RNA-Seq analysis and de novo transcriptome assembly of Coffea arabica and Coffea eugenioides : P0068

Coffee is one of the most important agricultural commodites in the world. Coffea arabica, an allotetraploid from a recent hybridization of two diploid species (C.canephora and C.eugenioides) is responsible for 70% of world commercial production. Recent studies have been done to analyse transcriptome data of Coffea spp. in order to improve our knowledge in genetics and gene expression of those species. However, there is few data on coffee fruit transcriptome. In this work, RNA-Seq of two organs (whole fruit - cherry stage and mature leaves) from allotetraploid C. arabica cv. Mundo Novo and its diploid ancestor C.eugenioides, were done using Illumina HiSeq 2000 (100 bp single-end sequences). We report the generation of near 10 million reads for C.arabica (4,747,049 of fruits and 6,156,750 reads of leaves) and 7,5 million reads for C.eugenioides (3,688,364 of fruits and 3,835,373 reads of leaves). De novo assembly and digital gene expression analyses was performed to generate a collection of 35,462 contigs from C.arabica cv. Mundo Novo (average length of 691 bp) and 36,935 contigs of C.eugenioides (average length of 701 bp). Funnctional annotation were performed by sequence comparison with public databases (NCBI-nr, Swiss-prot and PlantCyc). Our results showed the presence of prevalent genes and species-specific either in C.arabica or C.eugenioides that could explain particular characteristics of these two species. Characterization of Coffea spp. transcriptome provides an effective tool for better understanding for differential gene expression in Coffea spp., providing important clues of C.arabica gene evolution, as well as valuable information for coffee breeding. (Résumé d'auteur

Identification of Colletotrichum species associated with anthracnose disease of coffee in Vietnam

Colletotrichum gloeosporioides, C. acutatum, C. capsici and C. boninense associated with anthracnose disease on coffee (Coffea spp.) in Vietnam were identified based on morphology and DNA analysis. Phylogenetic analysis of DNA sequences from the internal transcribed spacer region of nuclear rDNA and a portion of mitochondrial small subunit rRNA were concordant and allowed good separation of the taxa. We found several Colletotrichum isolates of unknown species and their taxonomic position remains unresolved. The majority of Vietnamese isolates belonged to C. gloeosporioides and they grouped together with the coffee berry disease (CBD) fungus, C. kahawae. However, C. kahawae could be distinguished from the Vietnamese C. gloeosporioides isolates based on ammonium tartrate utilization, growth rate and pathogenictity. C. gloeosporioides isolates were more pathogenic on detached green berries than isolates of the other species, i.e. C. acutatum, C capsici and C. boninense. Some of the C. gloeosporioides isolates produced slightly sunken lesion on green berries resembling CBD symptoms but it did not destroy the bean. We did not find any evidence of the presence of C. kahawae in Vietnam

Analysis of nucleotide diversity in Coffea spp. (W153)

Marker-assisted selection becomes a reality for many crops; in perennial crops, the utilization of molecular markers in breeding programs can speed up genotype selection. However, the most important commercial coffee species - allotetraploid C. arabica - has a restrict number of available polymorphic markers, which is a consequence of the narrow genetic basis and low molecular variability among cultivars. In order to study the nucleotide diversity in C. arabica, as well in other diploid Coffea relatives, we sequenced PCR amplified fragments of nine genes in 20 Coffea genotypes: twelve C. arabica, including eight wild genotypes and four commercial cultivars; and eight C. canephora genotypes. Genotypes of C. eugenioides, C. racemosa and Psylanthus bengalensis were also included in this analysis. From a total of 9 Kb analyzed, we found 573 polymorphisms: 500 SNPs; 39 INDELs and 34 SSRs. In C. canephora genotypes, we detected 188 polymorphisms (frequency of 2.09/100bp). For C. arabica we obtained similar results: 144 polymorphism (frequency of 2.13/100bp). Most of the polymorphism found in C. arabica only reflected the differences between ancestral homeologs, and they were monomorphic among different genotypes. However, 19 % of these polymorphisms (27 SNPs) were interespecific for C. arabica, and 13 of them were fixed among genotypes. The strategy of this work reflects the importance in using a more diverse panel of genotypes in order to identify SNPs in C. arabica, pointing out that the exploitation of wild germplasm will be an important source of genetic variability. (Résumé d'auteur

Identification and characterization of genes involved in ABA perception and signal transduction in Coffea spp : S02P13

The Coffea genus represents a major agricultural commodity in world trade. Nowadays, drought and elevated temperatures are the major climatic limitations for coffee production. These variations also influence biochemical composition of beans, affecting directly the final cup quality. There is genetic variability within the Coffea genus that could be used to increase drought tolerance and generate coffee varieties better adapted to climatic variations. Abscisic acid (ABA) is a vital plant hormone acting as central regulator that protects plants against abiotic stresses such as drought. Recently, novel intracellular ABA receptors (PYL/RCARs) involved in ABA sensing and signaling have been identified in several species. A mechanism of ABA transduction has been proposed, involving PYR/PYL/RCARs receptors interacting with PP2Cs phosphatases and SnRK2 protein kinases. The goal of this study was to identify and characterize ortholog genes of this tripartite system in Coffea sp. For this purpose, protein sequences from Arabidopsis, citrus, rice, grape, and tomato were chosen as query to search ortholog genes in the coffee-sequence database. Using 51 PYR/PYL/RCAR sequences from those plant species, it was possible to identify 9 sequences for ABA receptors in coffee. Likewise, the 40 and 29 sequences query resulted in 6 and 9 similar sequences of PP2Cs and SnRK2 specific to ABA in Coffea sp. The 24 genes isolated, that belong to the tripartite system of the coffee's ABA pathway, showed in silico differential expression in tissues as leaves, seeds, roots and floral organs. Polymorphisms were found among the orthologs and homeologs genes. All analyses allowed the identification in C. arabica genome of sequences variations between the two ancestral diploid sub-genomes, C. canephora (CaCc) and C. eugenioides (CaCe). Further analyses will predict the functional effect of these polymorphisms in protein structure in different coffee species. All these evidences will also help us to identify the genetic determinism of drought tolerance essential to obtain molecular markers that could be used in coffeebreeding programs. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

A new root-knot nematode, Meloidogyne moensi n. sp. (Nematoda : Meloidogynidae), parasitizing Robusta coffee from Western Highlands, Vietnam

A new root-knot nematode, parasitizing Robusta coffee in Dak Lak Province, Western Highlands of Vietnam, is described as Meloidogyne moensi n. sp. Morphological and molecular analyses demonstrated that this species differs clearly from other previously described root-knot nematodes. Morphologically, the new species is characterized by a swollen body of females with a small posterior protuberance that elongated from ovoid to saccate; perineal patterns with smooth striae, continuous and low dorsal arch; lateral lines marked as a faint space or linear depression at junction of the dorsal and ventral striate; distinct phasmids; perivulval region free of striae; visible and wide tail terminus surrounding by concentric circles of striae; medial lips of females in dumbbell-shaped and slightly raised above lateral lips; female stylet is normally straight with posteriorly sloping stylet knobs; lip region of second stage juvenile (J2) is not annulated; medial lips and labial disc of J2 formed dumbbell shape; lateral lips are large and triangular; tail of J2 is conoid with rounded unstriated tail tip; distinct phasmids and hyaline; dilated rectum. Meloidogyne moensi n. sp. is most similar to M. africana, M. ottersoni by prominent posterior protuberance. Results of molecular analysis of rDNA sequences including the D2-D3 expansion regions of 28S rDNA, COI, and partial COII/16S rRNA of mitochondrial DNA support for the new species status

Physiological and molecular responses of diploid and tetraploid Carrizo Citrange under water Stress : S02P14

In citrus, the use rootstock promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. 'Carrizo' citrange (Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf), is one of the most popular rootstock in the Mediterranean basin. It is sensitive to drought and salt stress but confers tolerance to Tristeza virus, and promotes very good fruit quality. Previous studies have shown that doubled diploid (4x) 'Rangpur' lime (Citrus limonia, Osbeck) seedlings are more tolerant to water deficit than their respective diploid (2x). In the present work, we have characterized the water deficit tolerance in 2x and 4x 'Carrizo' citrange seedlings. Water deficit was applied for 35 days, followed by irrigation. Several physiological parameters were measured periodically during the experiment and samples were collected to investigate i) the activity of enzymes involved in detoxification processes, ii) the expression analysis of candidate genes involved in ABA biosynthesis, as well as iii) ABA and H2O2 production. Doubled diploid 'Carrizo' citrange seedlings were showed to be more drought tolerant than 2x. Water deficit caused a greater reduction in photosynthetic rates and stomatal conductance in 2x compared to 4x. Also higher ABA and H2O2 production were induced in 2x when compared to 4x. The better tolerance of 4x seedlings is discussed to the light of candidate genes expression analysis and activities of enzymes of detoxification. Work supported by CNPq and CAPES. (Texte intégral

Pest risk analysis for Bactrocera invadens : Guidelines on Pest Risk Analysis

Stripping related moisture damage has been recognized as one of the major pavement distresses since the early 1990s. The main objective of this study is to establish an effective test protocol to quantify moisture susceptibility of asphalt pavements. To this end, selective test methods (Texas Boiling test, Tensile Strength Ratio, Retained Stability, and Hamburg Wheel Test), and procedures based on surface chemistries and molecular-level mechanistic properties have been investigated in this study. Firstly, a comprehensive list of literature related to moisture damage in asphalts was reviewed. Based on the literature review, a detailed project plan and test matrix were developed. Binder samples originated from two different crude sources were collected. The moisture resistance related tests such as static contact angle measurements and Texas Boiling tests were conducted. Besides, asphalt binders’ nanomechanical properties using an Atomic Force Microscopy (AFM) and surface chemistries using a static contact were evaluated in the laboratory. Based on limited test data and analysis, it is concluded that there does not exist any single test method that all agencies are comfortable and equipped to follow in their daily work as each technique has some merits and demerits. However, the Texas Boiling test is found to be the simplest method that requires minimal time and resources. On the other hand, surface chemistry and atomic force microscope-based techniques are becoming popular among researchers and pavement professionals. Findings of this study are expected to help ARDOT in selecting an appropriate moisture resistance test method that is simple, reliable, and easy to implement in their routine work

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