Mirian Perez Maluf

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

BackgroundQuantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.ResultsThe expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), β-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.ConclusionOur data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.

Genetic diversity and structure of Ethiopian, Yemen and Brazilian Coffea arabica L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.

Genetic diversity of cultivated Coffea arabica inbred lines assessed by RAPD, AFLP and SSR marker systems

One of the greatest problems in Coffea arabica breeding is identifying precisely any inbred line, based only on botanical and agronomical descriptors, because of the reduced genetic variability of the species, close pedigree origin, which results in small phenotypic variation. Recently, molecular markers have been used for plant germplasm characterization and identification in several commercial species. This work evaluates the reliability of three marker systems: RAPD, AFLP and SSR, to characterize the genetic variability of commercially-used Coffea inbred lines developed by the Instituto Agronomico (IAC), and their potential for cultivar identification. All methods identified polymorphisms among the cultivars. The genetic diversity recognized by the methods is very similar, although is very narrow. RAPD and SSR marker systems grouped more efficiently the evaluated cultivars according to parental origin. None of the methods allowed inbred line identification. Therefore for varietal protection, it would be necessary using a combination of botanical, agronomical and molecular markers descriptors for precise cultivar identification.

Identification of coffee WRKY transcription factor genes and expression profiling in resistance responses to pathogens

In plants, WRKY proteins are a group of transcription factors existing as a gene superfamily that play important roles in regulation of defense response pathways. To assess the diversity of this protein family in coffee (Coffea spp.), data mining methods were used on a set of around 200,000 coffee expressed sequence tags. A total of 53 different putative WRKY genes were obtained, but only 22 unigenes encoding a protein with a WRKY domain were identified, eight of which are supported by full-length cDNA sequences. Alignment of WRKY domain sequences of the coffee unigenes and 72 Arabidopsis thaliana WRKY genes showed that the 22 coffee WRKY members were distributed among the main A. thaliana WRKY subgroups and shared conserved peptide domains. To assess the involvement of WRKY genes in coffee defense response pathways, their expression was analyzed under biotic (nematode and rust fungus infection), hormonal (salicylic acid, methyl-jasmonate), and wounding treatments, leaf senescence, and fruit development. Five members of WRKY groups IId and III were regulated only by pathogens and hormone treatments. Although a significant correlation of WRKY genes expression after MeJA and rust treatments was observed, expression of coffee genes involved in JA biosynthesis and lipoxygenase (EC 1.13.11.12) activity assays did not support the involvement of JA in the early coffee resistance responses to the rust pathogen. The five WRKY transcription factor members identified might play important roles as regulators of pathogen resistance responses and could be useful for improving coffee tolerance to various biotic stresses.

Caracterização de cultivares de Coffea arabica mediante utilização de descritores mínimos

Cerca de 70% do cafe produzido e comercializado mundialmente e oriundo de Coffea arabica. A especie apresenta base genetica estreita sendo as cultivares bastante aparentadas e originarias em sua maioria das tradicionais cultivares Tipica e Bourbon. Este trabalho foi desenvolvido com o objetivo de identificar a eficiencia de descritores minimos na caracterizacao de cultivares de cafeeiros e como diferenciadores entre cultivares a serem submetidas ao processo de protecao de cultivares no Brasil. Foram avaliadas trinta e oito caracteristicas botânicas ou tecnologicas das plantas, folhas, flores, frutos, sementes, assim como tres caracteristicas agronomicas. Utilizaram-se vinte e nove cultivares de cafeeiros selecionadas pelo Instituto Agronomico, sendo avaliadas trinta plantas de cada cultivar. Os resultados evidenciaram que apenas com a utilizacao das caracteristicas porte, cor do fruto, resistencia ao agente da ferrugem, Hemileia vastatrix e ciclo de maturacao e possivel obter uma discriminacao eficiente dos diferentes grupos de cultivares avaliadas. A cor das folhas jovens e o diâmetro da copa revelaram-se importantes descritores na discriminacao de cultivares do grupo Mundo Novo. Nao foi possivel, porem, identificar descritores eficientes na discriminacao das cultivares dos grupos Catuai Vermelho, Catuai Amarelo e Icatu Vermelho.

Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner–toward the identification of candidate genes for marker assisted-selection

BackgroundA successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach.ResultsThe microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars.ConclusionsOur results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

CaPrx, a Coffea arabica gene encoding a putative class III peroxidase induced by root-knot nematode infection.

Class III peroxidases (Prxs) are enzymes involved in a multitude of physiological and stress-related processes in plants. Here, we report on the characterization of a putative peroxidase-encoding gene from Coffea arabica (CaPrx) that is expressed in early stages of root-knot nematode (RKN) infection. CaPrx showed enhanced expression in coffee roots inoculated with RKN (at 12 h post-inoculation), but no significant difference in expression was observed between susceptible and resistant plants. Assays using transgenic tobacco plants harboring a promoter-β-glucuronidase (GUS) fusion revealed that the CaPrx promoter was exclusively active in the galls induced by RKN. In cross sections of galls, GUS staining was predominantly localized in giant cells. Up-regulation of GUS expression in roots of transgenic plants following RKN inoculation was observed within 16 h. Moreover, no increase in GUS expression after treatment with jasmonic acid was detected. Altogether, these results point to a putative role of this peroxidase in the general coffee response to RKN infection.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

n o 1.481, Caixa Postal 28, CEP 13012‑970 Campinas, SP, Brazil. E‑mail: mirian.maluf@embrapa.br Abstract - The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi‑quantitative and quantitative RT‑PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α‑galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish‑green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.

Genomic Tools for the Development of Engineered Meloidogyne-Resistant Coffee Cultivars

This chapter discuss major issues related to the development of transgenic Meloidogyne-resistant coffee cultivars. Initially, the relevance of engineering cultivars in vitro is highlighted in relation to the limitations found in traditional coffee-breeding programs. Given that potential approaches to develop transgenic cultivars are transferring genes that confer traditional plant resistance or anti-nematode results, this chapter discuss the selection process of genes candidates for transference, including resistance and general-defense genes, and proteinase inhibitors. The use of gene-silencing as an approach to modify gene expression during plant-nematode interaction, resulting in plant resistance, is also discussed. A review is presented on recent progresses on the functional characterization of coffee genes and nematode-responsive promoters. Finally, this chapter presents successful examples of engineered nematode-resistant cultivars in other plant species, which support the feasibility of these strategies for the development of transgenic coffee cultivars.