Genetic and physical mapping of the S<Subscript>H</Subscript>3 region that confers resistance to leaf rust in coffee tree (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Resistance to coffee leaf rust is conferred by S_H3, a major dominant gene that has been introgressed from a wild coffee species Coffea liberica (genome L) into the allotetraploid cultivated species, Coffea arabica (genome C^aE^a). As the first step toward the map-based cloning of the S_H3 gene, using a bacterial artificial chromosome (BAC) library, we describe the construction of a physical map in C. arabica spanning the resistance locus. This physical map consists in two homeologous BAC-contigs of 1,170 and 1,208 kb corresponding to the subgenomes C^a and E^a, respectively. Genetic analysis was performed using a single nucleotide polymorphism detection assay based on Sanger sequencing of amplicons. The C. liberica-derived chromosome segment that carries the S_H3 resistance gene appeared to be introgressed on the sub-genome C^a. The position of the S_H3 locus was delimited within an interval of 550 kb on the physical map. In addition, our results indicated a sixfold reduction in recombination frequency in the introgressed S_H3 region compared to the orthologous region in Coffea canephora.     

In situ hybridization identifies the diploid progenitor species of Coffea arabica (Rubiaceae)

 The most important commercial coffee species, Coffea arabica, which is cultivated in about 70% of the plantations world-wide, is the only tetraploid (2n=4x=44) species known in the genus. Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) were used to study the genome organization and evolution of this species. Labelled total genomic DNA from diploid species (C. eugenioides, C. congensis, C. canephora, C. liberica) closely related to C. arabica was separately used as a probe in combination with or without blocking DNA to the chromosome spreads of C. arabica. GISH discriminated between chromosomes of C. arabica only in the presence of an excess of unlabelled block DNA from the species not used as a probe. Among the range of different species combinations used, DNA from C. eugenioides strongly and preferentially labelled 22 chromosomes of the tetraploid C. arabica, while the remaining 22 chromosomes were labelled with C. congensis DNA. The similarity of observations between C. arabica and the two diploid species using two ribosomal genes with FISH with respect to metaphase chromosomes provided additional support to the GISH results. These results confirm the allopolyploid nature of C. arabica and show that C. congensis and C. eugenioides are the diploid progenitors of C. arabica. Received: 2 February 1998 / Accepted: 12 May 1998     

Inheritance of caffeine and heteroside contents in an interspecific cross between a cultivated coffee species Coffea liberica var dewevrei and a wild species caffeine-free C. pseudozanguebariae

 Coffee species originating from Africa, in particular the two major cultivated species C. arabica and C. canephora, usually contain caffeine in their beans, whereas almost all Malagasy coffee species are caffeine-free. However, one wild coffee species C. pseudozanguebariae, collected near the coast in south Kenya, is also caffeine-free. Beans of this species contain a specific heteroside diterpene (hereinafter referred to simply as heteroside) and give a bitter coffee beverage. We have investigated the inheritance of the caffeine and heteroside contents in the first and second generations of an interspecific cross between C. pseudozanguebariae and C. liberica var. dewevrei, for which the caffeine content is about 1% dmb (dry matter basis). The caffeine content of F₁ hybrids (0.2% dmb) was lower than the parental average (0.47% dmb). Caffeine and heteroside contents appeared to be under polygenic control with a strong genetic effect. Nevertheless, one major gene with two alleles seemed to be involved in the control of both compounds. Absence of caffeine was apparently controlled by one recessive gene. Heteroside content seemed to be controlled by one co-dominant gene, heterozygotes being intermediate between the two different groups of homozygotes. Received: 15 September 1997 / Accepted: 6 October 1997     

Ultrastructural Characterization of Oil Bodies in Different Coffea Species

In the coffee seed, the lipid component known as coffee oil is stored in the endosperm tissue as an energy reserve for germination and post-germination growth. This coffee constituent is present in the form of subcellular spherical oil bodies (“oleosomes”) in a typical size range of 0.2–2.5 μm. These particles are filled with an osmiophilic matrix of triglycerides, delimited by a single protein membrane, typical of oleaginous plant tissues. The object of this study is to characterize the morphology and distribution of oil bodies in different coffee species. In particular, we studied Indian samples of Coffea arabica, C. canephora, C. liberica, C. stenophylla and C. salvatrix. After appropriate fixation and preparation, the samples were examined and oil bodies characterized by optical microscopy and transmission electron microscopy. Oil bodies morphology, tissue distribution and size distribution were determined and several features of these subcellular structures were observed and discussed for the first time in the framework of a coffee inter-species comparative study.     

Biotechnological applications for the improvement of coffee (Coffea arabica L.)

Summary The important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F₁ hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.     

Lipid transfer proteins in coffee: isolation of Coffea orthologs,Coffea arabica homeologs,expression during coffee fruit development and promoter analysis in transgenic tobacco plants

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5′ deletions, were fused to the reporter gene β-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.     

Identification of attractant and repellent plants to coffee berry borer,Hypothenemus hampei

Colombia is one of the world's largest producers of coffee [Coffea arabica L. (Rubiaceae)]. The coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), is the main pest of coffee. This insect is controlled through an integrated pest management program that includes cultural, biological, and chemical control strategies. Despite research seeking CBB attractants and repellents, these potential management tools have not been successfully incorporated into control programs. This work proposes the use of plant functional diversity for CBB management, for which a number of plants related to coffee and weeds were selected. CBB preference to these plants was determined by olfactometry and volatile compounds emitted by them were identified. Field trials were performed to test CBB preference under field conditions. These trials determined the olfactory preference of CBB to coffee berries accompanied by material of the plants Crotalaria micans Link (Fabaceae), Lantana camara L. (Verbenaceae), Nicotiana tabacum L. (Solanaceae), Artemisia vulgaris L., Calendula officinalis L., Stevia rebaudiana (Bertoni) Bertoni, and Emilia sonchifolia (L.) DC. (all four Asteraceae). Under laboratory conditions N. tabacum, L. camara, and C. officinalis were identified as repellents for CBB in olfactometer assays, whereas E. sonchifolia functioned as attractant. Controlled field trials corroborated CBB repellency of N. tabacum and L. camara; both release volatile sesquiterpenes. Selected candidate attractants included E. sonchifolia plants, for showing attraction in the laboratory. The potential use of these plants in agroecological management of coffee plantations is discussed.     

Responses of two field-grown coffee species to drought and re-hydration

The gas exchange, parameters of chlorophyll fluorescence, contents of pigments, and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), as well as lipid peroxidation were investigated in two field-grown coffee species, Coffea arabica and C. liberica, exposed to drought and re-hydration. Drought caused a more pronounced inhibition of net photosynthetic rate in C. liberica compared to C. arabica. The de-epoxidation of xanthophyll cycle pigments at midday estimated by leaf reflectance was much higher in C. arabica than in C. liberica, but no significant change was found in response to drought. Under moderate drought, the activities of SOD and APX increased significantly only in C. arabica. The maximum photochemical efficiency of photosystem 2, PS2 (F_v/F_m) at predawn did not change and there was no lipid peroxidation during this time. Under severe drought F_v/F_m decreased and initial fluorescence (F₀) increased for both species, and SOD activity increased, APX activity remained relatively high, and malondialdehyde (MDA) accumulated in C. arabica, while APX decreased in C. liberica. The photosynthetic apparatus of C. arabica was completely recovered after 5 d of re-irrigation as indicated by the restoration of F_v/F_m to the control values. A lack of recovery upon rewatering of C. liberica indicated irreversible damage to PS2. Hence compared to C. liberica, C. arabica possesses a higher desiccation-induced antioxidative protection and higher portion of the total pigment pool used in photoprotection, which might aid alleviating photoinhibitory damage during desiccation and photosynthesis recovery when favourable conditions are restored.The research was financially supported by the project of the Chinese Academy of Sciences (KSCX2-SW-104).     

Effects of forest management on mating patterns,pollen flow and intergenerational transfer of genetic diversity in wild Arabica coffee (Coffea arabica L.) from Afromontane rainforests

Coffea arabica, the wild ancestor of all commercial Arabica coffee cultivars worldwide, is endemic to the montane rainforests of Ethiopia. These forests, which harbour the most important C. arabica gene pool, are threatened by increasing anthropogenic disturbance, potentially altering the mating patterns, pollen dispersal and maintenance of genetic diversity in C. arabica understorey populations. We genotyped 376 adult coffee shrubs and 418 progenies from three natural unmanaged, and three highly managed coffee populations, using 24 microsatellite markers. Mating system analysis of C. arabica yielded an overall multilocus outcrossing rate of 76%, which contrasts with the common knowledge that C. arabica is a predominantly selfing species. In highly managed coffee populations, paternity could be assigned to 78% of the progenies, whereas in the unmanaged natural coffee populations, only 57% of the progenies could be assigned to a father, indicating reduced long‐distance pollen dispersal in managed forests. Furthermore, the fraction of selfed progenies was significantly higher in managed (23%) than unmanaged (10%) coffee forests. Finally, the lack of spatial genetic structure in all studied populations suggests high seed dispersal in unmanaged populations, and intense berry harvesting and coffee planting in the managed populations. Our results imply that in situ conservation of the wild gene pool of C. arabica must focus on limiting intensification of coffee forest management, as decreased pollen dispersal and increased selfing in C. arabica in intensively managed populations may increase the risk of genetic erosion. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 76–88.     

Minimal growth in vitro conservation of coffee (Coffea spp.)

We have studied the influence of low concentrations of 6-benzyladenine on growth limitation, in order to preserve coffee germplasm through a microcutting collection. Concentrations of 0 M, 1.3 M and 4.4 M were compared in four species: Coffea congensis, C. canephora, C. liberica and C. racemosa. After six months, microcutting behaviour varied between the different treatments, and a species effect was observed. The slow growing species (C. liberica and C. congensis) needed 1.3 M; the others coffee species (C. canephora and C. racemosa) exhibited moderate caulogenesis on 6-benzyladenine-free medium. Zero and low concentrations did not affect survival rates. In conclusion 1.3 M seems most appropriate for conserving all four species.Abbreviation BA 6-benzyladenine     

Molecular characterisation and origin of the Coffea arabica L. genome

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period. Received: 6 June 1998 / Accepted: 10 November 1998     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.     

Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner

 A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype. The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein. Received: 7April 1999 / Revision received: 20 July 1999 / Accepted: 22 July 1999     

Introgression molecular analysis of a leaf rust resistance gene from <Emphasis Type="Italic">Coffea liberica</Emphasis> into <Emphasis Type="Italic">C. arabica</Emphasis> L.

Leaf rust caused by the fungus Hemileia vastatrix is the most devastating disease of arabica coffee (Coffea arabica). Therefore, developing leaf rust-resistant varieties has been a breeding objective of the highest priority in many countries. The purpose of the present work was to gain insight into the mechanism of introgression into C. arabica of a leaf rust resistance gene from C. liberica (i.e. S_H3 resistance factor) and to identify associated molecular markers. An F₂ progeny (i.e. 101 individuals) derived from a cross between Matari, an arabica accession and liberica-introgressed line S.288, was evaluated for resistance against three different races of H. vastatrix. The progeny segregated for the S_H3 gene in a 3:1 ratio, as expected for a single dominant gene. Amplified fragment length polymorphism analysis of a population subset using 80 different primer combinations revealed that at least half of the total polymorphism observed in the population is associated with introgression of C. liberica chromosome fragments. Furthermore, 15 primer combinations generating candidate marker bands associated with the S_H3 resistance gene were used to analyse the whole F₂ population. A total of 34 marker bands originating from S.288 and attributable to introgression were scored. None exhibited segregation distortion. Linkage analysis revealed only three distinct introgressed fragments corresponding to a total length of 52.8 cM. Twenty-one markers were strongly associated (LOD score >14) with the S_H3 gene and were grouped together in a single linkage group of 6.3 cM. The results are discussed in relation to the efficient use of genetic resources in arabica breeding.     

Isolation and characterization of nine microsatellite markers from Coffea arabica L., showing wide cross‐species amplifications

Genetic improvement of coffee (Coffea arabica L.) is constrained by low genetic diversity and lack of genetic markers, suitable screening tools, information on the genetic make‐up of available gene pool and long generation time. In this context, use of DNA markers such as microsatellites that provide high genetic‐resolution becomes highly desirable. Here, we report the development of nine new microsatellite markers from partial genomic library of an elite variety of Coffea arabica. The developed microsatellites revealed robust cross‐species amplifications in 17 related species of coffee, and their Polymorphic Information Content varied from 0 to 0.6, 0 to 0.78 and 0.67 to 0.90 for the arabica, robusta genotypes and species representatives, respectively. The data thus suggest their potential use as genetic markers for assessment of germplasm diversity and linkage analysis of coffee.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

Evidence of Intergenomic Relationships in Triploid Hybrids of Coffee (<Emphasis Type="Italic">Coffea sp.)</Emphasis> as Revealed by Meiotic Behavior and Genomic in Situ Hybridization

Interspecific hybrids involving the cultivated C. arabica (2n = 4x = 44, E^aE^aC^aC^a) and two related diploid species (2n = 2x = 22), C. eugenioides (EE) and C. liberica (LL), were produced and analyzed for their relative genome affinity using different complementary approaches, including chromosome association analysis, genomic in situ hybridization (GISH) and pollen fertility. The mean arm pairing frequency (c) and the relative affinity index (x) of triploid hybrids with known genome combinations were used as a measure of chromosome homology. Triploid hybrids were highly sterile as a result of meiotic abnormalities (fertility ranged from 1 to 15 %). Nevertheless, all hybrids exhibited a significant occurrence of genome affinities (x = 0.96 for E^aC^aE and 0.81 for E^aC^aL). Further analysis using the GISH approach revealed that C. eugenioides was more closely related to C. arabica than to C. liberica, which was in agreement with the ancestral history of the allotetraploid C. arabica. The absence of incompatibility barriers at the stylar level in the flowers of the triploid hybrids indicates the possibility of desirable gene transfer through breeding strategies.     

In Silico and Quantitative Analyses of the Putative FLC-like Homologue in Coffee (Coffea arabica L.)

The sequential pattern of coffee flowering is a major constraint that directly affects productivity, increases harvest costs, and generates a final product of lower quality for mixing dry fruits with ripe and unripe ones. The objective of this work was to identify and analyze one of the main genes involved in flowering regulation, FLOWERING LOCUS C (FLC) in coffee (Coffea arabica L.). The identification of this gene was conducted in silico using a coffee EST database (CAFEST) and bioinformatics tools. Quantitative PCR results suggest that the identified CaFLC-like homologue is directly involved in flowering regulation in coffee. This expands our knowledge on evolutionary conservation of flowering pathways in dicot species. The functional studies of CaFLC-like with mutants of a more tractable species will lead to a better understanding of the molecular regulation as well as the specific functions of each gene flowering during floral induction in coffee.