Resumo Poucas variedades de porta-enxertos são utilizadas para a limeira-ácida ‘Tahiti’, sendo que a diversificação se faz necessária, e o estudo das fases fenológicas de novas combinações copa/portaenxerto é importante para as estratégias de manejo do pomar. Objetivou-se avaliar a influência de diferentes porta-enxertos no somatório dos graus-dia (GD) e na fenologia das plantas, além da qualidade dos frutos de limeira-ácida ‘Tahiti IAC-5’, em Pindorama-SP. Foram marcados seis ramos em plantas enxertadas em 13 citrandarins (H), no limoeiro ‘Cravo’ (LC), no trifoliata ‘Flying Dragon’ (FD) e na tangerineira ‘Sunki’ (TS). A caracterização fenológica (antese à colheita) foi realizada utilizando-se de uma escala de notas. O acúmulo de GD (antese à colheita) foi determinado considerando-se a temperatura-base de 13 °C. Também foram avaliadas as características físicas (massa, altura, diâmetro e rendimento de suco) e químicas (acidez, sólidos solúveis totais e ratio) dos frutos. O delineamento experimental foi em blocos casualizados, com três repetições e uma planta por parcela. Os porta-enxertos estudados influenciaram no acúmulo de GD e na fenologia, identificando-se combinações precoces (H5, H73, H121, H135, H145, H152, FD - 1.558 a 1.676 GD e 135 a 146 dias) e mais tardias (H10, H14, H42, H47, H70, H150, H173, LC e TS - 1.694 a 1.829 GD e 148 a 157 dias) quanto à maturação de frutos. O ponto de colheita dos frutos de limeira-ácida ‘Tahiti’ enxertada no citrandarim H145 e no FD foi adiantado em torno de 20 dias em relação ao porta-enxerto padrão de LC. Os teores de sólidos solúveis totais e o tamanho dos frutos foram influenciados pelos porta-enxertos, entretanto, todas as combinações apresentaram frutos com características de qualidade dentro dos padrões comerciais.

Abstract Few rootstock varieties are used for ‘Tahiti’ acid lime; therefore, diversification is necessary and studying the phenological phases of new scion/rootstock combinations is important for orchard management strategies. The aim of this study was to evaluate the influence of different rootstocks on the sum of degree days (DD) and on the phenology of plants and also on the quality of ‘Tahiti’ acid lime fruits, in Pindorama, SP. Six branches were marked on plants grafted onto 13 citrandarins (H), ‘Rangpur’ lime (RL), ‘Flying Dragon’ trifoliate orange (FD) and ‘Sunki’ mandarin (SM). Phenological characterization (anthesis to harvest) was performed using a grading scale. DD accumulation (anthesis to harvest) was determined considering the base temperature of 13 °C. Physical (weight, height and diameter, juice yield) and chemical (acidity, total soluble solids and ratio) characteristics of fruits were also evaluated. The experimental design was in randomized blocks with three replicates and one plant per plot. The rootstocks under study influenced DD accumulation and phenology, with early (H5, H73, H121, H135, H145, H152, FD - 1,558 at 1,676 DD and 135 to 146 days) and later combinations (H10, H14, H42, H47, H70, H150, H173, RL and SM - 1,694 to 1,829 DD and 148 to 157 days) regarding fruit maturation. The harvest point of ‘Tahiti’ acid lime fruits grafted onto H145 citrandarin and FD was advanced about 20 days in relation to the standard rootstock, RL. Total soluble solids content and fruit size were influenced by rootstocks; however, all scion/rootstock combinations presented fruits with quality characteristics within commercial standards.

Abstract Citrus plants have been extremely affected by Huanglongbing (HLB) worldwide, causing economic losses. HLB disease causes disorders in citrus plants, leading to callose deposition in the phloem vessel sieve plates. Callose is synthesized by callose synthases, which are encoded by 12 genes (calS1– calS12)in Arabidopsis thaliana. We evaluated the expression of eight callose synthase genes from Citrus in hybrids between Citrus sunki and Poncirus trifoliata infected with HLB. The objective of this work was to identify possible tolerance loci combining the expression quantitative trait loci (eQTL) of different callose synthases and genetic Single-Nucleotide Polymorphism (SNP) maps of C. sunki and P. trifoliata. The expression data from all CscalS ranged widely among the hybrids. Furthermore, the data allowed the detection of 18 eQTL in the C. sunki map and 34 eQTL in the P. trifoliata map. In both maps, some eQTL for different CscalS were overlapped; thus, a single region could be associated with the regulation of more than one CscalS. The regions identified in this work can be interesting targets for future studies of Citrus breeding programs to manipulate callose synthesis during HLB infection.

RESUMO A mancha marrom de alternaria (MMA) é uma doença causada pelo fungo Alternaria alternata f. sp. citri, que causa lesões em frutos, folhas e ramos de citros. A ação do patógeno está diretamente relacionada à presença de receptores de toxina em genótipos suscetíveis. O objetivo deste estudo foi caracterizar uma população de híbridos de citros obtidos a partir de cruzamentos controlados entre a laranja Pêra de Abril e o híbrido de tangor Murcott x laranja Pêra (TM x LP 163) para resposta à MMA através da inoculação in vitro de esporos do fungo em folhas jovens destacadas. Um isolado do fungo foi obtido das lesões de frutos de tangor Murcott que exibiram sintomas de MMA. Duzentos e trinta e cinco híbridos foram avaliados, sendo que 70 (30%) apresentaram níveis diferentes de sintomas de doença em folhas destacadas após 72 horas da inoculação com o fungo e 165 (70%) foram assintomáticos. A frequência de segregação observada (165R:70S) e alta herdabilidade (h2g = 0,91) sugerem que poucos genes podem estar envolvidos no controle da herança da resistência à MMA em citros.

ABSTRACT Alternaria brown spot (ABS) disease is caused by the fungus of Alternaria alternata f. sp. citri, which causes injury in leaves, branches and fruits of citrus. The action of the pathogen is directly related to the presence of toxin receptors in susceptible genotypes. The objective of this study was to characterize a population of citrus hybrids obtained from controlled crosses between Pêra de Abril sweet orange and the hybrid of Murcott tangor x Pêra sweet orange (TM x LP 163) for response to ABS through the in vitro inoculation of fungal spores in young detached leaves. The fungus was isolated from the lesions of Murcott tangor fruits that exhibited ABS symptoms. Two hundred thirty-five hybrids were evaluated, and 70 (30%) showed different levels of disease symptoms on detached leaves after 72 hours of inoculation with the fungus, and 165 (70%) were asymptomatic. The frequency of segregation observed (165R:70S) and high level of heritability (h2g = 0.91) suggest that few genes may be involved in controlling the inheritance of ABS resistance in citrus.

RESUMO Plantar novos tipos de tangerinas é uma alternativa interessante para os citricultores brasileiros, devido ao crescente mercado interno. Assim, o objetivo deste estudo foi avaliar uma nova variedade de tangerina, a Fremont. A qualidade física e química da fruta e do suco (massa, cor da casca, rendimento de suco, acidez, teor de sólidos solúveis e ratio) foi avaliada, seguido por testes sensoriais para ordenar a diferença e preferência em relação às variedades clementina Nules e tangerina Ponkan. A tangerina Fremont produz frutos que atendem aos padrões exigidos pelo mercado consumidor brasileiro, com massa apropriada para a venda como fruta fresca, rendimento de suco adequado, maturação precoce a meia-estação. Além disso, teve a preferência, junto com a clementina Nules, quando comparado com a tangerina Ponkan, a principal variedade comercial no Brasil.

ABSTRACT Planting new types of tangerines is an interesting alternative for Brazilian citrus growers due to the increasing domestic market. Thus, the aim of this study was to evaluate a less exploited variety of tangerine, the Fremont. The physics and chemical quality of the fruit and juice (mass, peel color, juice yield, acidity, soluble solids and maturation index) was evaluated, followed by sensory testing for ranking of difference and preference to Nules clementine and Ponkan tangerine varieties. The Fremont tangerine produces fruits that meet the standards required by the Brazilian consumer market, with an appropriate mass for sale of the fresh fruit, a suitable juice yield, and early to mid-season maturation. Furthermore, et the preference, together with the Nules clementine, when compared to the Ponkan tangerine, wich is the main commercial variety in Brazil.

RESUMO A mancha marrom de alternária (MMA), causada por Alternaria alternata, que produz uma toxina específica ao hospedeiro (Host Specific Toxin – HST) é uma doença de grande importância nos pomares de tangerinas no mundo. A doença afeta tangerinas, alguns de seus híbridos interespecíficos e pomelos, sendo que no Brasil, as principais variedades plantadas, a tangerina Ponkan e o tangor Murcott, são altamente suscetíveis ao fungo, que também é saprofítico alternativo, se reproduz nos pomares, sendo seus esporos facilmente disseminados pelo vento. Para esse trabalho, foram selecionados híbridos de citros, obtidos por cruzamentos dirigidos, por apresentarem características agronômicas que os tornam potenciais novas variedades de laranjas e tangerinas, tanto para a citricultura industrial, quanto de mesa. Esses híbridos foram avaliados quanto à resposta à MMA por meio da inoculação com A. alternata in vitro e in vivo, uma etapa imprescindível para a liberação de novas variedades. Foram selecionados mais de trinta genótipos resistentes ou que apresentaram poucos sintomas da doença. A seleção de variedades resistentes à doença é de grande importância econômica, pois além de diminuir os custos de produção, aumentados sobremaneira com a utilização do controle químico, contribui para a diminuição de inóculo no campo.

ABSTRACT Alternaria brown spot (ABS), caused by Alternaria alternata, which produces a host-specific toxin (HST), is a very important disease in mandarin orchards worldwide. This disease affects mandarin, their hybrids and grapefruits. The main varieties cultivated in Brazil, Ponkan mandarin and Murcott tangor, are highly susceptible to the fungus, which is also an alternative saprophyte that reproduces in the orchards and has its spores easily spread by the wind. For this study, citrus hybrids obtained from directed crossing were selected for their agronomic traits, which make them potential new varieties of sweet orange and mandarins both for industrial citriculture and for fresh fruit market. These hybrids were evaluated for their response to ABS by means of A. alternata inoculation in vitro and in vivo, an indispensible step for the release of new varieties. More than 30 genotypes that were resistant or had few symptoms of the disease were selected. Selection of varieties resistant to the disease is of great economic importance, since it reduces the production costs, rather increased due to chemical control, and also contributes to the inoculum decrease in the field.

Desde 1997, o Centro APTA Citros Sylvio Moreira/IAC conduz um amplo programa de melhoramento genético de citros via cruzamentos dirigidos. Destes cruzamentos, alguns híbridos de tangerina com laranja vêm sendo selecionados para qualidade de fruta e potenciais novas variedades. Com isso, frutos de um híbrido entre tangor Murcott (TM) e laranja Pera (LP), denominado TMxLP 290, foram submetidos a análises físico-químicas e teste sensorial de aceitabilidade, a fim de avaliar a aceitação de tal variedade pelo mercado consumidor. Foram analisados os parâmetros físico-químicos: sólidos solúveis, acidez total titulável, ratio, rendimento de suco, índice de cor, número de sementes e massa do fruto. Aliado a essas análises, realizou-se o teste sensorial de aceitação com 50 provadores não treinados, que avaliaram a aceitabilidade do fruto e do suco do híbrido TMxLP 290, com o auxílio de uma escala hedônica de nove pontos. As amostras de suco e de fruto foram servidas em copos e pratos descartáveis, respectivamente, à temperatura ambiente e na presença de água potável para lavar o palato entre a degustação de uma amostra e outra. Tais avaliações permitiram constatar que as amostras processadas (suco) e in natura (fruto) apresentaram 84% e 81% de aprovação, respectivamente, evidenciando boa aceitação pelos provadores. Contudo, a intenção de compra por parte do consumidor foi excelente, sendo que 70% dos provadores comprariam o suco e 88% comprariam o fruto. Dessa forma, conclui-se que o híbrido TMxLP 290 é um produto com potencial para atender às expectativas do mercado consumidor.

Since 1997, the Centro APTA Citros Sylvio Moreira / IAC conducts a broad program of genetic improvement of citrus via controlled crosses. These crosses, some with hybrids of sweet orange and mandarin have been selected for fruit quality and potential new varieties. With this, the fruit of a hybrid between Murcott tangor (TM) and Pera sweet orange (LP), called TMxLP 290, underwent physical and chemical analysis and sensory acceptability test in order to evaluate the acceptance of such variety to the consumer market. We analyzed the physicochemical parameters of soluble solids, titratable acidity, ratio, juice yield, color index, number of seeds, fruit mass, but such tests alone are not sufficient to provide information about the product acceptance. Then allied to these analyzes, we carried out the sensory acceptance test with 50 untrained panelists, who evaluated the acceptability of the fruit and the juice of the hybrid TMxLP 290, with the aid of a nine-point hedonic scale. Samples of juice and fruit were served in disposable cups and plates, respectively, at room temperature and in the presence of potable water to rinse the palate between tasting samples and one another. Such evaluations have found that the samples processed (juice) and fresh (fruit) had 84% and 81% approval respectively, indicating good acceptance by the tasters. However, purchase intent among consumers was excellent, with 70% of the tasters would buy the juice and 88% would buy the fruit. Thus we conclude that the hybrid TMxLP 290 is a product with the potential to meet the expectations of the consumer market.

Embora a citricultura seja uma das mais importantes atividades econômicas no Brasil, ela está apoiada em um pequeno número de variedades. Este fato tem contribuído para sua vulnerabilidade, especial no que ser refere a fatores fitossanitários. A obtenção de um maior número de variedades, seja para a indústria seja para o mercado de fruta fresca, tem sido um dos principais objetivos dos programas de melhoramento. O melhoramento genético de citros tem sido ampliado nas últimas décadas em função da possibilidade de associação de ferramentas da biotecnologia e métodos clássicos de melhoramento. O uso de marcadores moleculares para a seleção precoce de plântulas zigóticas em cruzamentos controlados ampliou a possibilidade de seleção de maior número de novas combinações e, em consequência, o estabelecimento de grande número de híbridos em experimentos de campo. Quanto mais rápido novas ferramentas sejam incorporadas ao programa, mais rápido é a possibilidade de obter novos genótipos que possam ser testados como variedades. Características adequadas podem ser incorporadas ou mantidas, enquanto características indesejáveis podem ser excluídas ou minimizadas nos novos genótipos. Copas e porta-enxertos não podem ser considerados separadamente, e a compatibilidade de enxertia, qualidade de fruta e produtividade são características essenciais a serem avaliadas nos últimos estágios do programa. Mapeamento de características quantitativas (QTLs) tem propiciado os programas de melhoramento de várias espécies lenhosas perenes, e em citros é possível mapear várias características com herança quantitativa e qualitativa. A existências de mapas de ligação e QTLs, o desenvolvimento de bibliotecas de ESTs e BACs, assim como o sequenciamento completo do genoma de citros, ampliam a urgência para exploração de tais dados para ampliar os conhecimentos sobre genética do grupo. O volume crescente de informações de genomas de vários outros organismos tem ampliado novas abordagens procurando a integração entre melhoramento, genética e genoma. Seleção assistida por genoma envolve mais que sequenciamento de genes ou genomas completos e tem se tornado uma importante ferramenta nos programas de melhoramento de espécies perenes e anuais. Um grande volume de informações pode ser derivada da análise de genomas. O uso e benefício de tais informações dependerá da base genética do programa de melhoramento.

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

O objetivo deste trabalho foi obter plantas autotetraplóides de tangerina 'Ponkan', laranja 'Pêra-de-abril' e tangor 'Murcott', que serão usadas em cruzamentos com cultivares diplóides, visando à obtenção de indivíduos triplóides sem sementes. Utilizou-se o método de cultivo in vitro de segmentos de epicótilo em meio com colchicina (0,025, 0,05 e 0,1%), por diversos períodos (1, 2, 3, 7 e 14 dias), com subseqüente regeneração de brotações em meio sem a presença do alcalóide. As brotações foram microenxertadas in vitro e aclimatizadas em estufas. A determinação do nível de ploidia das plantas foi realizada por citometria de fluxo. A colchicina demonstrou ser tóxica aos explantes das três variedades, ocasionando redução significativa no número médio de brotações adventícias e aumento na porcentagem de explantes não-responsivos, em comparação com o controle. Entre as quatro plantas de laranja e uma de tangor obtidas, duas plantas de laranja e a de tangor, demonstraram ser autotetraplóides, apresentando folhas com maior espessura, arredondadas e coloração verde intensa. O método utilizado na duplicação cromossômica, com uso de colchicina, é eficiente em produzir plantas autotetraplóides de citros.

The objective of this work was to obtain autotetraploid plants of 'Murcott' tangor, 'Ponkan' mandarin and 'Pêra-de-abril' sweet orange to be used in crosses with diploid cultivars, aiming to produce triploid seedless hybrids. The methodology used was the in vitro culture of epicotyl segments in media containing different concentrations of colchicine (0.025, 0.05 and 0.1%), for several periods of time (1, 2, 3, 7 and 14 days), followed by the regeneration of adventitious shoots in culture media without the alkaloid. All shoots obtained were micro-grafted, acclimatized and transferred to a greenhouse. The evaluation of the ploidy level of the plants was performed by flow citometry technique. Colchicine was toxic to the explants of three varieties, and caused the reduction on the average number of shoots regenerated per explant as well as an increase in the percentage of non-responsive explants when comparing with the control plants. Among four plants of sweet orange obtained, two of them showed to be autotetraploid. The only plant of tangor obtained also showed to be autotetraploid. These plants presented thicker and roundisher leaves displaying deeper green color. The methodology used resulted efficient for obtaining tetraploid plants.

Citrus is the most important fruit crop in Brazil and Citrus tristeza virus (CTV) is considered one of the most important pathogens of citrus. Most citrus species and varieties are susceptible to CTV infection. However, Poncirus trifoliata, a close relative of citrus, is resistant to the virus. In order to better understand the responses of citrus plants to the infection of CTV, we constructed expressed sequence tag (EST) libraries with tissues collected from Poncirus trifoliata plants, inoculated or not with Citrus tristeza virus at 90 days after inoculation, grafted on Rangpur lime rootstocks. We generated 17,867 sequence tags from Poncirus trifoliata inoculated (8,926 reads) and not (8,941 reads) with a severe CTV isolate. A total of 2,782 TCs (Tentative Consensi sequences) were obtained using both cDNA libraries in a single clusterization procedure. By the in silico hybridization approach, 289 TCs were identified as differentially expressed in the two libraries. A total of 121 TCs were found to be overexpressed in plants infected with CTV and were grouped in 12 primary functional categories. The majority of them were associated with metabolism and defense response. Some others were related to lignin, ethylene biosynthesis and PR proteins. In general, the differentially expressed transcripts seem to be somehow involved in secondary plant response to CTV infection.

Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35% of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta- and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4% and 25.2%, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.