Cytogenetic and flow cytometry data expand knowledge of genome evolution in three <Emphasis Type="Italic">Coffea</Emphasis> species

Karyotype and nuclear 2C-value data are considered important in taxonomic and evolutionary approaches in Coffea. Still, new methods are needed to further support such studies, especially to determine the progenitors of Coffea arabica. In this work, new cytogenetic and flow cytometry data were used to compare Coffea arabica, Coffea canephora and Coffea congensis. These data corroborate the hypothesis that C. canephora and C. congensis originated from a single ancestor, whose basic chromosome number was x = 11. In agreement with the observations of other authors, the karyotype and mean 2C-values confirm that C. arabica is a true allotetraploid originating from two diploid Coffea species with similar genomes. Although C. canephora and C. congensis have been considered potential progenitors of C. arabica, karyotype comparison revealed that only one of these species may be parental to C. arabica. These accurate cytogenetic and flow cytometry data contribute to expand our knowledge of the Coffea genome, as well as of possible progenitors of C. arabica.     

Genome rearrangements derived from homoeologous recombination following allopolyploidy speciation in coffee

Allopolyploidization is widespread and has played a major role in flowering plant diversification. Genomic changes are common consequences of allopolyploidization, but their mechanisms of occurrence and dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen as a model to investigate genetic changes in allopolyploid using an approach that exploits next‐generation sequencing technologies. Genes affected by putative homoeolog loss were inferred by comparing the numbers of single‐nucleotide polymorphisms detected using RNA‐seq in individual accessions of C. arabica, and between accessions of its two diploid progenitor species for common sequence positions. Their physical locations were investigated and clusters of genes exhibiting homoeolog loss were identified. To validate these results, genome sequencing data were generated from one accession of C. arabica and further analyzed. Genomic rearrangements involving homoeologous exchanges appear to occur in C. arabica and to be a major source of genetic diversity. At least 5% of the C. arabica genes were inferred to have undergone homoeolog loss. The detection of a large number of homoeologous exchange events (HEEs) shared by all accessions of C. arabica strongly reinforces the assumption of a single allopolyploidization event. Furthermore, HEEs were specific to one or a few accessions, suggesting that HEE accumulates gradually. Our results provide evidence for the important role of HEE in allopolyploid genome evolution.     

Distribution of <Emphasis Type="Italic">Divo</Emphasis> in <Emphasis Type="Italic">Coffea</Emphasis> genomes,a poorly described family of angiosperm LTR-Retrotransposons

Coffea arabica (the Arabica coffee) is an allotetraploid species originating from a recent hybridization between two diploid species: C. canephora and C. eugenioides. Transposable elements can drive structural and functional variation during the process of hybridization and allopolyploid formation in plants. To learn more about the evolution of the C. arabica genome, we characterized and studied a new Copia LTR-Retrotransposon (LTR-RT) family in diploid and allotetraploid Coffea genomes called Divo. It is a complete and relatively compact LTR-RT element (~5 kb), carrying typical Gag and Pol Copia type domains. Reverse Trancriptase (RT) domain-based phylogeny demonstrated that Divo is a new and well-supported family in the Bianca lineage, but strictly restricted to dicotyledonous species. In C. canephora, Divo is expressed and showed a genomic distribution along gene rich and gene poor regions. The copy number, the molecular estimation of insertion time and the analysis at orthologous locations of insertions in diploid and allotetraploid coffee genomes suggest that Divo underwent a different and recent transposition activity in C. arabica and C. canephora when compared to C. eugenioides. The analysis of this novel LTR-RT family represents an important step toward uncovering the genome structure and evolution of C. arabica allotetraploid genome.     

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     

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     

Transcriptional Activity,Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes

Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences.     

First Coffea arabica karyogram showing that this species is a true allotetraploid

Evolutive studies have verified that Coffea arabica (2n = 44) is a natural segmental allopolyploid originated from a cross between two diploid (2n = 22) Coffea species. Data obtained by classical cytogenetic analyses showed that C. arabica chromosomes are small and morphologically similar, which hampers the karyogram assembly with well-identified homologue pairs. In the present study, the C. arabica complement was reanalysed using an improved cytogenetic protocol that allowed the obtention of high-quality prometaphasic and metaphasic chromosomes. The results showed that chromosomes are cytogenetically distinct (1, 2, 19, 20, 21 and 22) and identical (3–4, 5–6, 7–8, 9–10, 11–12, 13–14, 15–16 and 17–18), with regard to their total length, short and long arm sizes or chromosome classes. Our work suggests that C. arabica is a true non-segmental allotetraploid but originated from different species exhibiting similar and distinct chromosomes.     

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.     

Comparative sequence analyses indicate that Coffea (Asterids) and Vitis (Rosids) derive from the same paleo-hexaploid ancestral genome

The complete sequence of Vitis vinifera revealed that the rosid clade derives from a hexaploid ancestor. At present, no analysis of complete genome sequence is available for an asterid, the other large eudicot clade, which includes the economically important species potato, tomato and coffee. To elucidate the genomic history of asterids, we compared the sequence of an 800 kb region of diploid Coffea genome to the orthologous regions of V. vinifera, Populus trichocarpa and Arabidopsis thaliana. We found a very high level of collinearity between around 80 genes of the three rosid species and Coffea. Collinearity comparisons between orthologous and paralogous regions indicates that (1) the Coffea (and consequently all asterids) and rosids share the same hexaploid ancestor; (2) the diploidization process (loss of duplicated and redundant copies from the whole genome duplication) was very advanced in the most recent common ancestor of rosids and asterids. Finally, no additional polyploidization events were detected in the Coffea lineage. Differences in gene loss rates were detected among the three rosid species and linked to the divergence in protein sequences.     

Comparison between a coffee single copy chromosomal region and Arabidopsis duplicated counterparts evidenced high level synteny between the coffee genome and the ancestral Arabidopsis genome

The Arabidopsis thaliana genome sequence provides a catalogue of reference genes that can be used for comparative analysis of other species thereby facilitating map-based cloning in economically important crops. We made use of a coffee bacterial artificial chromosome (BAC) contig linked to the S_H3 leaf rust resistance gene to assess microsynteny between coffee (Coffea arabica L.) and Arabidopsis. Microsynteny was revealed and the matching counterparts to C. arabica contigs were seen to be scattered throughout four different syntenic segments of Arabidopsis on chromosomes (Ath) I, III, IV and V. Coffee BAC filter hybridizations were performed using coffee putative conserved orthologous sequences to Arabidopsis predicted genes located on the different Arabidopsis syntenic regions. The coffee BAC contig related to the S_H3 region was successfully consolidated and later on validated by fingerprinting. Furthermore, the anchoring markers appeared in same order on the coffee BAC contigs and in all Arabidopsis segments with the exception of a single inversion on AtIII and AtIV Arabidopsis segments. However, the S_H3 coffee region appears to be closer to the ancestral genome segment (before the divergence of Arabidopsis and coffee) than any of the duplicated counterparts in the present-day Arabidopsis genome. The genome duplication events at the origin of its Arabidopsis counterparts occurred most probably after the separation (i.e. 94 million years ago) of Euasterid (Coffee) and Eurosid (Arabidopsis).     

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.Communicated by J.W. Snape     

Detection of genetic diversity and selective gene introgression in coffee using RAPD markers

RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups — C. arabica var. typica and C. arabica var. bourbon — were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops.On study leave from: Universidad de San Carlos de Guatemala, Facultad de Agronomia, Ciudad Universitaria, Zona 12, Apartado Postal No. 1545, Guatemala, Central America     

Do domatia mediate mutualistic interactions between coffee plants and predatory mites?

Many plant species possess structures on their leaves that often harbour predatory or fungivorous mites. These so‐called domatia are thought to mediate a mutualistic interaction; the plant gains protection because mites decimate plant pathogenic fungi or herbivores, whereas the mites find shelter in the domatia. We tested this hypothesis using two species of coffee (Coffea spp.) plants that posses domatia consisting of small cavities at the underside of the leaves, and which often harbour mites. We assessed densities of domatia, of the predatory mite Iphiseiodes zuluagai Denmark and Muma (Acari: Phytoseiidae) and of herbivorous mites Oligonychus ilicis (McGregor) (Acari: Tetranychidae) and Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) on Coffea arabica L. (Rubiaceae) and Coffea canephora Pierre in the field. Over a period of 50 days, C. arabica harboured on average 7.5 times more predatory mites and 0.4–0.66 fewer prey mites than C. canephora. Hence, the higher density of predatory mites on C. arabica could not be explained by higher densities of prey. However, the density of domatia on C. arabica was on average 1.65 times higher than on C. canephora, and within each species, leaves with higher densities of domatia also harboured more predators. This suggests a positive effect of domatia on predatory mites. In the laboratory, survival of adult female predatory mites on leaves of C. arabica with open domatia was indeed significantly higher than on leaves with closed domatia. Hence, predatory mites benefited from the domatia. However, plants with higher densities of domatia did not harbour fewer herbivores. Taken together, our study only provides partial evidence for a mutualistic interaction between coffee plants and predatory mites, mediated by domatia.     

Introgression into the allotetraploid coffee ( Coffea arabica L.): segregation and recombination of the C. canephora genome in the tetraploid interspecific hybrid ( C. arabicax C. canephora)

Transfer of desired characters from the diploid relative species such as Coffea canephora into the cultivated allotetraploid coffee species (Coffea arabica L.) is essential to the continued improvement of varieties. Behaviour of the C. canephora genome and its interaction with the C. arabica genome were investigated in tetraploid interspecific hybrids (C. arabica×C. canephora 4x) resulting from a cross between an accession of C. arabica and a tetraploid plant of C. canephora obtained following colchicine treatment. Segregation and co-segregation of restriction fragment length polymorphism (RFLP) and microsatellite loci-markers were studied in two BC₁ populations. These two populations of 28 and 45 individuals, respectively, resulted from the backcross of two tetraploid F₁ plants to C. arabica. The presence in BC₁ plants of specific C. canephora markers was scored for 24 loci (11 RFLP and 13 microsatellites) distributed on at least 7 of the 11 linkage groups identified in C. canephora. At almost all loci analysed, the segregation of C. canephora alleles transmitted by the (C. arabica×C. canephora 4x) hybrids conformed to the expected ratio assuming random chromosome segregation and the absence of selection. The recombination fractions of C. canephora chromosome segments were estimated for seven marker intervals, and compared with the recombination fractions previously observed in C. canephora for the equivalent marker intervals. The recombination frequencies estimated in both plant materials were rather similar, suggesting that recombination in the (C. arabica×C. canephora 4x) hybrid is not significantly restricted by the genetic differentiation between chromosomes belonging to the different genomes. The hybrid (C. arabica×C. canephora 4x) therefore appeared particularly favourable to intergenomic recombination events and gene introgressions. Received: 26 March 2001 / Accepted: 29 June 2001     

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.