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

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.

>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.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-09-14T18:22:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese Juliano Dutra Schmitz.pdf: 9582623 bytes, checksum: f579b0458e2ab6f4ae5579715de842ea (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-15T19:46:43Z (GMT) No. of bitstreams: 2 Tese Juliano Dutra Schmitz.pdf: 9582623 bytes, checksum: f579b0458e2ab6f4ae5579715de842ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-15T19:46:43Z (GMT). No. of bitstreams: 2 Tese Juliano Dutra Schmitz.pdf: 9582623 bytes, checksum: f579b0458e2ab6f4ae5579715de842ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-03 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / A macieira (Malus X domestica Borkh.) apresenta anomalias fisiológicas quando cultivada em regiões de inverno ameno, onde o frio hibernal é insuficiente para superação da dormência. Assim, na presente tese foram estudados três temas de pesquisa. No tema 1 estudou-se a distribuição e fenologia da brotação e crescimento inicial da ramificação primaveril; No tema 2 estudou-se a brotação primaveril através da determinação do status hídrico de gemas laterais e da condutância hidráulica do xilema. O tema 3 realizou-se a análise do comportamento da brotação primaveril de uma cultivar de baixo exigência em frio cultivada em inverno ameno. Para isso, foram montados dois dispositivos experimentais: Experimento 1: realizado em Montpellier/França, onde foram estudadas quatro cultivares de macieira, com diferentes requerimentos em frio (‘Condessa’, ‘Granny Smith’, ‘Royal Gala’ e ‘Starkrimson’), submetidas a dois regimes térmicos (inverno frio, condições naturais de Montpellier; e inverno ameno, temperatura controlada em casa-de-vegetação). Experimento 2: realizado com a cultivar de baixo requerimento em frio ‘Eva’ sob regime térmico hibernal ameno (condição natural de Capão do leão/Brasil). A partir do experimento 1, dois artigos foram redigidos . Conclui-se a partir dos resultados obtidos (artigos 1 e 2) que as temperaturas hibernais têm o principal efeito na distribuição da ramificação ao longo do eixo principal e no tempo para brotação; a presença de folha das plantas submetidas ao regime térmico de inverno ameno não afeta a distribu ição de ramos prolépticos vegetativos; a cultivar exerce efeito no crescimento da ramificação. Com relação ao status hídrico, conclui-se que durante o inverno (período de dormência) as gemas laterais permanecem hidraulicamente isoladas do eixo principal; assim como o potencial de brotação está relacionado a um efeito ramo inteiro (todo eixo principal) do que ao potencial individual de cada gema lateral. Através do experimento 2, um artigo foi elaborado, tendo por objetivo testar a hipótese que a posição em que a gema lateral está localizada sobre o eixo principal têm efeito na brotação primaveril, no conteúdo de água e tamanho das mesmas. Pode-se concluir deste estudo que uma semana antes a brotação, as gemas localizadas na zona distal possuem maior potencial de crescimento (maior frequência de brotação e menor tempo médio para brotação), além de apresentarem maior umidade ponderal e tamanho. / The apple tree (Malus X domestica Borkh.) presents morphological and physiological anomalies when grown in mild winter climates with insufficient winter chilling to overcome winter dormancy. Symptoms are typically delayed and erratic budburst, entailing desynchronized flowering and fruitset and poor agronomic performances. This thesis aimed at gaining more insights on the following issues. Firstly, what are the effects of winter temperatures on axillary burdburst and bud outgrowth, and what are the respective effects of winter temperatures and cultivar?, and secondly, is there a link between the temperature-dependent budburst and bud water status? Works were done in France and Brazil. In France, experiments were carried out in controlled conditions on four apple cultivars characterized by either high chilling (‘Granny Smith’, ‘Royal Gala’, ‘Starkrimson’) or low chilling (‘Condessa’) requirements and were submitted to outdoor-cold and greenhouse-mild winter temperatures. We showed that the actual shoot architecture and budburst resulted from an ordered sequence of events with a pivotal role of winter temperatures on the dormancy completion of individual lateral buds. Endogenous factors related to the cultivar branching pattern overtook the temperature effect on the lateral bud outgrowth. Furthermore, the delayed senescence and subsequent leaf persistence during winter, characterizing the apple tree in the mild winter temperature conditions, had only a weak effect on the topological distribution of budburst and lateral outgrowth. The analyses of bud water status were done on distal buds only, characterized by high budburst frequency in cold winter conditions. We showed that, from endodormancy to the pre-budburst stage, xylem conductance at the stem-to-bud junction did not show consistent changes across cultivars and winter temperature treatments. Bud water potential had negative values, between -4.35 and -2.24 MPa, depending on cultivars and winter temperature treatments. Moreover, whatever the cultivar, there were no significant trends across dates for the effects of winter temperatures on bud water potential and relative water content without a consistent relationship with actual spring budburst frequency. These results suggested that lateral buds were hydraulically isolated from the parent stem during winter until a few days before budburst. The other set of experiments was carried out in Brazil, under mild winter conditions, on the low chilling apple cultivar ‘Eva’. The objectives were to gain more insights on the effect of the position of the over-wintering lateral bud along the whole-parent shoot on bud size and water content. Results highlighted that distal buds were larger and had a higher water content than proximal buds with a strong increase of water content a week before spring budburst. It was concluded that the acrotonic pattern of budburst was mainly established during ecodormancy. As a whole, we showed that spring budburst seemed more related to a whole-shoot effect than to the water status of the individual bud during winter dormancy. Our study substantiated the importance of the whole shoot as an integrated morphological and physiological unit in driving budburst and further growth.