Paulo Ricardo Dias de Oliveira

Erratum to: Reference genes for transcriptional analysis of flowering and fruit ripening stages in apple (Malus × domestica Borkh.)

Apple (Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple “Gala” cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple.

Resgate de macieiras antigas no Rio Grande do Sul: uma opção para a manutenção da diversidade genética

Nesse cenario, os programas de melhoramento conduzidos no Brasil e a introducao de novas cvs. sempre mantinham como meta final a obtencao de materiais geneticos semelhantes as caracteristicas da cv. Gala e/ou Fuji, procuravam materiais com maturacao no periodo entre as duas cvs. principais e tinham como alvo a resistencia a sarna da macieira (Venturia inaequalis) utilizando fontes de resistencia semelhantes aos utilizados em outros programas deste tipo no exterior. Nesta situacao, a preocupacao inicial estava centrada no impacto que teria a introducao de uma doenca quarentenaria, numa populacao geneticamente homogenea de macieiras, principalmente porque, nessa epoca, havia importacao macica de porta-enxertos e de plantas enxertadas de diversos paises. Felizmente isto nao ocorreu. Todavia, o surgimento da epidemia da mancha foliar da Gala (Colletotrichum gloeosporioides), iniciada a partir de 1990, e que causou perdas elevadas em toda a regiao produtora de macas no Pais, somada a constatacao da quebra de resistencia a sarna da macieira das cvs. que utilizavam o gene Vf na Europa, deu um alerta aos pesquisadores da Embrapa Uva e Vinho quanto a necessidade de buscar estrategias alternativas que garantissem maior variabilidade para a cultura. Contribuiram para reforcar este tipo de preocupacao a analise e o acompanhamento do sistema produtivo da maca, iniciado a partir de 1997 por uma equipe multidisciplinar que veio discutir e pesquisar os sistemas de Producao Integrada de Macas (PIM) e, posteriormente, a producao orgânica de macas. Nessa epoca, esta discussao ja ocorria nos eventos de Producao Integrada de Frutas na Europa e relatavam-se os primeiros resultados dos esforcos feitos para resgatar as fontes de variabilidade abandonadas ou perdidas durante o processo de melhoramento. Citavase, tambem, a organizacao de uma rede de pesquisa formada por diversos paises europeus a qual tinha a funcao de avaliar macieiras antigas das suas regioes que tivessem potencial para serem utilizadas como cvs. regionais e/ou como fontes de germoplasma para os programas de melhoramento e para estudos de resistencia duravel para as duas doencas mais importantes na Europa: a sarna e o oidio da macieira. As primeiras acoes destinadas ao resgate das cvs. antigas disponiveis no Sul do Brasil foram feitas pela Embrapa Uva e Vinho em 2002, percorrendo-se propriedades rurais e pomares caseiros nas cidades do norte e nordeste do Rio Grande do Sul. Esses locais foram identificados com auxilio das informacoes de produtores e de agentes da Extensao Rural (Emater/RS) e neles verificava-se a sobrevivencia de plantas de idade superior a 60 anos que ainda produziam mesmo sem

Análise da diversidade genética por AFLP e identificação de marcadores associados à resistência a doenças em videira

The grapevine cultivars A 1976, CG 87746, CNPUV 154-27, Crimson Seedless, Gota de Ouro, Italia, Seyve Villard 12327 and Seyve Villard 12375 were analyzed to establish the genetic relationship and to identify markers associated with resistance to downy mildew (Plasmopara viticola) and powdery mildew (Uncinula necator). The amplified fragment lenght polymorphism (AFLP) analysis comprise the steps of digestion, ligation, preamplification and amplification. The amplified fragments were separeted on a 7% denaturing polyacrylamide gel (7M urea) and silver stained. The similarity was estimated using Jaccards coefficient and the clustering was estimated by UPGMA. A similarity matrix was generated with a good fit for aggregation (r = 0,84). The clusters corresponded to origin and botany classification of the cultivars. Fifteen markers were associated with resistance, eight for downy mildew and seven for powdery mildew.

Structural genomics and transcriptional characterization of the Dormancy-Associated MADS-box genes during bud dormancy progression in apple

The molecular control of bud dormancy establishment and release is still not well understood, although some genes have already been demonstrated to play important roles in this process. The dormancy-associated MADS-box (DAM) genes were first identified in the peach EVERGROWING locus and are considered the main regulators of bud dormancy control. In this work, the apple (Malus × domestica Borkh.), a perennial plant adapted to temperate climates that displays cycles of growth and bud dormancy, was screened for the presence of DAM genes. The candidate genes retrieved were characterized in comparison to DAM genes from other species. Four of them (MdDAM1–4) are structurally very similar to the reported DAM genes. When apple genomic segments containing these candidates were compared to the peach EVERGROWING locus, a highly conserved noncoding region was detected inside their largest intron. Similar sequences were also identified inside introns of apricot and pear DAM genes. Organ expression patterns revealed that MdDAM1–4 are mainly expressed in dormant buds and seeds, with low transcript accumulation in vegetative structures. In addition, the MdDAM genes showed seasonally oscillating patterns of steady-state messenger RNA (mRNA) levels and were downregulated by artificial chilling. Motif analyses in the promoter and in the intronic conserved region of the MdDAM genes disclosed some clues to the regulation of the expression patterns observed. Possible roles for the conserved intronic sequence in dormancy regulation are discussed.

Identification of a Simple Sequence Repeat molecular-marker set for large-scale analyses of pear germplasm

Simple Sequence Repeats (SSR) are molecular markers suitable to assess the genetic variation of germplasm resources; however, large-scale SSR use requires protocol optimization. The present work aimed to identify SSR markers, developed for pear and other fruit species that are effective in characterizing pear germplasm collections and in demonstrating their use in providing support for genetic breeding programs. From a total of 62 SSR markers investigated, 23 yielding reproducible and polymorphic patterns were used to genotype a sample of 42 pear accessions of the Brazilian Pear Germplasm Bank (PGB). When compared to these 23 SSR markers, a subset of eleven markers, selected based on He, PIC and PId, was used to distinguish individual accessions and perform cluster analysis with similar efficacy. Genetic diversity analysis clustered the European, Japanese and Chinese accessions in distinct groups. This markers subset constitutes a valuable tool for several applications related to pear genetic resources management and breeding.

Uso prático de marcadores moleculares para seleção assistida no melhoramento de uvas de mesa apirênicas

The inheritance of seedlessness in grapevine is based on a complex genetic system, where the expression of three independently inherited recessive genes is controlled by a dominant regulator gene (sdI). In a previous study, Lahogue et al. (1998) identified a random amplified polymorphic DNA marker, tightly linked to the sdI gene and developed a codominant SCAR (Sequence Characterized Amplified Region) marker named SCC8, that allows the distinction of seeded and seedless plants in a segregating progeny. The aim of the present work was to evaluate the usefullness of the SCAR marker SCC8 for assisted selection of the seedlessness character in grape breeding. According to our results, the use of the SCC8 marker is economically viable and the consequences of its use in the grapevine breeding program at Embrapa Uva e Vinho are discussed.

ESTÁDIOS DE DESENVOLVIMENTO DE EMBRIÕES NA OBTENÇÃO DE PLANTAS EM CRUZAMENTOS ENTRE GENITORES APIRENOS DE VIDEIRA

It was investigated the effect of the growth stage of rescued embryos on plant obtention from crosses between seedless grape parents. Two methods for in vitro culture were compared. Method 1 (M1): seed traces were cultured 60 days in ER culture medium. Method 2 (M2): procedure of M1 followed by additional 30 days in MS culture medium. The stage of embryo growth was established at the end of this period, when the embryos were rescued and transfered to WP culture medium. Four classes of embryo stages were indentified: globular, heart, torpedo and undefined. The globular stage, the most frequently found, produced the largest quantity of recovered plants, though it had the lowest capacity of originating plants. The torpedo stage was the most efficient in producing plants. M2 promoted the largest amount of germinated embryos and recovered plants. As the embryo growth stage affects the plant obtention, the rescue of embryos in an advanced growth stage promotes a higher capacity of producing plants. Therefore the use of techniques that favour the rescuing of embryos in an advanced growth stage can increase the obtention of plants.

Evolutionary diversification of galactinol synthases in Rosaceae: adaptive roles of galactinol and raffinose during apple bud dormancy

Galactinol and raffinose act together to protect dormant buds against limited availability of winter water; the apple galactinol synthases MdGolS1 and MdGolS2 are responsible for their seasonal accumulation during dormancy.

HIBRIDAÇÕES CONTROLADAS INTER E INTRAESPECÍFICAS PARA O MELHORAMENTO GENÉTICO DE PORTA-ENXERTOS DE PEREIRA

The objective of the study was to characterize the pollen compatibility of pear with quince (interspecific crosses) and between species of pear. It was used: ‘Packham’s Triumph’ (female parental) and ‘William’s’ and ‘Clapp’s Favorite’, ‘Maca’ and ‘Portugal’ (male parental). It was determined fruit set (%) in the field at 40 days after crosses and the in vivo germination. It was obtained a higher fruit set in the crossing ‘Packham’s Triumph’ x ‘Clapp’s Favorite’. In interspecific crosses, the highest fruit set was obtained with ‘Packham’s Triumph’ x ‘Portugal’’. In in vivo germination, the developmental stage of the pollen tube showed significant variations between the combinations, but the pollen tube did not penetrate the embryo sac. As it was observed fructification in all crossing is possible the existence of compatibility among genotypes. The results indicated that the pollen donor cultivars of the species P. communis are compatible with ‘Packham’s Triumph’. Other studies should be conducted with the pollen of quince to confirm this compatibility.