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.

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.

Philosophiae Doctor - PhD / The domesticated apple (Malus x domestica Borkh.) has been distributed into diverse climatic conditions worldwide for commercial production of fruit. Apple trees need exposure to cold temperatures, referred to as chill unit (CU) accumulation during winter, in order for budbreak to occur promptly and uniformly after winter. In warmer production areas the application of dormancy breaking chemicals has enabled successful production of high chilling requiring apple cultivars in suboptimal environmental conditions. In the Western Cape region of South Africa it is common orchard practice to apply dormancy breaking chemicals after winter in order to stimulate vegetative growth. If this is not done prolonged dormancy symptoms (PDS) are experienced which include extended rest, less synchronised breaking of buds and reduced branching. An increasing awareness of both global warming and the negative effects associated with the use of chemical sprays (for both pest and disease resistance and growth regulation) has resulted in the need to breed cultivars better adapted to current and future environmental conditions. The breeding of new cultivars using conventional breeding methods is a time consuming process, especially in perennial tree species with a long juvenile phase such as apple. The implementation of marker-assistedbreeding (MAB) and selection (MAS) will enable the selection of favourable genotypes at a very early seedling stage. Although markers linked to genes involved in disease resistance for a variety of known apple pathogens have been identified and are already in use in breeding programs, the genetic determinants of dormancy related characteristics residing within the bud itself (endodormancy) are poorly understood. This hampers the genetic improvement of such characters. Although this study focused on time of initial vegetative budbreak IVB, there are various other characteristics that can be associated with dormancy, such as position and number of budbreak and budbreak duration. The unravelling of the genetic basis of complex traits such as dormancy, can be done through the construction of a genetic linkage map followed by the identification of genomic regions, known as quantitative trait loci (QTL), that can be ssociated with the trait of interest. This study involved the construction of genetic linkage maps for two mapping pedigrees where the low chilling requiring cultivar ‘Anna’ was used as common male parent in crosses with the higher chilling requiring ‘Golden Delicious’ and ‘Sharpe’s Early’. A third mapping pedigree, with ‘Golden Delicious’ as female parent and ‘Prima’ as male parent, was also included. Maps consisted of transferable SSR markers only, facilitating the alignment with the proposed apple reference map (Silfverberg-Dilworth et al., 2006) and adherence to the common LG numbering system now being used for apple genetic linkage maps (Maliepaard et al., 1998). A number of newly developed EST-SSR markers are reported, some of which are candidates for filling large gaps between adjacent SSR markers on the apple reference map. An interactive database was developed to successfully manage the large amount of data generated during this investigation. A selective mapping, or bin mapping strategy (Vision et al., 2000) was developed for two of the three mapping populations in order to facilitate the incorporation and positioning of newly developed markers onto existing genetic linkage maps. This involves the screening of new markers on a small subset of the population, drastically reducing the cost and time involved. Genetic linkage maps constructed allowed for the detection of 18 putative QTLs affecting the time of IVB. Four of these QTLs co-localize with previously identified QTLs. A QTL identified on LG 8 confirms a previously identified QTL (Segura et al., 2007), while one of the QTLs identified on LG 9 might coincide with a QTL identified on the corresponding LG 3 of the genetic linkage map constructed by Conner et al. (1998). Two QTLs identified on LG 10 might coincide with markers found to co-segregate with time of budbreak in an earlier study conducted by Lawson et al. (1995). An additional 14 QTLs involved in time of IVB have been identified. We proposed the testing of four markers in a validation study conducted on a second mapping pedigree derived from a cross between ‘Anna’ and ‘Golden Delicious’. These markers are CH04a12, CH04c06y, CH01h01 and A267. Not only do these markers show significant levels of association with the time of IVB, but segregation of parental alleles from the cultivar ‘Anna’ for two of these markers, CH04c06y and CH01h01, were found to be associated with the time of IVB in different genetic backgrounds. The identification of markers closely associated with time of IVB will facilitate the implementation of MAS in breeding programs in order to breed cultivars that are better adapted to local climatic conditions.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-05T15:03:53Z No. of bitstreams: 1 PGCS16MA132.pdf: 848698 bytes, checksum: 5897528fb935abd45691ddc9a7e601c1 (MD5) / Made available in DSpace on 2018-03-05T15:03:53Z (GMT). No. of bitstreams: 1 PGCS16MA132.pdf: 848698 bytes, checksum: 5897528fb935abd45691ddc9a7e601c1 (MD5) Previous issue date: 2016-01-19 / Capes / 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

<p>Циљ савременог гајења јабуке је да се у засаду велике густине садње, заснованом са садницама високог квалитета које рађају већ у години садње, и савременим сортиментом који укључује и сорте склоне алтернативној родности, успоставе редовни и високи приноси који ће се кретати од 50-70 t/ha и добар квалитет плодова. Мера која је од највећег значаја за регулисање родности јесте хемијско проређивање цветова и плодова јабуке.<br />Циљ обављених истраживања је да се испита утицај хемијског проређивања цветова и плодова на заметање, квалитет плодова и родност пупољака сорти јабуке као и да се испита интеракција између минералне исхране азотом и хемијског проређивања. Резултати истраживања били би смернице за хемијско проређивање плодова у агроеколошким условима Србије, са тежњом да у скорој будућности постану део законске регулативе и припадајућих правилника за интегралну производњу, као и развој побољшаних формулација препарата за проређивање.<br />На основу испитивања проређивања цветова може се закључити да сорте јабуке бребурн и златни делишес различито реагују на примену ATS и KTS. Примена 3% ATS и 1,5% KTS код сорте бребурн изазива јаке ожеготине на листовима које могу бити узрок смањеној маси плода и слабијем образовању родних пупољака. Код сорте златни делишес ATS концентрације 3% не испољава негативно дејство на крупноћу плодова и родност пупољака, али са друге стране није ефикаснији од ATS нижих концентрација, док KTS има знатно слабије дејство.<br />Ефикасност препарата на бази NAA у смањењу заметања и приноса сорте јабуке бребурн није зависила од примењене концентрације, док је маса плода у свим варијантама била веома велика, те се за практичну примену могу препоручити ниже концентрације, до 10 &mu;l/l NAA. Сви третмани са NAA у распону концентрација од 8 до 12 &mu;l/l били су једнако ефикасни у редуковању заметања и приноса плодова, док је BA испољио слабију ефикасност у проређивању сорте јабуке камспур. Родност пупољака у третманима проређивања плодова сорти бребурн и камспур није сразмерна повећању концентрације препарата, већ у великој мери зависи од године испитивања.<br />Значајна интеракција је утврђена између фактора ђубрење и проређивање за параметре заметања плодова. Интеракција ова два фактора огледа се у томе да се у режиму интензивног ђубрења азотним ђубривима концентрација препарата на бази NAA и BA треба повећати како би се постигао задовољавајући ниво проређивања.<br />Додавање нафтенских киселина нафтилсирћетној киселини и бензиладенину значајно доприноси повећању просечне масе плода сорте јабуке златни делишес у односу на формулације без нафтенских киселина, као последица биолошке активности нафтенских киселина у смеши. Запажено је повећање чврстине плода у варијантама где је примењена формулација BA са нафтенским киселинама у односу на формулације без<br />нафтенских киселина.<br />Датум прихватања теме од стране Сената:<br />ДП<br />30.5.2013.<br />Чланови комисије:</p> / <p>Cilj savremenog gajenja jabuke je da se u zasadu velike gustine sadnje, zasnovanom sa sadnicama visokog kvaliteta koje rađaju već u godini sadnje, i savremenim sortimentom koji uključuje i sorte sklone alternativnoj rodnosti, uspostave redovni i visoki prinosi koji će se kretati od 50-70 t/ha i dobar kvalitet plodova. Mera koja je od najvećeg značaja za regulisanje rodnosti jeste hemijsko proređivanje cvetova i plodova jabuke.<br />Cilj obavljenih istraživanja je da se ispita uticaj hemijskog proređivanja cvetova i plodova na zametanje, kvalitet plodova i rodnost pupoljaka sorti jabuke kao i da se ispita interakcija između mineralne ishrane azotom i hemijskog proređivanja. Rezultati istraživanja bili bi smernice za hemijsko proređivanje plodova u agroekološkim uslovima Srbije, sa težnjom da u skoroj budućnosti postanu deo zakonske regulative i pripadajućih pravilnika za integralnu proizvodnju, kao i razvoj poboljšanih formulacija preparata za proređivanje.<br />Na osnovu ispitivanja proređivanja cvetova može se zaključiti da sorte jabuke breburn i zlatni delišes različito reaguju na primenu ATS i KTS. Primena 3% ATS i 1,5% KTS kod sorte breburn izaziva jake ožegotine na listovima koje mogu biti uzrok smanjenoj masi ploda i slabijem obrazovanju rodnih pupoljaka. Kod sorte zlatni delišes ATS koncentracije 3% ne ispoljava negativno dejstvo na krupnoću plodova i rodnost pupoljaka, ali sa druge strane nije efikasniji od ATS nižih koncentracija, dok KTS ima znatno slabije dejstvo.<br />Efikasnost preparata na bazi NAA u smanjenju zametanja i prinosa sorte jabuke breburn nije zavisila od primenjene koncentracije, dok je masa ploda u svim varijantama bila veoma velika, te se za praktičnu primenu mogu preporučiti niže koncentracije, do 10 &mu;l/l NAA. Svi tretmani sa NAA u rasponu koncentracija od 8 do 12 &mu;l/l bili su jednako efikasni u redukovanju zametanja i prinosa plodova, dok je BA ispoljio slabiju efikasnost u proređivanju sorte jabuke kamspur. Rodnost pupoljaka u tretmanima proređivanja plodova sorti breburn i kamspur nije srazmerna povećanju koncentracije preparata, već u velikoj meri zavisi od godine ispitivanja.<br />Značajna interakcija je utvrđena između faktora đubrenje i proređivanje za parametre zametanja plodova. Interakcija ova dva faktora ogleda se u tome da se u režimu intenzivnog đubrenja azotnim đubrivima koncentracija preparata na bazi NAA i BA treba povećati kako bi se postigao zadovoljavajući nivo proređivanja.<br />Dodavanje naftenskih kiselina naftilsirćetnoj kiselini i benziladeninu značajno doprinosi povećanju prosečne mase ploda sorte jabuke zlatni delišes u odnosu na formulacije bez naftenskih kiselina, kao posledica biološke aktivnosti naftenskih kiselina u smeši. Zapaženo je povećanje čvrstine ploda u varijantama gde je primenjena formulacija BA sa naftenskim kiselinama u odnosu na formulacije bez<br />naftenskih kiselina.<br />Datum prihvatanja teme od strane Senata:<br />DP<br />30.5.2013.<br />Članovi komisije:</p> / <p>The goal of modern apple growing in high density orchards, established with high-quality nursery trees which bear fruits in the year of planting, often including varieties prone to biennial bearing is to achieve high and regular yields that range from 50 to 70 t/ha with good fruit quality. Chemical thinning of flowers and fruits is a technical practice which is of great importance for crop regulation.<br />The aim of the research was to investigate the effect of chemical thinning of apple flowers and fruits on fruit set, fruit quality and bearing potential of apple buds as well as to examine the interaction between the nitrogen supply and chemical thinning treatments. The research will result with guidelines for the chemical thinning in agroecological conditions of Serbia, aiming to become a part of the legislation and associated regulations for integrated production in the near future. Testing of improved formulations of thinning chemicals will be included into expe rimental plan.<br />Considering the results derived from flower thinning experiments, it could be concluded that cv. Braeburn and Golden Delicious react differently to the application of ATS and KTS. Application of 3% ATS and 1.5% of KTS in cv Braeburn causes severe leaf burns that may be causing reduced fruit weight and a decrease in bearing potential. In cv Golden Delicious ATS applied at the concentration of 3% does not exhibit a negative effect on fruit size and bearing potential, but at the other side is not more efficient than ATS at lower concentrations, whereas KTS has a much weaker thinning effect.<br />The efficacy of NAA in reducing fruit set and yield of apple variety Braeburn is not dependent on the concentration applied, while fruit size was very large within the range of NAA concentrations. Therefore, for the practical application, concentrations lower than 10 ml/l NAA would be recommended. All treatments with NAA at concentrations ranging from 8 to 12 ml/l were equally effective in reducing fruit set and yield, while the BA exhibited decreased efficiency in thinning apple variety Camspur. Fertility of buds in Braeburn and Camspur varieties was not proportional to the increase in concentration of chemical, but to a large extent depends on the conditions of the experimental year.<br />An interaction between nitrogen supply and chemical thinning significantly affected fruit set in a way that concentration of NAA and BA for fruit thinning should be increased in order to achieve a satisfactory level of thinning at a higher level of nitrogen supply.<br />Adding of naphthenic acids to NAA and BA significantly contributed to the increase of average fruit weight of apple variety Golden Delicious compared to same chemical formulation without naphthenic acids, as a result of the biological activity of naphthenic acids in the mixture. An increase of fruit firmness was detected in thinning treatments where naphthenic acids were added to BA compared BA applied alone.</p>

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.

<p>&nbsp / </p> <p align="left">The domesticated apple (<i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">x </font><i><font face="TimesNewRomanPS-ItalicMT">domestica </font><font face="TimesNewRomanPSMT">Borkh.), belonging to the </font><i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">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.&nbsp / 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.</font></i></i></i></p>

<p>&nbsp / </p> <p align="left">The domesticated apple (<i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">x </font><i><font face="TimesNewRomanPS-ItalicMT">domestica </font><font face="TimesNewRomanPSMT">Borkh.), belonging to the </font><i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">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.&nbsp / 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.</font></i></i></i></p>

Philosophiae Doctor - PhD / The domesticated apple (Malus x domestica Borkh), belonging to the Malusgenus 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 / South Africa