Evaluation of Somatic Embryogenesis Ability in Robusta Coffee (Coffea Canephora Pierre)

Embriogenesis somatik diharapkan sebagai metode perbanyakan tanaman yang sangat efektif pada kopi. Evaluasi dua jenis proses embriogenesis somatik, yaitu proses langsung dan tidak langsung akan bermanfaat untuk menggambarkan kemampuan proliferasi sel. Penelitian untuk mengevaluasi embriogenesis somatik kopi Robusta (Coffea canephora) yang mempunyai tingkat keragaman genetik tinggi telah dilakukan di Nestlé R&D Centre Tours, Perancis. Bahan tanam menggunakan kopi Robusta koleksi Nestle Perancis dan tiga klon koleksi Pusat Penelitian Kopi dan Kakao Indonesia (Puslitkoka). Tiga aspek, yaitu proses embriogenesis, keragaman embriogenesis dan kemantapan embriogenesis dievaluasi dalam penelitian ini. Hasil penelitian menunjukkan bahwa baik embriogenesis somatik langsung maupun tidak langsung dapat diamati. Penelitian ini menunjukkan bahwa kedua proses embriogenesis somatik tersebut merupakan dua mekanisme yang berbeda. Dalam penelitian ini ditunjukkan bahwa kemampuan embriognesis somatik tergantung pada genotipe, baik antar maupun di dalam kelompok genetik kopi Robusta, yaitu Congolese,Guinean dan Conillon. Lebih lanjut diketahui bahwa kedua proses embriogenesis somatik tersebut stabil terhadap indukan sebagai sumber eksplan. Kemampuan embriogenesis somatik tidak langsung ketiga klon Puslitkoka (BP409, BP961 dan Q121) sangat beragam, sehingga memberikan harapan adanya pola segregasi yang baik berdasarkan kemampuan embriogenesis somatik tidak langsung pada populasi yang dibuat dari silangan klon tersebut

Identification of Quantitative Trait Loci Determining Vegetative Growth Traits in Coffea Canephor

Recently the use of molecular markers has been successfully applied for some crops. For coffee, new opportunities have been opened since Nestlé R&D Centre in collaboration with ICCRI completed the first genetic map of Coffea canephora. This study was aimed both to evaluate the phenotypic trait and also to identify the quantitative trait loci (QTLs) controlling the vegetative growth in Robusta coffee. Present study used three C. canephora populations and six genetic maps developed based on these populations using simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) markers. A total of 17 different quantitative data were used for the detection of QTLs on each of three populations. Present result showed that most of these traits were not heritable. The nine vegetative traits have been identified and distributed over seven different linkage groups. Due to some QTLs determining one given trait were overlapping on the same linkage group and were coming from the same favourable parent, a total of 19 QTLs detected for vegetative traits might finally be considered as only 12 QTLs involved. However, only two of them were shared for different traits. One involved for the number/length of primary branches and width of the canopy while the other for length of internodes and width of canopy. These two QTLs might determine the size of the tree canopy in this species

Functional analysis of CcDREB1D promoter region from two genotypes of Coffea canephora through genetic transformation of Nicotiana tabacum : S03P08

Although some studies in plant physiology resulted in a better understanding of the mechanisms involved in drought tolerance in coffee, knowledge about the metabolic and molecular changes involved in the response of the coffee plant to water deficit conditions is still scarce. Recent studies permitted the identification of several candidate genes presenting differential expression between genotypes contrasting (tolerant vs. susceptible) to this trait. In many higher plants, DREB genes were shown to be involved in the transduction pathways of water stress. Previous results showed that CcDREB1D gene expression increased under drought stress in leaves of drought-tolerant clone 14 but not in those of the drought-susceptible clone 22 of Coffea canephora. By sequencing the DREB1D promoter regions of these clones, several nucleic polymorphisms ("single nucleotide polymorphism" [SNP] and insertion/deletion [INDELs]) were found. In order to know if these polymorphisms could explain the differences of DREB1D gene expression observed between the clones 14 and 22 of C. canephora., 5 'deletions of several alleles of the CcDREB1D promoter regions were made and cloned in the binary vector pBI101 in order to analyze their ability to control the expression of the uidA reporter gene in transgenic tobacco (Nicotiana tabacum) plants. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

Molecular Genetic Diversity Study of Forest Coffee Tree (Coffea arabica L.) Populations in Ethiopia: Implications for Conservation and Breeding

Coffee provides one of the most widely drunk beverages in the world, and is a very important source of foreign exchange income for many countries. Coffea arabica, which contributes over 70 percent of the world's coffee productions, is characterized by a low genetic diversity, attributed to its allopolyploidy origin, reproductive biology and evolution. C. arabica has originated in the southwest rain forests of Ethiopia, where it is grown under four different systems, namely forest coffee, small holders coffee, semi plantation coffee and plantation coffee. Genetic diversity of the forest coffee (C. arabica) gene pool in Ethiopia is being lost at an alarming rate because of habitat destruction (deforestation), competition from other cash crops and replacement by invariable disease resistant coffee cultivars. This study focused on molecular genetic diversity study of forest coffee populations in Ethiopia using PCR based DNA markers such as random amplified polymorphic DNA (RAPD), inverse sequence-tagged repeat (ISTR), inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) or microsatellites. The objectives of the study are to estimate the extent and distribution of molecular genetic diversity of forest coffee and to design conservation strategies for it’s sustainable use in future coffee breeding. In this study, considerable samples of forest coffee collected from four coffee growing regions (provinces) of Ethiopia were analysed. The results indicate that moderate genetic diversity exists within and among few forest coffee populations, which need due attention from a conservation and breeding point of view. The cluster analysis revealed that most of the samples from the same region (province) were grouped together which could be attributed to presence of substantial gene flow between adjacent populations in each region in the form of young coffee plants through transplantation by man. In addition wild animals such as monkeys also play a significant role in coffee trees gene flow between adjacent populations. The overall variation of the forest coffee is found to reside in few populations from each region. Therefore, considering few populations from each region for either in situ or ex situ conservation may preserve most of the variation within the species. For instance, Welega-2, Ilubabor-2, Jima-2 and Bench Maji-2 populations should be given higher priority. In addition, some populations or genotypes have displayed unique amplification profiles particularly for RAPD and ISTR markers. Whether these unique bands are linked to any of the important agronomic traits and serve in marker assisted selections in future coffee breeding requires further investigations

Analysis of CcDREB1D promoter region from drought-tolerant and susceptible clones of Coffea canephora by homologous genetic transformation of Coffea arabica

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREBID gene in several Coffee genotypes. The promoter and coding regions of DREBID gene were cloned and sequenced from 16 coffee plants (10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed a high conservation of DREB1 D proteins among the homologous sequences due to the low level of diversity and the high number of synonymous mutations and neutral changes which represents the majority of sequence variations. However, several nucleic polymorphisms ("single nucleotide polymorphism" and insertion/deletion [InDels]) were found in the coffee DREBID promoters. A comparison of predicted cis-acting elements for all the promoter sequences signaled the loss of some regulatory DNA elements. The sequence variation and the loss of some regulatory DNA elements could explain the differences of DREBID gene expression previously observed in leaves of drought tolerant (clone 14) and susceptible (clone 22) clones of C. canephora. In fact, both clones 14 and 22, have one same CcDREBID allelic sequence (hp15), and diverge at a second allele. Thus, the CcDREBID allele in the tolerant 14 (hp16) was considered to be the favorable/tolerant allele and the allele in 22 (hp17) was inferior/sensitive. The capacity of CcDREBID promoter to control the expression of the uidA reporter gene is under evaluation in transgenic plants of Coffee arabica cv. caturra stably transformed by Agrobacterium tumefaciens mediated gene transfer procedure. Caturra transgenic embryos were placed on a clean bench and subjected to dehydration tests. Preliminary results of bioassays checking GUS (/3-glucuronidase) activities indicate that the observed sequence variations have a direct role in the regulation of CcDREBID expression. The proximal promoter of CcDREBID for the three alleles tested (hp15, hp16 and hp17) equally induced the uidA gene expression, however, expression of uidA under control of the complete CcDREBID promoter was significantly induced in the tolerant allele (hp16) in response to the osmotic stress, whereas, it was not significantly upregulated for the common (hp15) and sensitive alleles (hp17). These results also evidence that the sequence variation present at the first -700 by of CcDREBID promoter do not interfere the regulation activity of the promoter, probably due to the non-overlapping of SNPs and cis-regulatory elements. Though, the higher sequence variation and co-occurrence of SNPs and cis-regulatory elements observed between -700 and -1500 by seems to affect the regulation of CcDREBID promoter in response to drought stress.Support: CAPES COFECUB, INCT-Café, CNPq and ConsOrcio Pesquisa Café. (Texte intégral

Analysis of DREB1D gene sequence in different Coffea genotypes : S03P07

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREB1D gene in several Coffea genotypes. The promoter and coding regions of this gene were cloned and sequenced from 16 coffee plants (including 10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed that the DREB1D-coding sequence was highly conserved within coffee plants. However, several nucleic polymorphisms ("single nucleotide polymorphism" [SNP] and insertion/deletion [INDELs]) were found in the coffee DREB1D promoter regions. These polymorphisms could explained the differences of DREB1D gene expression levels previously observed in leaves of drought tolerant and susceptible clones of C. canephora. These polymorphisms also allowed the identification of different haplotypes like orthologous sequence variants (OSVs) of C. canephora and C. eugenioides as well as homologous single-nucleotide variants (HSVs) for C. arabica subgenomes (C. canephora and C. eugenioides) that could be used to develop allele and homoeologous specific markers for this locus. Work is now under way to evaluate the capacity of DREB1D promoter regions to control the expression of the uidA reporter gene in transgenic coffee plants. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

Analysis of CcDREB1D promoter region from drought-tolerant and susceptible clones of Coffea canephora by homologous genetic transformation of Coffea arabica

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREBID gene in several Coffee genotypes. The promoter and coding regions of DREBID gene were cloned and sequenced from 16 coffee plants (10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed a high conservation of DREB1 D proteins among the homologous sequences due to the low level of diversity and the high number of synonymous mutations and neutral changes which represents the majority of sequence variations. However, several nucleic polymorphisms ("single nucleotide polymorphism" and insertion/deletion [InDels]) were found in the coffee DREBID promoters. A comparison of predicted cis-acting elements for all the promoter sequences signaled the loss of some regulatory DNA elements. The sequence variation and the loss of some regulatory DNA elements could explain the differences of DREBID gene expression previously observed in leaves of drought tolerant (clone 14) and susceptible (clone 22) clones of C. canephora. In fact, both clones 14 and 22, have one same CcDREBID allelic sequence (hp15), and diverge at a second allele. Thus, the CcDREBID allele in the tolerant 14 (hp16) was considered to be the favorable/tolerant allele and the allele in 22 (hp17) was inferior/sensitive. The capacity of CcDREBID promoter to control the expression of the uidA reporter gene is under evaluation in transgenic plants of Coffee arabica cv. caturra stably transformed by Agrobacterium tumefaciens mediated gene transfer procedure. Caturra transgenic embryos were placed on a clean bench and subjected to dehydration tests. Preliminary results of bioassays checking GUS (/3-glucuronidase) activities indicate that the observed sequence variations have a direct role in the regulation of CcDREBID expression. The proximal promoter of CcDREBID for the three alleles tested (hp15, hp16 and hp17) equally induced the uidA gene expression, however, expression of uidA under control of the complete CcDREBID promoter was significantly induced in the tolerant allele (hp16) in response to the osmotic stress, whereas, it was not significantly upregulated for the common (hp15) and sensitive alleles (hp17). These results also evidence that the sequence variation present at the first -700 by of CcDREBID promoter do not interfere the regulation activity of the promoter, probably due to the non-overlapping of SNPs and cis-regulatory elements. Though, the higher sequence variation and co-occurrence of SNPs and cis-regulatory elements observed between -700 and -1500 by seems to affect the regulation of CcDREBID promoter in response to drought stress.Support: CAPES COFECUB, INCT-Café, CNPq and ConsOrcio Pesquisa Café. (Texte intégral

A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors

Functional analysis of CcDREB1D promoter region from haplotypes of Coffea canephora through genetic transformation of Nicotiana tabacum

Recent studies in coffee resulted in the identification of many candidate genes for drought tolerance characterized by differential expression profiles in leaves of drought-tolerant and susceptible clones of Coffea canephora Conilon. Among those are found the genes involved in the water stress response pathway, such as CcDREB1D (coding for DREB transcription factors) that showed higher expression under water stress conditions in the leaves of clone 14 (droughttolerant) than in those of the clone 22 (drought-sensitive). After sequencing, nucleic polymorphisms were identified in the promoter regions of CcDREB1D gene of clones 14 and 22, indicating the presence of three haplotypes (15, 16 and 17) of this promoter. With the aim of studying the participation of these polymorphisms in the response to drought, several genetic constructions of the CcDREB1D promoter regions were made in the pBI101 binary vector and tested via genetic transformation of Nicotiana tabacum cv. SRI, by evaluating the capacity of such fragments in controlling the expression of the ?-glucoronidase (uidA) reporter gene. For the constructions containing the longest versions (D) of the pDREB1D, a basal expression of uidA gene was observed in both leaves and roots of T0 plants grown without drought stress. To see if CcDREB1D haplotypes would respond to abiotic stresses, T0 tobacco plants were submitted to dehydration and elevated temperature assays, and subsequently analyzed for the expression of the uidA reporter gene by GUS histochemical tests and real-time quantitative PCR (RT-qPCR). A slight induction of the uidA gene was confirmed in the leaves of T0 plants transformed with pD22-hp17D. However, gene expression levels were much lower than those measured in plants transformed with the positive control (pBI121). No induction of the reporter gene was observed in plants transformed with the different constructions containing the other haplotypes (15 and 16) of the CcDREB1D promoter. Altogether, these results showed that (i) the pDREB1D promoters of C. canephora are weak in tobacco and that (ii) the haplotype 17 of this promoter, derived from C. canephora clone 22, was induced with abiotic stresses in the tobacco leaves. This indicates that the molecular mechanisms implicated in the regulation of the gene expression in response to drought are (at least partially) conserved between coffee and tobacco plants and that the functioning of the pDREB1D promoters from coffee clones 14 and 22 is different in tobacco, suggesting that the polymorphisms previously identified are important in regulating these promoters. (Texte intégral