Mariângela Cristofani-Yaly

Frequency and distribution of microsatellites from ESTs of citrus

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.

Global gene expression of Poncirus trifoliata , Citrus sunki and their hybrids under infection of Phytophthora parasitica

BackgroundGummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica.ResultsThree pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plantsConclusionsTwenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to P. parasitica in Citrus.

Development of genetic maps of the citrus varieties 'Murcott' tangor and 'Pera' sweet orange by using fluorescent AFLP markers.

The progeny of 87 BC1 hybrids of ‘Murcott’ tangor and ‘Pêra’ sweet orange, genotyped with fluorescent amplified fragment length polymorphism (fAFLP) markers, was used for the construction of genetic maps for both citrus varieties. Mapping strategies, considering the progeny as a result of backcrossing and cross-pollination, were exploited in Mapmaker 2.0 (LOD score ≥ 3.0 and 9 ≤ 0.40) and JoinMap 3.0 software (LOD score ≥ 3.0 and 9 ≤ 0.25), respectively. Genetic map distances (in cM) between the linked fAFLPs were estimated, in both packages, by the Kosambi’s function. Maps of both parents were constructed in Mapmaker with 121 of the 202 fAFLP markers showing 1:1 Mendelian segregation rates (’Murcott’ map: 65 fAFLPs, average distance between them 29.5 cM, divided into 9 linkage groups (LGs), total size 1651.47 cM; ’Pêra’ map: 55 fAFLPs, average distance between them 31.9 cM, divided into 5 LGs, total size 1596.2 cM). The second ’Murcott’ map, constructed through linkage analysis of 347 fAFLP markers with 3:1 or 1:1 segregation rates by using JoinMap, resulted in the linkage of 227 markers with an average distance of 4.25 cM among them, divided into 9 LGs of 845 cM. fAFLP loci showing distorted segregation and/or clustered were observed in different LGs of the maps generated by all the software. The use of the ’Murcott’ tangor and ’Pêra’ sweet orange genetic maps in research on identification of citrus QRLs (quantitative resistance loci) toXylella fastidiosa and QTLs (quantitative trait loci) related to the productivity and quality of the juice, respectively, is discussed.

Breeding, genetic and genomic of citrus for disease resistance

Embora a citricultura seja uma das mais importantes atividades economicas no Brasil, ela esta apoiada em um pequeno numero de variedades. Este fato tem contribuido para sua vulnerabilidade, especial no que ser refere a fatores fitossanitarios. A obtencao de um maior numero de variedades, seja para a industria seja para o mercado de fruta fresca, tem sido um dos principais objetivos dos programas de melhoramento. O melhoramento genetico de citros tem sido ampliado nas ultimas decadas em funcao da possibilidade de associacao de ferramentas da biotecnologia e metodos classicos de melhoramento. O uso de marcadores moleculares para a selecao precoce de plântulas zigoticas em cruzamentos controlados ampliou a possibilidade de selecao de maior numero de novas combinacoes e, em consequencia, o estabelecimento de grande numero de hibridos em experimentos de campo. Quanto mais rapido novas ferramentas sejam incorporadas ao programa, mais rapido e a possibilidade de obter novos genotipos que possam ser testados como variedades. Caracteristicas adequadas podem ser incorporadas ou mantidas, enquanto caracteristicas indesejaveis podem ser excluidas ou minimizadas nos novos genotipos. Copas e porta-enxertos nao podem ser considerados separadamente, e a compatibilidade de enxertia, qualidade de fruta e produtividade sao caracteristicas essenciais a serem avaliadas nos ultimos estagios do programa. Mapeamento de caracteristicas quantitativas (QTLs) tem propiciado os programas de melhoramento de varias especies lenhosas perenes, e em citros e possivel mapear varias caracteristicas com heranca quantitativa e qualitativa. A existencias de mapas de ligacao e QTLs, o desenvolvimento de bibliotecas de ESTs e BACs, assim como o sequenciamento completo do genoma de citros, ampliam a urgencia para exploracao de tais dados para ampliar os conhecimentos sobre genetica do grupo. O volume crescente de informacoes de genomas de varios outros organismos tem ampliado novas abordagens procurando a integracao entre melhoramento, genetica e genoma. Selecao assistida por genoma envolve mais que sequenciamento de genes ou genomas completos e tem se tornado uma importante ferramenta nos programas de melhoramento de especies perenes e anuais. Um grande volume de informacoes pode ser derivada da analise de genomas. O uso e beneficio de tais informacoes dependera da base genetica do programa de melhoramento.

Differential expression of genes identified from Poncirus trifoliata tissue inoculated with CTV through EST analysis and in silico hybridization

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.

In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

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.

Genome wide selection in Citrus breeding

Genome wide selection (GWS) is essential for the genetic improvement of perennial species such as Citrus because of its ability to increase gain per unit time and to enable the efficient selection of characteristics with low heritability. This study assessed GWS efficiency in a population of Citrus and compared it with selection based on phenotypic data. A total of 180 individual trees from a cross between Pera sweet orange (Citrus sinensis Osbeck) and Murcott tangor (Citrus sinensis Osbeck x Citrus reticulata Blanco) were evaluated for 10 characteristics related to fruit quality. The hybrids were genotyped using 5287 DArT_seqTM (diversity arrays technology) molecular markers and their effects on phenotypes were predicted using the random regression - best linear unbiased predictor (rr-BLUP) method. The predictive ability, prediction bias, and accuracy of GWS were estimated to verify its effectiveness for phenotype prediction. The proportion of genetic variance explained by the markers was also computed. The heritability of the traits, as determined by markers, was 16-28%. The predictive ability of these markers ranged from 0.53 to 0.64, and the regression coefficients between predicted and observed phenotypes were close to unity. Over 35% of the genetic variance was accounted for by the markers. Accuracy estimates with GWS were lower than those obtained by phenotypic analysis; however, GWS was superior in terms of genetic gain per unit time. Thus, GWS may be useful for Citrus breeding as it can predict phenotypes early and accurately, and reduce the length of the selection cycle. This study demonstrates the feasibility of genomic selection in Citrus.

FREMONT - IAC 543: TANGERINE WITH POTENTIAL FOR THE BRAZILIAN MARKET

Plantar novos tipos de tangerinas e 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 fisica e quimica da fruta e do suco (massa, cor da casca, rendimento de suco, acidez, teor de solidos soluveis e ratio) foi avaliada, seguido por testes sensoriais para ordenar a diferenca e preferencia em relacao as variedades clementina Nules e tangerina Ponkan. A tangerina Fremont produz frutos que atendem aos padroes exigidos pelo mercado consumidor brasileiro, com massa apropriada para a venda como fruta fresca, rendimento de suco adequado, maturacao precoce a meia-estacao. Alem disso, teve a preferencia, junto com a clementina Nules, quando comparado com a tangerina Ponkan, a principal variedade comercial no Brasil.

Screening of genomic libraries.

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs.

Sensory acceptance and postharvest quality of a new hybrid of citrus: TMxLP 16

Planting new types of sweet orange is an interesting alternative for the Brazilian citrus growers as this domestic market continues to grow. Thus, the aim of this study was to evaluate the acceptance and storage of a new hybrid between Murcott tangor and Pêra sweet orange, TMxLP 16. The treatments tested were: storage of fruits with and without wax, at room temperature (21 ± 2 °C and relative humidity (RH) = 60 ± 5%) and at cold temperature (10 ± 2 °C and RH = 90 ± 5%). Before storage samples were sensory tested for acceptance. Physicochemical qualities of the fruit and juice [mass, seeds, juice yield, acidity, soluble solids and ratio of soluble solids to titratable acidity (SS/TA)] were performed, before and after storage. The TMxLP 16 hybrid has an appropriate mass required for sale per fruit, a suitable juice yield, and early-mid season maturation, with high sensory acceptance, all characteristics which meet the standards required by the Brazilian consumer market. The results of this investigation showed that fruit stored at room temperature lost considerable weight. These results indicate a trend towards a loss of quality in fruits stored under ambient conditions and not coated with wax, which reaffirms the need for the use of refrigeration and waxy coverage for postharvest conservation of citrus fruits in natura.