabalho foi investigar o perfil gênico diferencial entre cultivares de macieiras contrastantes para requerimento de frio. As cultivares selecionadas foram Gala e sua mutante espontânea Castel Gala, as quais apresentam alto e baixo requerimento de frio, respectivamente. A técnica de hibridização supressiva subtrativa (SSH) permitiu a identificação de 28 genes candidatos à regulação da dormência. Análises de RT-qPCR foram realizadas visando a validação da expressão diferencial dos genes selecionados, assim como caracterizá-los transcricionalmente em três cultivares distintas durante um ciclo de crescimento e de dormência. Dos 28 genes candidatos, 17 apresentaram o mesmo perfil diferencial identificado por SSH. Um acúmulo sazonal de transcritos durante o inverno foi identificado para alguns genes e as cultivares de maior requerimento de frio apresentaram acúmulo de transcritos por mais tempo. Este perfil permitiu-nos sugerir que estes genes podem estar atuando na regulação dos processos de dormência e de aclimatação ao frio. Dos 17 genes validados, aqueles codificadores de proteínas DAM, desidrinas, GAST1, LTI65, NAC, histonas variantes H2A.Z e RAP2.12 apresentaram os maiores contrastes transcricionais entre as cultivares analisadas durante o inverno e constituem-se como fortes candidatos a participantes do processo de progressão da dormência em macieiras. Finalmente, a família de genes codificadores de desidrinas de macieira teve seus membros identificados e caracterizados transcricionalmente. Análises in silico permitiram a identificação de oito modelos gênicos preditos de desidrinas no genoma de macieira. As cadeias peptídicas deduzidas foram classificadas conforme a presença dos segmentos conservados YnSKn. Um perfil sazonal de regulação da expressão foi identificado, com a presença de um pico de acúmulo de transcritos durante o inverno, o que sugere a presença de um mecanismo similar de regulação entre genes de desidrinas de macieira.
Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related with the release from a bud dormancy process. This process is defined as the plant inability to initiate growth from meristems under favorable conditions and molecular information about dormancy control in apple trees is limited. The aim of the present work was to investigate the differential gene expression profiles between apple tree cultivars contrasting in chilling requirement for breaking dormancy. The selected apple cultivars were Gala and its derived bud sport Castel Gala, which displays high and low chilling requirement, respectively. A suppression subtractive hybridization (SSH) assay yielded 28 candidate genes putatively associated to dormancy cycling. RT-qPCR analyses were performed in order to validate the differential expression profiles and also to transcriptionally characterize the selected genes in three distinct apple tree cultivars during a growth to dormancy cycle. Among the 28 candidate genes, 17 confirmed the differential expression profile predicted by SSH. A seasonal transcript accumulation during the winter was identified to some genes, with high chilling requirement cultivars presenting higher levels of transcripts. This profile allowed us to suggest that these genes may be acting on dormancy regulation and cold acclimation. Out of the 17 candidate genes, those coding for DAM, dehydrins, GAST1, LTI65, NAC, histone variants H2A.Z and RAP2.12 displayed major differences in gene expression between cultivars through the winter and are strong candidates to play key roles on dormancy progression in apple trees. Finally, we identified and transcriptionally characterized the dehydrin gene family in apple trees. In silico analyses allowed us to identify eight predicted gene models for dehydrins in the apple genome. Deduced polypeptides were classified according to the presence of the conserved YnSKn segments. A seasonal regulation of gene expression was observed, with higher transcript accumulation during the winter. This data suggests that a similar mechanism of transcript regulation is acting through the apple dehydrin genes.

A macieira (Malus x domestica Borkh.), uma das frutíferas mais importantes das regiões de clima temperado, é caracterizada pela cessação de crescimento visível durante o inverno, processo este chamado de dormência. A dormência de gemas permite que a planta sobreviva às baixas temperaturas e é determinante para a eficiência na produção de maçãs. Entender o processo de dormência, assim como seus mecanismos de controle, tornou-se fundamental para contornar as perdas na produção, seja por meio de técnicas de manejo ou pela geração de variedades comerciais melhor adaptadas às regiões de cultivo. A enzima galactinol sintase (GolS) catalisa a primeira etapa da via de síntese de oligossacarídeos da família da rafinose, cujo acúmulo em resposta a estresses abióticos é fato bem conhecido. Em trabalho anterior, nosso grupo mostrou um acúmulo de transcritos constrastante de quatro genes MdGolS durante o inverno, sugerindo-se uma possível função adaptativa desses genes durante a dormência de gemas em macieira. Pelo presente trabalho, tem-se como objetivo quantificar o acúmulo de quatro proteínas MdGolS em gemas apicais da macieira Fuji Standard visando confirmar os perfis transcricionais previamente obtidos. Anticorpos policlonais capazes de reconhecer as quatro MdGolS foram separadamente produzidos para emprego na técnica de Western blot Análise por Dot blot permitiu-nos mostrar que os quatro conjuntos de anticorpos são específicos para o seu respectivo peptídeo, sem reações cruzadas. Cinco protocolos de extração de proteínas foram testados e otimizados para a obtenção de maiores quantidades de proteínas de gemas fechadas de macieira. Dois desses extratos foram escolhidos para realizar a técnica de Western blot. Proteínas totais foram quantificadas pelo método de Bradford e 20 μg foi considerada a quantidade mais adequada para os ensaios. O anticorpo MdGolS1 permitiu a detecção de sinais em ambos os extratos, mas nenhum no tamanho esperado para a respectiva proteína, isto é, com aproximadamente 38 kDa. Os anticorpos MdGolS2, MdGolS3 e MdGolS4 não permitiram detectar quaisquer sinais ou, ainda, sinais fracos que não puderam ser relacionados com o perfil transcricional previamente obtido. Estratégias alternativas, em nível proteico, para validar os níveis aumentados de transcritos de GolS anteriormente observados são discutidas.
Apple tree (Malus x domestica Borkh), one of the world’s most important fruit crops in temperate regions, is characterized by the cessation of visible growth during winter, a process called dormancy. Bud dormancy allows plant survival under low temperatures and it is crucial for the efficiency of apple production. The knowledge about the dormancy process, as well as its control mechanisms, became an essential approach to circumvent production losses either through management techniques or by the generation of new commercial varieties better adapted to each regional cultivation environment. The galactinol synthase (GolS) enzyme catalyzes the first step in the synthesis pathway of the raffinose family of oligosaccharides, whose accumulation in response to abiotic stress is well known. A previous work of our group allowed us to show contrasting transcript levels of four MdGolS during winter, suggesting a possible adaptative role for some of these genes during bud dormancy. The present work aims to quantify MdGolS protein accumulation in apical buds of the Fuji Standard apple tree cultivar in order to verify and associate with transcriptional profiles previously obtained. Polyclonal antibodies that recognize the four MdGolS produced to perform Western blot assays. Dot blot analysis revealed that the four sets of antibodies were specific to their respective peptides, without cross-reactions. Five protein extraction protocols were tested and optimized for obtaining greater amounts of proteins from apple apical buds. Two of these extracts were chosen to perform Western blot assays. Total protein was quantified using the Bradford method and 20 μg was found to be the most adequate for the assay. MdGolS1 antibody allowed the detection of signals in both extracts, but none in the expected protein mass, i.e., approximately 38 kDa. MdGolS2, MdGolS3 and MdGolS4 antibodies allowed us to detect only weak or no signals, that could not be correlated to the transcript profile previously obtained. Alternative strategies to validate the previously observed increased GolS transcript levels at the protein level are discussed.

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Abscission is a natural self-regulatory mechanism whereby fruit trees shed part of the fruitlets, and is an important agricultural event from the farmer’s point of view because it directly affects the final size and quality of the commodity. In spite of this self-regulatory mechanism, fruit trees set too many fruitlets negatively affecting not only the final quality, but also the returning bloom. To avoid these effects, farmers perform blossom or fruitlet thinning to adjust crop load and ensure a satisfactory fruit quality at harvest for commercial purposes. Chemical thinning is nowadays a common orchard practice in some apple cultivars but its effects on fruit trees depend on environmental factors and genotypes. A widely employed chemical thinner, the carbaryl (Sevin), has been withdrawn from the market and replaced with benzyladenine (BA), a cytokinin that has a milder effect both on the tree and human health. At molecular level, abscission is a coordinated event under the control of auxin and ethylene. Both hormones play their role at the abscission zone (AZ) level. Auxin flux through the AZ target cell layers inhibits the onset of shedding whereas ethylene causes the up-regulation of degrading cell wall enzymes causing the cell separation and the fruitlet shedding. Nevertheless, no information is available about the signalling that causes the decreasing auxin flow and the gain in ethylene sensitivity in AZ. In Arabidopsis thaliana, the isolation of mutants with defects in floral organ abscission allowed the identification and characterization of genes involved in AZ differentiation and/or the signalling pathways. A model has been proposed by characterizing genes encoding receptors and ligands involved in the transfer of the shedding signal. In apple, a model to explain the fruitlet physiological drop is still missing as well as the isolation and characterization of genes involved in abscission. Although most of the researchers focused their attention mainly on the effect of chemicals on fruit quality and returning bloom, few of them considered also the dynamics of the transcriptomic changes occurring as consequence of the abscission induction. A working hypothesis taking into account the carbohydrate status within the fruitlet population and between fruitlets and shoots has been proposed. The recent development of massive gene approaches, based on microarray technologies, allowed to deeply investigate many fundamental biological process from a transcriptomic point of view. A recent work carried out in tomato investigated the genes involved in auxin homeostasis, as a consequence of IAA depletion during flower abscission induction. The model proposed represents a powerful platform for further analyzing the putative regulatory abscission-related genes involved in the gaining of ethylene sensitivity of the AZ. In the present dissertation, two different approaches have been adopted to investigate the early phases of apple fruitlet abscission: 1) A transcriptomic approach to isolate genes involved in the early steps of abscission, and 2) the characterization of volatile organic compounds (VOCs) emitted during abscission induction. 1) Apple fruitlet abscission was induced using BA as thinner. Fruitlets differing in size and position within the cluster were collected over a 4-day time-course, after BA treatment, and their gene expression profiles were analyzed, separately in cortex and seeds, by means of a newly released 30K oligonucleotide microarray. The analysis of the transcriptomic profiles of abscising and non-abscising fruitlets was tested for statistical association with abscission potential to identify molecular signatures strictly related to fruit destiny. Reactive oxygen species (ROS) and carbohydrates (glucose, fructose, sucrose, sorbitol, and starch) were also measured. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via ROS/sugar and hormones signalling crosstalks, and followed by a block of embryogenesis and the consequent activation of the abscission zone. 2) Fruitlets of two cultivars (Golden Delicious and Red Chief) with different abscission potentials treated with two different thinning agents (BA and metamitron) were analyzed by means of proton transfer reaction mass spectrometer (PTR-MS) over a 10-day time-course looking for volatile organic compounds (VOCs) differentially emitted. Results pointed out that isoprene is more abundantly emitted by the abscising fruitlets in both cultivars. A relationship between isoprene emission and ABA content in the fruit cortex was also pointed out, along with a specific activation of the corresponding biosynthetic genes. A delayed transcriptional activation of a biosynthetic gene involved in a key step of methyl-erythritol phosphate (MEP) pathway, supplying the precursors for both isoprene and ABA biosynthesis, was also shown. According to the main findings, a role for isoprene as a ROS-detoxifying mechanism mediated and transcriptionally controlled by ABA may be hypothesized. Future perspectives will be focused on all the research lines herein adopted. The biological function of the genes identified by means of the transcriptomic approach will be studied at the cellular level, with in situ hybridization, and their expression pattern further validated. Attention will be focused in particular on transcription factors and other regulatory elements involved in hormonal cross-talk. The abscission-related VOCs (i.e. isoprene) will be validated to assess the possible applications in fruit load prediction systems, in order to fine-tune the use of thinning chemicals according to a more-environment-friendly agriculture.
L'abscissione è un meccanismo auto-regolativo per cui gli alberi da frutto rilasciano naturalmente parte dei frutticini ed è un evento agronomico importante dal punto di vista del produttore perché incide sulla qualità del raccolto a maturazione. Nonostante questo meccanismo auto-regolativo, gli alberi da frutto trattengono troppi frutticini, influendo negativamente non solo sulla dimensione e la qualità finale dei frutti, ma anche sulla fioritura nell’anno successivo. Per evitare questi effetti negativi, i coltivatori utilizzano comunemente diradanti chimici che agiscono su fiori o frutticini, allo scopo di regolare il carico iniziale ed ottenere così una qualità della frutta corrispondente alle esigenze di mercato. In melo il diradamento chimico è una pratica comune la cui efficacia sugli alberi da frutto è dipendente, purtroppo, da fattori ambientali e dai diversi genotipi. Un diradante chimico ampiamente utilizzato, il carbaryl (Sevin), è stato ritirato dal mercato e parzialmente sostituito con la benziladenina (BA), una citochinina che ha un effetto più contenuto sull'albero e sulla salute umana. A livello molecolare, l'abscissione è un processo coordinato principalmente dall'auxina e dall'etilene. Entrambi questi ormoni svolgono il loro ruolo a livello della zona di abscissione (AZ). Il flusso continuo dell'auxina attraverso la AZ inibisce il processo di abscissione, mentre l'etilene induce una regolazione positiva degli enzimi degradanti la parete cellulare provocando la separazione delle cellule della AZ e la caduta dei frutticini. Le informazioni riguardanti il segnale che causa la diminuzione del flusso di auxina e l'aumento nella sensibilità all'etilene nella AZ sono tuttavia ancora parziali e piuttosto carenti. In Arabidopsis thaliana l'isolamento di mutanti con difetti nel processo di abscissione dei fiori ha permesso l'individuazione di geni coinvolti o nel processo di differenziazione della AZ o nella via di trasduzione del segnale. Per quanto riguarda la cascola fisiologica dei frutticini in melo, le informazioni riguardanti il segnale che genera l’evento abscissione sono tuttora carenti e le collezioni di geni legati a tale fenomeno sono ancora molto parziali. La maggior parte delle ricerche, infatti, si è concentrata principalmente sullo studio dell’effetto di prodotti chimici sulla qualità della frutta e sulla fioritura, mentre solamente pochi studi hanno considerato la dinamica dei cambiamenti trascrizionali conseguente all'induzione dell’abscissione. Sulla base dei dati disponibili, è stata proposta l’ipotesi che considera lo stato nutrizionale all’interno della popolazione di frutticini e fra i fruitticini ed i germogli vegetativi come segnale necessario per l’attivazione dell’abscissione. Recentemente, lo sviluppo di approcci trascrittomici di carattere massale, basato sulle tecnologie microarray, ha consentito di studiare in maniera più approfondita questo processo biologico. Un recente studio effettuato in pomodoro ha permesso di studiare geni coinvolti nel mantenimento dell’omeostasi dell'auxina a livello di AZ in seguito ad una diminuzione del flusso della stessa durante l'induzione dell’abscissione nel fiore. Il modello proposto rappresenta un punto di partenza molto rilevante per identificare altri geni coinvolti nella regolazione dell’abscissione e nella sensibilizzazione dell’AZ all'etilene. In questa tesi sono stati impiegati due differenti approcci per studiare l’abscissione in melo: 1) Un approccio massale trascrittomico per isolare i geni strettamente coinvolti nelle prime fasi induttive dell’abscissione e 2) lo studio di composti organici volatili (VOCs) emessi durante l’induzione dell’abscissione. 1) L'abscissione di frutticini di melo è stata indotta usando la BA come agente diradante. Frutticini differenti per dimensione e posizione all'interno del corimbo sono stati raccolti entro i quattro giorni dal trattamento. L'espressione genica è stata analizzata per mezzo di un vetrino 30K recentemente sviluppato. L'analisi dei profili trascrizionali dei frutticini cascolanti e non cascolanti è stata esaminata allo scopo di identificare marcatori molecolari associati al destino del frutto. Il livello di specie reattive dell’ossigeno (ROS) e di alcuni carboidrati (glucosio, fruttosio, saccarosio, sorbitolo e amido) è stato misurato nella cortex degli stessi campioni. Un modello ipotetico per l’abscissione di frutticini di melo è stato ottenuto unendo i dati trascrittomici e metabolomici disponibili. Secondo questo modello, il trattamento con la BA amplificherebbe lo stress nutrizionale già in atto all'interno dell'albero, il quale viene percepito soprattutto dalla cortex di frutticini il cui sviluppo viene quindi bloccato. Nei frutti più deboli, questo stress viene quindi percepito a livello del seme. La traduzione di questo stress avviene probabilmente attraverso il crosstalk tra ROS, zuccheri e ormoni ed è seguito da un blocco dell'embriogenesi e dall'attivazione della AZ. 2) Frutticini di due diverse cultivar (Golden Delicious e Red Chief) con differente potenziale di abscissione trattati con due differenti diradanti chimici (BA e metamitron) sono stati analizzati per mezzo del PTR-MS (proton transfer reaction mass-spectrometer), entro i dieci giorni dal trattamento, allo scopo di identificare composti organici volatili (VOCs) associati all’abscissione. I risultati hanno evidenziato che i frutticini con potenziale di abscissione maggiore in entrambe le cultivar emettono più isoprene rispetto ai frutti persistenti. E’ stata inoltre evidenziata una correlazione significativa tra emissioni di isoprene e contenuto di ABA della cortex, parallelamente all’attivazione specifica dei rispettivi geni biosintetici. Successivamente avviene l’attivazione ritardata dei geni coinvolti nei passaggi chiave della via del metileritritolo fosfato (MEP), che fornisce i precursori per la biosintesi sia del volatile che dell’ormone. Secondo questi risultati, si può ipotizzare per l’isoprene un ruolo di detossificatore di ROS, la cui attivazione è mediata e controllata a livello trascrizionale dall’ABA. Le prospettive future di questa ricerca saranno focalizzate su tutte le linee di ricerca finora perseguite. La funzione biologica dei geni identificati tramite l’approccio trascrittomico sarà ulteriormente studiata a livello cellulare, tramite ibridazioni in situ, e i loro profili di espressione genica saranno ulteriormente validati. Particolare attenzione sarà prestata ai fattori di trascrizione e agli altri elementi regolativi coinvolti nel cross-talk ormonale. Per quanto attiene i composti volatili (isoprene) saranno validati i risultati finora ottenuti allo scopo di verificare possibili applicazioni pratiche in sistemi previsionali che consentano di predire il livello di carica fruttifera e, quindi, di dosare i trattamenti diradanti nell’ottica di un’agricoltura sostenibile.

Apple storage in controlled atmosphere (CA) can increase the marketing window of apples but at the same time it is an expensive practice and on the basis of cultivar, season, harvest time and storage condition (low O2, low/high CO2, low temperature) apples can develop different physiological disorders. It appears as a darkened area due to necrosis of hypodermal cells especially in Granny Smith and Red Delicious cultivars. Superficial scald annually causes the major economic loss to apple growers worldwide. It can arise after a relative long period (2-4 months) of cold storage with an increase in severity when apples are removed from storage and are leaved at room temperature (ca 7 dd). It is also influenced by different preharvest factors including fruit ripening at harvest, environmental conditions during growth, ethylene action or fruit mineral content. Superficial scald is associated with the accumulation of volatiles, in particular of α-farnesene, the biosynthesis of which is influenced by ethylene. This compound can be oxidized along with the storage period to a group of molecules called conjugated trienols. The accumulation of these oxidative products can induce metabolic dysfunction and cell death. Different strategies are provided to successfully inhibit scald development. The most common techniques in use combine storage in control atmosphere (CA) with treatments with the antioxidant diphenylamine (DPA), or with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception and the most effective molecule to control scald, or application of an initial low oxygen stress (ILOS), followed by CA storage. In 2011 the use of DPA has been banned in Europe for health concerns, 1-MCP is effective but has high costs, while ILOS is difficult to apply requiring more research for its optimization and apples cannot be stored for long periods. The identification of the molecular mechanism involved in the regulation of superficial scald development may allow the possibility to predict at harvest, before CA storage, which apple batches may probably develop the disorder allowing rational storage strategies with significant economic gains. The aim of this work was to provide a preliminary characterization of the molecular factors putatively involved in scald development associated with ethylene action and ROS metabolism, two actors that seem to have a role in scald occurrence, in peels of apples, cv Granny Smith, treated or not with 1-MCP or DPA and stored in controlled atmosphere for 1, 3 or 6 months. For this purpose gene families involved in the maintenance of ROS homeostasis were identified in the apple genome. In particular ROPs and ancillary proteins ROP-GEFs, -GAPs and -GDIs, RBOHs (NADPH oxidases involved in the generation of ROS at the apoplastic levels) and PLsDα (involved togheter with ROPs in the regulation of RBOHs activity) were identified. It is known in fact that Arabidopsis cells during oxygen deprivation activate a mechanism controlled by negative feedback regulation which involves ROPs, ROP-GAPs, RBOH and H2O2, and it is termed the ROP-GAP rheostat. Expression analyses on the identified genes evaluated by real-time PCR allowed the identification of 2 ROPs, 7 ROP-GEFs, 8 ROP-GAPs, 2 RBOHs and 2 PLsDα de-repressed in samples treated with 1-MCP and in most cases with a minor extent, by DPA treatment. The expression of the same genes was evaluated by qPCR on peels of apples subjected to short-time treatments with a saturating concentration (100 ppm) of ethylene for 4 and 24 hours showing that ethylene negatively regulates many of the apple ROP-GAP rheostat genes including RBOHs and PLsDα. Then analyses of content of malonyldialdehyde, a marker of lipid peroxidation, H2O2, ascorbate and glutathione, suggested that cells of untreated apples, which showed scald symptoms in the 97% of cases after 6 months of storage, perceived the oxidative stress associated with the loss of H2O2 homeostasis. On the contrary samples treated with 1-MCP showed higher levels of H2O2 thus maintaining the H2O2 homeostasis. This finding was further confirmed by the up-regulation of transcription of alcohol dehydrogenase, a marker of H2O2 response. The subcellular localization of H2O2, obtained by means of cerium chloride reaction through transmission electron microscopy, revealed higher levels of H2O2 in the apoplast of apple peels treated with 1-MCP compared to control ones, confirming a role of RBOHs and their regulative system on control of apoplastic H2O2 homeostasis. Finally RNA-seq analyses on the same samples, allowed to construct an heatmap that highlighted in samples treated with 1-MCP a co-regulation between genes of the ROP-GAP rheostat and of the ROP system, and genes belonging to the ascorbate, dehydroascorbate reductase and thioredoxin families, involved respectively in the detoxification or protection of thiol groups by ROS action. These results demonstrate for the first time that during cold stress ethylene induces in apples that develop scald a disruption of ROS homeostasis caused by the loss of ROP-GAP rheostat and RBOHs regulation, thus provoking a different transcriptional response of genes involved in ROS detoxification.
La conservazione delle mele per lunghi periodi è resa possibile grazie all’introduzione di tecniche quali la conservazione in atmosfera controllata e permette ai produttori di aumentare la finestra temporale di commercializzazione di questo frutto sul mercato. È una procedura costosa che può causare a seconda delle varietà di mele conservate, della stagionalità, del periodo di raccolta dei frutti e delle condizioni di conservazione applicate (bassa concentrazione di O2, bassa/alta concentrazione di CO2, bassa temperatura) la comparsa di diversi disordini fisiologici. Tra i disordini più comuni che colpiscono le mele prodotte e vendute in Italia vi è il riscaldo superficiale che si manifesta come un’area necrotica a livello della buccia nelle varietà sensibili quali Granny Smith e Red Delicious. Il riscaldo superficiale causa la maggior perdita di mele e di conseguenza il maggior danno economico ai produttori di tutto il mondo. Il riscaldo insorge in seguito a periodi di conservazione a basse temperature relativamente lunghi (2-4 mesi) e successiva conservazione a temperatura ambiente (ca 7 gg) dopo l’uscita dalle celle. È un disordine la cui comparsa è influenzata anche da fattori indipendenti dalla conservazione quali lo stadio di maturazione dei frutti alla raccolta, le condizioni ambientali durante la crescita, l’azione dell’etilene o ancora il contenuto minerario. Ad oggi studi fisiologici e biochimici su Granny Smith hanno evidenziato come l’α-farnesene, un volatile presente nella buccia delle mele il cui processo finale di biosintesi è influenzato dall’etilene, possa andare incontro ad un processo di ossidazione quando le mele vengono poste nelle celle di conservazione in atmosfera controllata. L’accumulo dei prodotti ossidativi derivanti, tra cui i trienoli coniugati porterebbe alla degenerazione del tessuto. Diverse strategie sono state adottate negli anni per prevenire la comparsa dei sintomi del riscaldo tra cui l’impiego dell’antiossidante difenilamina (DPA), dell’inibitore della percezione dell’etilene 1-metilciclopropene (1-MCP) o l’applicazione di un iniziale stress a basso ossigeno (ILOS -initial low oxygen stress-) durante le prime settimane di conservazione. L’impiego del DPA è stato proibito in Europa dal 2011, mentre i trattamenti con 1-MCP assicurano il controllo del riscaldo ma hanno costi elevati, infine lo stress iniziale a basso ossigeno non permette una conservazione per lunghi periodi e necessita di continui monitoraggi per evitare che le mele sviluppino disordini legati allo stress da basso ossigeno. Conoscere i meccanismi molecolari che regolano la comparsa e lo sviluppo dei sintomi del riscaldo potrebbe permettere di identificare alla raccolta le partite di mele soggette alla manifestazione del disordine, così da individuare quali partite conservare o meno e garantire un guadagno economico al produttore. Lo scopo di questo lavoro è stato quindi cercare di caratterizzare in maniera preliminare su campioni di bucce di mele della varietà Granny Smith, trattate o meno con 1-MCP o DPA e conservate in atmosfera controllata per 1, 3 o 6 mesi, i possibili fattori molecolari coinvolti nel riscaldo, in associazione con l’attività dell’etilene e del metabolismo ROS, due agenti che dai dati in letteratura sembrano avere un ruolo nello sviluppo del riscaldo. A questo scopo sono state caratterizzate in melo le famiglie geniche coinvolte nel mantenimento dell’omeostasi dei ROS. In particolare le ROP e le proteine accessorie ROP-GEF, -GAP e -GDI, le RBOH (NADPH ossidasi coinvolte nella produzione dei ROS a livello apoplastico) e le PLDα (coinvolte nella regolazione dell’attivazione delle RBOH insieme alle ROP) in quanto è noto che in Arabidopsis, in condizioni di basso ossigeno, le cellule attivano un meccanismo regolativo a feedback negativo che coinvolge in generale ROP, ROP-GAP, RBOH e H2O2, e prende il nome di reostato ROP-GAP. Tramite real-time PCR sono state analizzate le espressioni trascrizionali dei geni identificati, individuando 2 ROP, 7 ROP-GEF, 8 ROP-GAP, 2 RBOH e 2 PLDα che vengono de-repressi nei campioni trattati con 1-MCP, e in maniera minore anche dal trattamento con DPA. Trattamenti di 4h e 24h con etilene esogeno hanno permesso di dimostrare che alcuni di questi geni de-repressi in presenza di 1-MCP vengono effettivamente regolati in maniera negativa dall’etilene. Successivamente le analisi effettuate sul contenuto in malonildialdeide, un marcatore della perossidazione lipidica, in H2O2, ascorbato e glutatione, suggeriscono che le cellule delle mele non trattate, che nel 97% dei casi hanno manifestato riscaldo alla fuoriuscita dalle celle, presentino una situazione di stress associata alla perdita dell’omeostasi dell’H2O2 che viene invece mantenuta nei campioni trattati con 1-MCP i quali presentano anche un aumento dei livelli trascrizionali di alcol deidrogenasi (ADH), un marcatore della risposta all’H2O2. La localizzazione subcellulare dell’H2O2, determinata col cerio cloruro tramite microscopia elettronica, ha rilevato poi maggiori livelli di H2O2 a livello dell’apoplasto nelle bucce dei campioni trattati con 1-MCP rispetto al controllo, confermando quindi un ruolo delle RBOH e del loro sistema regolativo nel mantenimento di livelli omoeostatici di H2O2 nell’apoplasto. Infine in seguito ad un’analisi RNA-seq sugli stessi campioni, è stato possibile costruire una heatmap che ha evidenziato solo nei campioni trattati con 1-MCP una evidente co-regolazione tra i geni identificati del reostato ROP-GAP, e in generale del sistema ROP, e le sequenze geniche appartenenti alle famiglie delle ascorbato perossidasi, monodeidroascorbato reduttasi e tioredossine coinvolte rispettivamente nella detossificazione e nella protezione dei gruppi tiolici dall’azione dei ROS. Nell’insieme i risultati ottenuti dimostrano per la prima volta che durante lo stress da freddo la presenza dell’etilene induce nei campioni che manifestano riscaldo una perdita dell’omeostasi dell’H2O2 causata dalla mancata regolazione del reostato ROP-GAP e delle RBOH, che porta all’attivazione di una diversa risposta trascrizionale dei geni coivolti nella detossificazione dei ROS.

L'objectif de la thèse était d'étudier l'existence d'une coordination entre les croissances aériennes et racinaires chez le pommier, en prenant en compte une possible variation intra spécifique. Deux génotypes hybrides créés à l'INRA ont été étudiés à deux stades de développement. D'une part, des arbres ont été observés au verger, sur leurs propres racines (SPR) et greffés sur M9. La topologie des systèmes aériens des deux arbres par modalité a été décrite pour trois années de croissance, et les durées de croissance des pousses moyennes et longues ont été observées au cours de deux saisons de végétation. Les émissions racinaires ont été observées grâce à des plans d'observation de 1 m2, placés au pied de chaque arbre. D'autre part, de jeunes plants issus de culture in vitro des deux génotypes ont été observés sur leurs propres racines, dans des minirhizotrons placés en conditions contrôlées. La croissance des systèmes aérien et racinaire a été observée deux fois par semaine pendant 4 mois, sur deux séries de 5 plants pour chaque génotype. L'analyse des croissances aériennes au verger a mis en évidence des différences architecturales entre génotypes et les effets du greffage sur le développement aérien. Les deux génotypes diffèrent par le nombre de rameaux développés, leur floribondité et le taux de nouaison associé, X3305 étant moins ramifié, plus floribond et plus régulier en floraison. Outre une plus grande précocité d'entrée en production, le greffage produit (i) une modification de la composition raméale des arbres, en particulier pour les positions intra arbre des inflorescences, (ii) une augmentation des fréquences de retour à fleurs et, (iii) une augmentation de la mortalité des rameaux axillaires en fonction de l'âge du rameau. Généralement, le nombre de racines par arbre a été proportionnel au nombre d'unités de croissance aériennes à l'échelle de l'arbre, cette relation étant différente entre génotypes. Comme attendu, la production des racines a été plus importante chez les arbres SPR que chez ceux greffés sur M9. Les nouvelles racines ont été émises au cours de deux périodes principales, au printemps, généralement après la phase de croissance aérienne, et en période automnale. L'occupation du sol a progressé en profondeur chaque printemps mais également entre les deux années d'observation. In fine, les arbres SPR semblent avoir mieux réparti leurs émissions racinaires dans le temps et en profondeur que ceux greffés. Chez les jeunes plants, les nouvelles feuilles et les surfaces foliaires se sont développées selon un rythme caractérisé par des auto-corrélations significatives, alternativement positives et négatives. Les caractéristiques de ramification des racines primaires et la morphologie des racines secondaires ont présenté des différences entre génotypes: chez X3305, les racines secondaires ont été plus nombreuses et ont eu une vitesse de croissance plus lente que chez X6407. Le nombre de nouvelles racines secondaires a également suivi un rythme caractéristique du génotype et qui alterne avec l'expansion des surfaces foliaires. En conclusion, l'existence d'une coordination entre les systèmes aériens et racinaires a été confirmée chez les deux génotypes de pommier et à deux stades de développement, bien que les échelles de temps de l'alternance des émissions d'organes aient été très différentes. L'analyse de la structure 3D des systèmes racinaires au verger et l'étude des interactions génotype x milieu sur des plants miniaturisés pourraient constituer une suite logique à ce travail

Orientador: Miriam Dupas Hubinger
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este estudo visou formular uma cobertura comestível à base de fécula de mandioca, cera de carnaúba e ácido esteárico ou palmítico, avaliando suas propriedades e seus efeitos quando aplicados em maçãs minimamente processadas. Em uma primeira etapa, foram realizados delineamentos Plackett-Burman para seleção das seguintes variáveis para formulação das coberturas: porcentagem de fécula de mandioca (1 a 3% p/p), porcentagem de glicerol (0 a 2% p/p), razão entre cera de carnaúba e ácido graxo (0,16:0,84 a 0,64:0,36% p/p), velocidade (8000 a 16000 rpm) e tempo de emulsificação (1 a 5 minutos). Destas variáveis, a porcentagem de fécula de mandioca (2 a 4% p/p), porcentagem de glicerol (1 a 3% p/p) e razão cera de carnaúba: ácido graxo (0:0 a 0,40:0,60% p/p) foram selecionadas para compor delineamentos compostos centrais rotacionais (DCCR) 2³, um para cera de carnaúba e ácido esteárico e outro para cera de carnaúba e ácido palmítico, fixando o tempo e a velocidade de emulsificação em 3 minutos e 12.000 rpm, respectivamente. Os delineamentos visaram a seleção de formulações de coberturas através da avaliação da estabilidade à cremeação e tamanho médio das gotas lipídicas das soluções filmogênicas, propriedades mecânicas, cor, solubilidade e umidade dos filmes formados, taxa respiratória de fatias de maçã com coberturas e resistência ao vapor de água das coberturas aplicadas sobre cilindros de maçã. Quatro formulações com ácido esteárico na região otimizada foram selecionadas para a etapa de validação. A formulação composta por 3% (p/p) de fécula de mandioca, 1,5% (p/p) de glicerol, 0,2% (p/p) de cera de carnaúba e 0,8% (p/p) de ácido esteárico foi selecionada para a etapa seguinte, pois apresentou coberturas com boas propriedades de barreira, boa estabilidade e distribuição de lipídios na emulsão, filmes com boas propriedades ópticas, mecânicas, térmicas, físicas e estruturais. No estudo da vida útil de maçãs minimamente processadas, foram aplicados nas amostras os tratamentos: Controle (amostras apenas sanitizadas); ACAA (imersão em 1% de ácido cítrico e 1,5% de ácido ascórbico); FM (imersão em solução de ácidos cítrico e ascórbico e cobertura à base de 3% de fécula de mandioca e 1,5% de glicerol) e FMC (imersão em solução de ácidos cítrico e ascórbico e cobertura selecionada na validação do planejamento experimental). Os efeitos dos tratamentos sobre a taxa respiratória, perda de peso, propriedades mecânicas, cor, sólidos solúveis totais, pH, acidez total titulável, vitamina C, atividade de água, umidade, estrutura celular, crescimento microbiano e aceitação sensorial das fatias de maçã estocadas a 5 °C durante 12 dias, além da determinação composição gasosa das embalagens foram avaliados. A aplicação de ácidos cítrico e ascórbico foi eficiente na redução do escurecimento enzimático, e sua associação com a cobertura à base de fécula de mandioca e lipídios promoveu eficaz diminuição da taxa respiratória e da perda de peso e de vitamina C, manutenção das propriedades mecânicas e da estrutura celular. A adição de lipídios no tratamento FMC não alterou o sabor e aroma das amostras, alcançando uma vida útil de 5 dias atestada sensorialmente. As amostras ACAA e FM não diferiram estatisticamente do controle na maioria das análises, apresentando apenas manutenção da coloração e menores perdas de vitamina C devido ao uso de agentes antioxidantes, obtendo também uma vida útil de 5 dias. O controle apresentou grande escurecimento enzimático e perda de textura, sendo rejeitado sensorialmente após 1 dia de estocagem
Abstract: This study aimed at formulating a cassava starch, carnauba wax and stearic acid or palmitic acid - based edible coating, and evaluating their properties and their effects when applied to minimally processed apples. In a first step, Plackett-Burman designs were performed for the following variables selection of coating formulations: cassava starch concentration (1 - 3% w/w), glycerol percentage (0 - 2% w/w), carnauba wax and fatty acid ratio (0.16:0.84 ¿ 0.64:0.36% w/w), stirring speed (8000 - 16000 rpm) and emulsification time (1-5 minutes). The variables cassava starch concentration (2-4% w/w), glycerol percentage (1- 3% w/w) and carnauba wax: fatty acid ratio (0:0 to 0.40:0.60% w/w) were selected to compose two composite central rotational designs 2³ (CCRD), one for carnauba wax and stearic acid and the other for carnauba wax and palmitic acid, setting the emulsification time and stirring speed in 3 minutes and 12,000 rpm, respectively. The designs aimed at the coating formulations selection by evaluating physical stability and average lipid particle size of filmogenic solutions, mechanical properties, color, solubility and moisture of films, respiration rate of coated apple slices, surface solid density and water vapor resistance of coatings applied to apple cylinders. Four formulations with stearic acid in the optimized region were selected for the validation procedure. The formulation elaborated with 3% (w/w) of cassava starch, 1.5% (w/w) of glycerol, 0.2% (w/w) of carnauba wax and 0.8% (w/w ) of stearic acid was selected for the shelf life study, since it showed coatings with good barrier properties, good stability and distribution of lipids in the emulsion and films with good optical, mechanical, thermal, physical and structural properties. In the shelf life study of minimally processed apples, the following treatments were applied in samples: control (samples only sanitized); ACAA (immersion in 1% citric acid and 1.5% ascorbic acid), FM (immersion in citric and ascorbic acids solution and coating with 3% cassava starch and 1.5% glycerol) and FMC (immersion in citric and ascorbic acid solution and coating selected in the experimental design validation). The treatments effects on respiration rate, weight loss, mechanical properties, color, soluble solids, pH, total acidity, vitamin C, water activity, moisture, cell structure, microbial growth and sensory acceptance of sliced apples stored at 5 °C for 12 days, and the determination of packaging gas composition were evaluated. The citric and ascorbic acid application was effective in reducing enzymatic browning, and its association with the cassava starch and lipids based coating promoted effective decrease in respiration rate, weight and vitamin C loss, maintenance of mechanical properties and cellular structure. The lipids addition to FMC treatment did not affect taste or aroma of samples, achieving a shelf life of 5 days sensory attested. The ACAA and FM samples did not differ statistically from the control in most analysis, except on color maintenance and reduction of vitamin C loss due to the use of antioxidants, also obtaining a shelf life of 5 days. The control showed great enzymatic browning and texture loss, and was sensory rejected after 1 day of storage
Doutorado
Engenharia de Alimentos
Doutor em Engenharia de Alimentos

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico.
Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought.

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Phosphorus (P) fertilization in apple orchards has received less attention than nitrogen (N) and potassium (K) fertilization. In Brazil there is no information about apple response to P application to the soil. Thus, the objective of this study was to assess the effect of P application to the soil on some parameters related to fruit quality as well as to fruit mineral composition. The experiment was conducted in a commercial orchard in São Joaquim, SC, during the growing seasons of 2012/2013, 2013/2014 and 2014/2015. It was used the cultivar ‘Fuji Suprema’ grafted over Marubakaido/M9 rootstock, in a high tree density system, planted on an Haplumbrept. Treatments were arranged in a complete randomized block design, with five replicaitions. Each experimental unit had seven trees spaced 4.2 x 1.2 m, but only the five central trees were used for evaluations. Treatments consisted of P2O5 rates (0, 40, 80, 120 and 160 kg ha-1 year-1), applied annually from 2011, in the form of triple superphosphate, after harvesting, on soil surface, without incorporation, centralized along the planting line. Fruits were harvested 15 days before commercial harvest, and we collected two samples of 15 fruits from each treatment. One sample was evaluated immediately after harvest and the other one was cold stored in controlled atmosphere chambers for six months before evaluation. The parameters related to fruit quality assessed at harvest and after six months of storage were: pH, soluble solid (SS), titratable acidity (TA),flesh firmness and skin color. Ca, Mg, K, N and P contents were determined in the fruits once a year. Fruit quality data were statistically analyzed using analysis of variance and the means were compared using Tukey test (p < 0,05). Analysis of variance and regression analysis were performed on fruit mineral content data to determine the effects of P rates. Fruit attributes (pH, SS, AT and flesh firmness), at both determining times, were not affected by P application to the soil. In relation to fruit color attributes, only the parameter h° of the redder side of the fruit, determined at harvest, was affected by P addition to the soil. The contents of Ca, Mg, K, N and P were not affected by P addition to the soil. The P content in the soil increased with P addition, and the layer of 0-10 cm depth presented the highest increases. Thus, the lack of response of ‘Fuji’ fruits to soil P addition indicates that phosphorus fertilization is not needed to increase fruit quality on established apple orchards
A adubação com fósforo (P) em pomares de macieira tem recebido menos atenção que a adubação com nitrogênio (N) e potássio (K). No Brasil existem poucas informações sobre a resposta da cultura à aplicação de P para as condições de solo e regiões onde a macieira é cultivada. Assim, o objetivo deste trabalho foi avaliar o efeito da aplicação de P nos parâmetros de qualidade no momento da colheita e após armazenamento, e a composição mineral de frutos de maçã 'Fuji'. O experimento foi instalado em 2010 e conduzido em um pomar comercial no município de São Joaquim, SC, durante as estações de crescimento de 2012/2013, 2013/2014 e 2014/2015. Usou-se a cultivar ‘Fuji Suprema’ sobre o porta-enxerto Marubakaido/M9, num sistema de alta densidade de plantio, em um Cambissolo Húmico. Foi utilizado o delineamento experimental de blocos ao acaso, com cinco repetições. As unidades experimentais foram compostas por sete plantas, espaçadas em 4,2 x 1,2 m, porém, apenas as cinco plantas centrais foram avaliadas. Os tratamentos consistiram de doses de P2O5 (0, 40, 80, 120 e 160 kg ha-1), aplicados anualmente a partir do ano de 2011, na forma de superfosfato triplo, após a colheita dos frutos, sobre a superfície do solo e sem incorporação, centralizada junto à linha de plantio. Os frutos foram colhidos 15 dias antes da colheita comercial, sendo coletadas duas amostras de 15 frutos de cada unidade experimental. Uma amostra foi avaliada logo após a colheita e a outra armazenada em câmaras frigorificas com atmosfera controlada por seis meses. Os parâmetros relacionados com a qualidade de frutos, tanto na colheita quanto após seis meses de armazenamento, foram: pH, sólidos solúveis (SS), acidez titulável (AT), firmeza de polpa e cor do fundo da epiderme. Uma vez por ano foi avaliado o teor de Ca, Mg, K, N e P nos frutos. Os dados das variáveis da qualidade de frutos foram submetidos à análise de variância (ANOVA) e teste de comparação de médias (Tukey; p < 0,05); os atributos minerais foram submetidos à ANOVA e de regressão (p < 0,05) para determinar os efeitos das doses de P. Os atributos físico-químicos dos frutos, como pH, AT, SS e firmeza de polpa, na colheita e após seis meses de armazenamento, não foram influenciados pela aplicação de P ao solo, na média dos anos. Nos atributos de coloração dos frutos, somente a média do ângulo h° do lado mais vermelho do fruto foi afetado, reduzindo a coloração dos frutos com a adição de P ao solo. Os teores de Ca, Mg, K, N e P nos frutos, não sofreram influência da adição de P ao solo. Os teores de P no solo aumentaram com a adição de P, sendo a camada de 0-10 cm a que apresentou os maiores aumentos. Os dados obtidos indicam que os atributos relacionados com a qualidade dos frutos da cultivar ‘Fuji Suprema’ não são afetados pela adição de P ao solo

The effects of AVG, an inhibitor of ethylene synthesis, in combination with MCP or heat treatment (HT) on quality traits of several apple cultivars after harvest (AH) or cold storage (ACS), and the involvement of ethylene in the regulation of SDH activity during the last weeks of fruit development were studied. AVG was applied to Royal Gala, Lodi, Senshu, Redchief Delicious and Red Fuji trees 4 weeks before normal harvest (H1). Control and AVG-treated (AVG) fruit were harvested at H1 and treated with MCP or air-heated. Fruit were ripened at room temperature (RT) AH or ACS. Some AVG fruit were harvested at H1 and 1 to 2 weeks after H1 (H2), or at H2 only. Ethylene production (EP), respiration rate (RR), firmness, starch index (SI), titratable acidity (TA), volatile production (VP) and AAT activity, among others, were measured AH and ACS. Peel and cortex of Gala were alcohol-fed and ester production quantified. EP and SDH presence and activity were measured at various harvest dates on control and AVG Lodi, Red Delicious and Fuji apples. AVG plus MCP was more effective in reducing HEP, RR, firmness and TA loss than either treatment alone; it did not provide further control on SI and did not repress Gala red skin color development more than AVG alone, though it consistently repressed VP. AVG plus HT was generally more effective than single treatments in reducing HEP, RR and firmness loss during storage. It was not different than the single treatments on TA, SI, and VP. The effect of AVG plus HT on fruit quality ACS was cultivar-dependent. AVG plus HT was not enough to maintain the quality of the earlyharvest cultivars, and it did not improve fruit quality of late-harvest cultivars. The effects of AVG plus MCP but not of AVG plus HT were evident at H2. Precursor availability was the major factor limiting VP, suggested by the low VP when RR was low, the increase in ester production in alcohol-fed samples, and the lack of correlation between AAT and ester production. SDH activity or expression was not affected by a reduction in ethylene production.

Die Effektivität der Züchtung von Apfel (Malus ×domestica BORKH.) sowie anderen mehrjährigen hölzernen Obstkulturen hängt stark von der langen juvenilen Phase der Sämlinge ab. Die juvenile Phase bei Apfelsämlingen dauert meist fünf bis zehn Jahre. Während dieser Phase sind die züchtungsrelevante Bildung von Blüten und Früchten und die Selektion von Nachkommen anhand adulter Merkmale ausgeschlossen. Die Juvenilität verlängert somit einzelne Kreuzungszyklen drastisch, was einen hohen Zeit- und Kostenaufwand für die Züchter bedeutet. Das Rapid Cycle Breeding System von Apfel ist eine neue Züchtungstechnik, die eine beschleunigte Introgression von Zielgenen, vor allem Resistenzgenen aus Wildarten, in etablierte Sorten ermöglicht. Im Gegensatz zu agrotechnischen Methoden, verkürzt diese Technik die juvenile Phase der Apfelsämlinge auf wenige Wochen. Der Einsatz frühblühender transgener Linien, welche das FRUITFULL-Homolog BpMADS4 der europäischen Silberbirke (Betula pendula Roth.) über-exprimierten, ermöglichte die Realisierung einer Kreuzungsgeneration pro Jahr. Das derzeitige Rapid Cycle Breeding System stützt sich auf die 35S::BpMADS4-Linie T1190 als Modellpflanze, deren T-DNA auf der Kopplungsgruppe 4 lokalisiert wurde. Für die Selektion von Null-Segreganten im finalen Schritt des Kreuzungsprogramms ist es entscheidend, dass blühinduzierendes Transgen und eingekreuzte Zielgene auf nicht-homologen Chromosomen lokalisiert sind. Zur Erhöhung der Flexibilität des derzeitigen Rapid Cycle Breeding Systems im Hinblick auf die Realisierung verschiedenster Züchtungsziele befasste sich die vorliegende Arbeit mit drei Themenschwerpunkten: (1) Erweiterung des Spektrums an frühblühenden 35S::BpMADS4-Linien, welche sich in der Kopplungsgruppe der T-DNA-Insertion und Apfelsorte von T1190 unterscheiden. (2) Beurteilung der Eignung frühblühender 35S::BpMADS4-Linien für das Rapid Cycle Breeding im Rahmen eines Kreuzungsprogramms mit der Apfelschorf-resistenten Sorte 'Regia' und der Feuerbrand-resistenten Wildart-Akzession MAL0045 von Malus fusca (Raf.) Schneid. (3) Vermeidung negativer Auswirkungen der konstitutiven Überexpression von BpMADS4 auf den Habitus und die Blütenmorphologie durch Verwendung eines induzierbaren Promotors. Mit Hilfe des hitzeinduzierbaren Gmhsp17.5-E-Promotors aus Soja (Glycine max) sollte ein temporäres RNAi-Silencing des apfeleigenen Blüh-repressors MdTFL1 und damit gezielt eine frühe Blüte induziert werden. Zusammenfassend zeigten die Ergebnisse der vorliegenden Arbeit, dass: (1) BpMADS4 erfolgreich in den Apfelsorten 'Gala Mitchgla' und 'Santana' über-exprimiert werden konnte. Mit Hilfe eines Genome Walking-Verfahrens wurden T-DNA-Insertionen in neun der elf untersuchten 35S::BpMADS4-Linien der Apfelsorten 'Pinova', 'Gala', 'Gala Mitchgla', 'Santana' und 'PNS', einem Sämling von 'Pinova', identifiziert. Als Ergänzung zu T1190 sind frühblühende Linien mit BpMADS4 auf Kopplungsgruppe 2, 5, 7, 8, 9, 10, 14, 15 und 16 verfügbar. Sequenzanalysen zeigten analog zu Ergebnissen von T1190, dass die Agrobacterium-vermittelte Transformation zu Deletionen der T-DNA (bis zu 245 bp) und im Genom (bis zu 361 bp) führte. Im Gegensatz zu T1190 wurden bei den drei transgenen Linien von 'PNS' T1189, T1236 und T1240 Vektorbackbone-Insertionen im Genom identifiziert. Entsprechende Analysen der anderen Linien sind noch ausstehend. Die konstitutive Überexpression von BpMADS4 induzierte analog zu T1190 eine Verkürzung der juvenilen Phase auf nur wenige Wochen in allen elf 35S::BpMADS4-Linien. Dabei bewirkte die permanente Blütenbildung eine drastische Stauchung des vegetativen Sprosswachstums und gehäuftes Auftreten von Blütenanomalien. (2) Trotz signifikanter Unterschiede in Blütenmorphologie und Pollenfunktionalität der 35S::BpMADS4-Linien zu T1190 bzw. ihren nicht-transgenen Kontrollen war deren Blütenfertilität nicht eingeschränkt. In einem Kreuzungsprogramm mit M. fusca und 'Regia' konnte gezeigt werden, dass die Linien mit T1190 vergleichbare Frucht-ansatzraten hatten. Das Programm zielte auf die Introgression der Resistenzgene gegenüber Apfelschorf (Rvi2, Rvi4) und der rosigen Apfelfaltenlaus (Sd1) aus 'Regia' und des Feuerbrand-QTLs von M. fusca oder 'Regia' (FB-F7, FB-Mfu) ab. Aus 149 F1-Sämlingen wurden mittels Marker-gestützter Selektion 37 Sämlinge identifiziert, die BpMADS4 in Kombination mit mindestens einem Resistenzmarker aufwiesen. Acht frühblühende transgene F1-Sämlinge mit interessanter Kombination eingekreuzter Resistenzen wurden für Kreuzungen mit 'Golden Delicious' ausgewählt. Die F1-Sämlinge zeigten vergleichbare Fruchtansätze zu den entsprechenden frühblühenden 35S::BpMADS4-Linien. Mittels Marker-gestützter Selektion konnten aus 17 BC1-Sämlingen sechs BpMADS4-überexprimierende Sämlinge selektiert werden, die jedoch nur einen Teil der eingekreuzten Resistenzen vorwiesen. Letztlich ermöglichte die Kombination aus frühblühenden 35S::BpMADS4-Linien und Marker-gestützter Selektion der Nachkommen die Realisierung einer Kreuzungsgeneration pro Jahr. (3) Für ein temporäres Silencing der MdTFL1-Gene MdTFL1-1 und MdTFL1-2 in Apfel das duale Promotorkonstrukt 619pD1-HSP::MdTFL1-1-HSP generiert wurde, bei dem zwei Gmhsp17.5-E-Sequenzen ein 323 bp CDS-Fragment von MdTFL1-1 flankierten. Es wurden fünf transgene Linien der Apfelsorten 'Pinova' und 'Gala' erzeugt. Gen-expressionsstudien bestätigten, dass ein einmaliger Hitzeschock bei 42°C für 1 h bis 2 h den Gmhsp17.5-E-Promotor erfolgreiche aktivierte. Diese Aktivierung induzierte nur ein kurzzeitiges Silencing beider MdTFL1-Gene in den transgenen Linien, welches keinen Effekt auf die Transkription von MdFT1, dem Antagonisten von MdTFL1 hatte. Multiple Hitzeschockbehandlungen über einen Zeitraum von 28 d waren weder ausreichend, ein langfristiges Silencing von MdTFL1 noch eine frühe Blüte in den transgenen Linien zu induzieren. Negative Auswirkungen des MdTFL1-Silencing auf den Habitus konnten nicht bestätig werden. Mögliche Faktoren des Ausbleibens einer frühen Blüte wurden diskutiert. Interessanterweise zeigten Genexpressionsstudien, dass die Hitzeschock-Behand-lungen die Transkription von MdTFL1-1 und MdTFL1-2 gleichermaßen in transgenen und nicht-transgenen Pflanzen unterdrückten. In silico-Analysen identifizierten cis-regulatorische Hitzeschock-Elemente sowie flankierende TATA- und CCAAT-Box-Motive in den Promotorsequenzen beider Gene, welche bei der Reaktion pflanzlicher Gene auf Hitzestress eine Rolle spielen. Zusammenfassend zeigten die Untersuchungen dieser Arbeit, dass aus phänotypischer Sicht alle charakterisierten elf 35S::BpMADS4-Linien sich für das Rapid Cycle Breeding eignen. Vom molekulargenetischen Standpunkt aus können die transgenen Linien erst nach Untersuchung auf Vektorbackbone-Insertionen bzw. nach deren Mapping im Genom für das System verwendet werden. Die Eignung der acht frühblühenden F1-Sämlinge als Vorzuchtmaterial kann erst nach Durchführung entsprechender Resistenzstudien beurteilt werden. Im Gegensatz zu den frühblühenden 35S::BpMADS4-Linien hatten die HSP::MdTFL1-1-Linien keinen Nutzen für das Rapid Cycle Breeding. Dennoch belegten die Ergebnisse dieser Arbeit eine Hauptrolle beider MdTFL1-Gene in der pflanzlichen Hitzestressantwort.

Cette etude constitue la mise au point d'un systeme efficace de regeneration pour l'etude de la variation somaclonale (organogenese apres callogenese) ou pour l'utilisation dans les techniques modernes d'amelioration genetique (organogenese directe). La premiere partie consiste en l'amelioration du microbouturage et de l'enracinement in vitro afin de disposer d'explants en bonnes conditions physiologiques pour les etudes de callogenese et d'organogenese. La callogenese a ensuite ete mise au point et optimisee sur differents types d'explants. Les cals ont ete caracterises morphologiquement et histologiquement. On les a repartis en 5 classes. La presence d'auxine est necessaire a l'induction, et l'utilisation d'une cytokinine ameliore le taux de callogenese. Plusieurs facteurs ont ete testes: hormonaux, trophiques, physiques et physiologiques. Une etude particuliere a porte sur l'entretien des cals dans des conditions destabilisantes: cultures en presence de 2,4-d pendant un temps eleve. Enfin, un troisieme type de resultats porte sur l'obtention de neoformations a partir de cals ou directement sur limbes. Un large effet genotype a ete note: les varietes porte-greffes sont plus recalcitrantes que les varietes greffons. Le facteur hormonal semble primordial dans tous les cas, mais d'autres facteurs peuvent entrer en jeu. L'organogenese a ete induite sur des cals de racines, des cals primaires ou secondaires de limbes et des cals primaires d'entre-nuds. La regeneration directe a partir de limbes a pu etre obtenue sur: gala, golden delicious, granny smith et cepiland. La variete greffon gala donne la meilleure reponse; un traitement permet en particulier d'obtenir 100% d'explants organogenes. Une etude histologique a ete menee pour suivre l'origine de l'organogenese. Mille plantes neoformees a partir d'explants de gala ont ete acclimatees avec succes et seront plantees au ch

El bitter pit és la fisiopatia més important en molts cultivars de pomes. No obstant, no existeix una estratègia completament efectiva per al seu control, per aquest motiu, un mètode de predicció que identifiqui anys i plantacions amb un alt potencial de desenvolupar la fisiopatia permetrà evitar pèrdues econòmiques, especialment durant la conservació i confecció. L’objectiu principal de la present tesi doctoral va ser la posada a punt d’un sistema de predicció de la incidència del bitter pit en plantacions de poma ‘Golden Smoothee’. Per això, es van investigar diferents mètodes basats en tres tecnologies diferents: i) l’anàlisi mineralògic de fruits en diferent època (en estadis primerencs i a recol·lecció), ii) la inducció de símptomes (infiltració de Mg, banys amb etefón, embossat de fruits i mètode passiu) i iii ) l’espectroscopía VIS/NIR. Els diferents mètodes es van avaluar en diferents períodes de creixement del fruit. Paral·lelament, es va avaluar i quantificar l’eficàcia de diferents estratègies per mitigar l’aparició de bitter pit basades en aplicacions de CaCl2 a pre i postcollita (aplicacions radiculares, foliars i banys en poscosecha). L’anàlisi primerenc de Ca en fruit a 60 dies després de la floració (DDPF) va mostrar una precisió en la predicció similar o millor que l’anàlisi a recol·lecció. Es va definir un valor de referència de 60 DDPF de 11 mg de Ca 100 g-1 de pes fresc, per sobre del qual es minimitza la incidència de bitter pit. La majoria de mètodes basats en induir símptomes, a excepció de l’embossat, van mostrar eficàcia a partir dels 40 dies abans de la recol·lecció (DAR), amb una correlació amb el bitter pit de postcollita del 70-80%. La espectroscopía VIS/NIR va mostrar resultats poc satisfactoris per a la predicció del bitter pit, però va ser capaç de discriminar fruits afectats quan els símptomes eren visibles a postcollita. Finalment, es va disenyar un model de predicció per al bitter pit basat en l’anàlisi de Ca en fruit a 60 DDPF i el mètode passiu a partir de 40 DAR. Respecte la mitigació del bitter pit, els resultats obtinguts en els anys amb alta incidència van mostrar una reducció del 20% a un 12%, 8% o 3% mitjançant aplicacions foliars, banys de postcollita o la combinació d’ambdues, respectivament, per tant, les aplicacions foliars de CaCl2 així com els banys postcollita serien pràctiques a recomanar en els casos d’alt risc de bitter pit.
El bitter pit es la fisiopaía más importante en muchos cultivares de manzana. Sin embargo, no existe una estrategia de control completamente efectiva, por lo que un método de predicción que identifique años y plantaciones con alto potencial de desarrollar la fisiopatía permitirá evitar pérdidas económicas, especialmente durante la conservación y confección. El objetivo principal de la presente tesis doctoral fue la puesta a punto de un sistema de predicción de la incidencia de bitter pit en plantaciones de manzanas ‘Golden Smoothee’. Para ello, se investigaron diferentes métodos de predicción basados en tres tecnologías distintas: i) el análisis mineralógico de fruto (en estadios tempranos y en recolección), ii) la inducción de síntomas (infiltración de Mg, baños con etefón, embolsado de frutos y método pasivo) y iii) la espectroscopía VIS/NIR. Los distintos métodos se evaluaron en diferentes períodos de crecimiento del fruto. Paralelamente, se evaluó y cuantificó la eficacia de distintas estrategias para la mitigación del bitter pit basadas en aportaciones de CaCl2 en pre y poscosecha (aplicaciones radiculares, foliares y baños en poscosecha). El análisis temprano de Ca en fruto a 60 días después de plena floración (DDPF) mostró una precisión de predicción similar o mejor que el análisis de Ca en recolección. Se definió un umbral de referencia a 60 DDPF de 11 mg Ca 100 g-1 de peso fresco, por encima del cual se minimizó el riesgo de aparición del bitter pit. La mayoría de métodos basados en inducir síntomas, a excepción del embolsado de frutos, mostraron eficacia a partir de los 40 días antes de recolección (DAR), con una correlación con el bitter pit de poscosecha del 70-80%. La espectroscopía VIS/NIR mostró resultados poco satisfactorios para la predicción del bitter pit, sin embargo, sí fue capaz de discriminar frutos afectados cuando los síntomas eran visibles en poscosecha. Finalmente, se diseñó un modelo de predicción del bitter pit basado en el análisis de Ca en fruto a 60 DDPF y el método pasivo a partir de 40 DAR. Respecto la mitigación del bitter pit, los resultados obtenidos en años con alta incidencia mostraron una reducción de un 20% a un 12%, 8% o 3% mediante aplicaciones foliares, baños en poscosecha o la combinación de ambas, respectivamente, por lo que tanto las aplicaciones foliares de CaCl2 como los baños poscosecha serían prácticas a recomendar en el caso de riesgo de bitter pit.
Bitter pit is the most important physiological disorder in many apple cultivars. However, there is no a completely effective control strategy, therefore, a prediction method that identifies years and orchards with high potential to develop bitter pit will allow reducing economic losses, especially during storage and fruit packing. The main objective of this PhD thesis was the development of a system to predict the incidence of bitter pit for ‘Golden Smoothee’ apple orchards. For this, different methods to predict bitter pit based on three different technologies were investigated: i) mineral analysis (at early stages and at harvest period, ii) induction of symptoms (Mg infiltration, dips with etephon solution, bagging of fruit and passive method) and iii) VIS/NIR spectrophotometry. The different methods were tested in different fruit growth stages. At the same time, the efficacy of different strategies based on CaCl2 applications at pre- and postharvest (fertigation, foliar and postharvest dips) to mitigate bitter pit incidence, were evaluated and quantified. The accuracy of mineral analysis at early development fruit after 60 days after full bloom (DAFB) was better or equal than Ca analysis at harvest. A reference threshold at 60 DAFB of 11 mg Ca 100 g-1 fresh weight was defined. Values equal or higher indicated a low risk of bitter pit. Most methods based on inducing symptoms, with the exception of bagging fruit, showed efficacy from 40 days before harvest (DBH), with a correlation with bitter pit at postharvest of 70-80%. VIS/NIR spectrophotometry showed unsatisfactory results for bitter pit prediction, however, it was able to discriminate affected apples when the symptoms were visible at postharvest. Finally, a bitter pit prediction model based on the analysis of Ca in fruitlet at 60 DAFB and the passive method from 40 DBH was designed. Regarding bitter pit mitigation, the results obtained in seasons with a high incidence showed a reduction from 20% to 12%, 8% or 3% using Ca sprays, postharvest dips or the combination of both, respectively. Therefore, Ca sprays and postharvest dips in CaCl2 solutions are recommended practices when there is a diagnostic of high risk of bitter pit.

A cultura da macieira no Brasil ocupa uma área de 35 mil hectares, concentrada nos estados do Sul do país. Apesar do alto patamar técnico atingido, a cultura sofre o ataque e perdas por inúmeras doenças, dentre as quais, incluem-se a Fuligem e a Sujeira de Mosca (F&SM). Classificadas como doenças secundárias, reduzem o valor de comercialização dos frutos in natura, pela formação de manchas escuras na epiderme dos frutos como resultado da colonização epífita por fungos na superfície das maçãs. No Brasil, dados referentes à biologia de F&SM restringem-se a testes de eficiência de fungicidas ou provêm de estudos com outros patossistemas. O objetivo deste trabalho foi o de elucidar aspectos epidemiológicos destas doenças, fornecendo as bases para o manejo integrado. Foram realizados estudos de disponibilidade, dinâmica e controle de inóculo, de análise de distribuição espacial, de modelagem do progresso temporal e uma avaliação de um modelo de previsão de F&SM na região Nordeste do Rio Grande do Sul. As pesquisas foram realizadas nos ciclos 2006/07 e 2007/08 em Vacaria-RS. Foi verificado que o inóculo de F&SM esteve disponível durante todo o período de formação dos frutos, sugerindo que ciclos secundários são responsáveis pelo incremento da doença. A incidência de F&SM apresentou relação positiva com a intensidade das chuvas (mm/chuva e mm/h), e com o somatório de horas de molhamento foliar (R2≥0,85; P<0,05), sendo que as primeiras infecções foram estabelecidas desde os 30 primeiros dias após a queda das pétalas. A utilização de oxicloreto de cobre (0,5%) e hidróxido de cobre (0,5%) no período dormente de macieiras ‘Pink Lady®’ reduziu os danos de F&SM no momento da colheita, com controle de 33% e 53%, respectivamente. O regime pluviométrico nos três meses que antecederam à colheita influenciou a incidência de F&SM sem, no entanto, afetar o padrão agregado destas doenças (D>1). O modelo monomolecular apresentou o melhor ajuste ao progresso temporal, com R*2>0,90 independentemente do ciclo ou tratamento. Aplicações de tiofanato metílico + captana seguindo o critério do sistema de alerta, em combinação com o uso de poda verde, possibilitou, no ciclo 2007/08, uma redução de 66% no número de aplicações com fungicidas, sem afetar o controle quando comparado aos tratamentos preventivos.
Apple production in Brazil is located at the southern states and comprises an area of around 35 thousand hectares. In spite of the advanced technology in use, crop physiological stresses and disease are responsible to severe economical losses, in which Sooty Blotch and Flyspeck (SBFS) are of secondary importance, among other diseases. Losses by SBFS are due to fruit blemishes that are caused by growth of the fungi on the apple cuticule. In Brazil, epidemiological knowledge on SBFS is very limited and restricted to fungicide testing studies. The aim of this work was to study key epidemiological aspects and management strategies for SBFS control at the conditions of northeastern production of Rio Grande do Sul State. Field experiments were conducted in 2006/07 and 2007/08 seasons at Vacaria, RS. A series of experiments were planned to study inoculum and infections dynamics driven by environmental conditions, disease reduction by winter treatments, disease spatial patterns and assessment of management strategies for disease control by evaluating usefulness of a warning system and effect of cultural practices and fungicide treatments. Disease incidence was observed in fruits exposed during all infection windows demonstrating inoculum availability during all fruits stages. Disease incidence showed a linear relationship with precipitation rates (mm/rain and mm/h) and leaf wetness duration (R²≥0,85; P=0,05) measured during each window. The use of cooper oxycloret (0,5%) or cooper hydroxid (0,5%) sprays during dormant stages allowed reduction of 33% and 53%, respectively, in the SBFS incidence at harvest. Spatial analysis using beta-nominal distribution and geoestatistics showed disease aggregation among samplins units, being stronger aggregated when decreasing the number of plants in the sampling units. The monomolecular model best described SBFS incidence temporal progress (R*²>0,90) regardless of the fungicides treatments or season. Fungicide applications with a mix of thiophanate-methyl and captana following a disease warning system, in association with summer pruning allowed reduction of 66% in the number of sprays when compared to preventive treatment with the same control efficiency.

A macieira necessita de estímulo do frio para a queda das folhas ao final do ciclo, à dormência hibernal e a sua quebra desta. A não ocorrência adequada de frio no período do inverno resulta em brotação e florescimento irregular, resultando em desenvolvimento vegetativo e reprodutivo deficientes. Os objetivos deste trabalho foram de caracterizar indivíduos da população F1 para caracteres associados ao requerimento de frio hibernal como data de brotação e floração, construir um mapa genético de ligação em população clonal F1 e mapear QTLs associados a estes caracteres. Os genótipos da população F1 de estudo segregaram para os caracteres fenotípicos de brotação vegetativa e floração. Os mapas genéticos dos genitores ‘M13/91’ e ‘Fred Hough’ foram obtidos com 729 e 711 marcadores funcionais do tipo SNP, respectivamente. Dezessete grupos de ligação foram obtidos para cada genitor, cobrindo 1361 cM e 1066 cM para ‘M13/91’ e ‘Fred Hough’, respectivamente. As posições dos marcadores mapeados estão consistentes com o mapa consenso. Na extremidade do grupo de ligação 9, QTLs majoritários, possivelmente associados a fatores genéticos importantes para as características avaliadas, foram identificados nos mapas de ambos os genitores. Nesta região foram mapeados quatro marcadores genéticos significativamente ligados aos QTLs majoritários para brotação vegetativa e floração, explicando de 32,3% a 73,4% da variação fenotípica observada, respectivamente. Estes resultados sustentam a hipótese de que a extremidade do grupo de ligação 9 possui fatores genéticos associados ao controle do tempo de brotação e floração regulado pela exposição ao frio. Nesta região encontram-se genes de transcritos preditos com grande similaridade ao gene Flowering Locus C de Arabidopsis thaliana.
Apple trees require cold stimulus for leaf shedding at the end of the growth cycle and for winter dormancy. The non-occurrence of the appropriate cold condition during the winter causes irregular sprouting and flowering, resulting in deficient vegetative and reproductive growth. This study aimed to characterize individuals from a F1 population derived from ‘Fred Hough’ and ‘M13/91’ for cold requirement associated traits, like vegetative bud burst and flowering, to develop a genetic linkage map for both genitors and identify QTLs associated with these traits. The F1 population showed segregation for time for vegetative sprouting and time for flowering. A total of 729 and 711 functional SNP type markers were used to generate a map for each parent. For both parents, 17 linkage groups were obtained; covering 1361 cM and 1066 cM for ‘M13/91’ and ‘Fred Hough’, respectively. The position of the SNP loci in the obtained maps is consistent with the genomic sequence. At the end of linkage group 9, major QTLs genetically associated with the agronomical traits evaluated were identified for both genitors. In this region of the linkage group 9, four SNP markers were significantly associated with the major QTLs for vegetative bud burst and time of flowering, explaining 32,3% to 73,4% of the total phenotypic variation observed. Our results demonstrate that the same region of linkage group 9 contains important genetic determinants for the control of time of bud burst and flowering. Moreover, assessing the genomic sequence in this region, two predicted transcripts with similarity to the Flowering Locus C from Arabidopsis thaliana were identified. 1Master of

In two Italian sites, multiaxis trees slightly reduced primary axis length and secondary axis length of newly grafted trees, and increased the number of secondary shoots. The total length, node production, and total dry matter gain were proportional to the number of axis. Growth of both primary and secondary shoots, and dry matter accumulation, have been found to be also well related to rootstock vigour. A great variability in axillary shoot production was recorded among different environments. Grafted trees had higher primary growth, secondary axis growth, and dry matter gain than chip budded trees. Stem water potential measured in the second year after grafting was not affected by rootstocks or number of leaders. Measurements performed in New Zealand (Hawke’s Bay) during the second year after grafting revealed that both final length and growth rate of primary and secondary axis were related to the rootstock rather than to the training system. Dwarfing rootstocks reduced the number of long vegetative shoots and increased the proportion of less vigorous shoots.