Introgressive hybridization between the allotetraploid Coffea arabica and one of its diploid ancestors, Coffea canephora, in an exceptional sympatric zone in New Caledonia.

The importance of introgressive hybridization in plant evolution has long been recognized. Nevertheless, information on gene flow between allopolyploids and their diploid relatives is very limited, even though gene flow could play a major role in polyploid establishment and evolution. Here, we investigated the processes governing hybrid formation and introgression between the allotetraploid Coffea arabica and one of its ancestral diploid progenitors, C. canephora, in a sympatric zone of New Caledonia. The occurrence of a large assortment of hybridization events between the 2 coffee species is clearly established. First-generation hybrids (F1) and post-F1 hybrids were characterized. The involvement of unreduced gametes of C. canephora is suggested, because tetraploid F1 hybrid plants were detected. Moreover, although bidirectional mating was observed, only unidirectional gene flow from C. canephora to C. arabica was noted in post-F1 hybrids. Most of the collected post-F1 hybrid plants exhibited a high level of introgression, and the frequency of introgression observed among the different analyzed loci was homogeneous, suggesting no significant counterselection against introgressions from C. canephora. Overall, the New Caledonian central mountains appear to be a highly favourable environment for introgressive hybridization and a genetic diversity center for C. arabica.

Introgression molecular analysis of a leaf rust resistance gene from Coffea liberica into C. arabica 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(H)3 resistance factor) and to identify associated molecular markers. An F(2) 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(H)3 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(H)3 resistance gene were used to analyse the whole F(2) 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(H)3 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.

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (Coffea arabica 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.

Gene introgression into Coffea arabica by way of triploid hybrids (C. arabica x C. canephora).

Interspecific triploid hybrid plants between the tetraploid species Coffea arabica L. and the diploid species C. canephora P. were backcrossed to C. arabica. Although characterised by a low production and an important fruit dropping, all attempted crosses (ie, 6) generated BC(1) progenies. Flow cytometric analysis of the nuclear DNA content revealed that most of the BC1 individuals were nearly tetraploid. Among the male gametes produced by the interspecific triploid hybrids, those presenting a high number of chromosomes appeared strongly favoured. Only pollen mother cells having nearly 22 chromosomes were effective, the others leading to deficient endosperm and fruit dropping. Molecular markers (ie, microsatellite and AFLP) combined with evaluations of morphological characteristics and resistance to leaf rust were applied to verify the occurrence of gene transfer from C. canephora into C. arabica, and to estimate the amount of introgression present in BC(1) individuals. The results reveal a strong deficiency in the C. canephroa alleles indicating a severe counter-selection against the introgression of genetic material from C. canephora into C. arabica by way of triploid hybrids. However, introgressants displaying desirable traits such as a high resistance to leaf rust were obtained. The low level of introgression could be an advantage by facilitating the recovery of the recurrent parent and possibly reducing the number of required backcrosses. On the other hand, this could be a limitation when attempting the transfer of a complex trait or several simply inherited traits.

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. arabicax C. canephora 4 x) 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 BC(1) populations. These two populations of 28 and 45 individuals, respectively, resulted from the backcross of two tetraploid F(1)plants to C. arabica. The presence in BC(1) 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. arabicax C. canephora 4 x) hybrids conformed to the expected ratio assuming random chromosome segregation and the absence of selection. The recombination fractions of C. canephorachromosome 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. arabicax C. canephora 4 x) hybrid is not significantly restricted by the genetic differentiation between chromosomes belonging to the different genomes. The hybrid ( C. arabicax C. canephora 4 x) therefore appeared particularly favourable to intergenomic recombination events and gene introgressions.

Genetic linkage map of Coffea canephora: effect of segregation distortion and analysis of recombination rate in male and female meioses.

Two complementary segregating plant populations of Coffea canephora were produced from the same clone. One population (DH) comprised 92 doubled haploids derived from female gametes, while the other population (TC) was a test cross consisting of 44 individuals derived from male gametes. Based on the DH population, a genetic linkage map comprising 160 loci was constructed. Eleven linkage groups that putatively correspond to the 11 gametic chromosomes of C. canephora were identified. The mapped loci included more than 40 specific sequence-tagged site markers, either single-copy RFLP probes or microsatellites, that could serve as standard landmarks in coffee-genome analyses. Furthermore, comparisons for segregation distortion and recombination frequency between the two populations were performed. Although segregation distortions were observed in both populations, the frequency of loci exhibiting a very pronounced degree of distortion was especially high in the DH population. This observation is consistent with the hypothesis of strong zygotic selection among the DH population. The recombination frequencies in both populations were found to be almost indistinguishable. These results offer evidence in favour of the lack of significant sex differences in recombination in C. canephora.

Origin, diversity and evolution of NBS-type disease-resistance gene homologues in coffee trees (Coffea L.).

The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.

Single-locus inheritance in the allotetraploid Coffea arabica L. and interspecific hybrid C. arabica x C. canephora.

Molecular cytogenetic analysis has indicated that Coffea arabica is an amphidiploid formed from the hybridization between two closely related diploid progenitor species, C. canephora and C. eugenioides. Our aim was to determine the mode of inheritance in C. arabica and in a tetraploid interspecific hybrid (called arabusta) between C. arabica and C. canephora as revealed by segregation analyses of restriction fragment length polymorphism (RFLP) loci markers. The observed RFLP allele segregations in an F(2) progeny of C. arabica conform to disomic inheritance as expected, with regular bivalent pairing of homologous chromosomes in the F1 hybrid. In contrast, RFLP loci followed tetrasomic inheritance in the arabusta interspecific hybrid, although bivalents have been reported to predominate greatly at meiosis in its hybrid. These results suggest that homologous chromosomes do not pair in C. arabica, not as a consequence of structural differentiation, but because of the functioning of pairing regulating factors. Moreover, the arabusta hybrid seems to offer the possibility of gene exchange between the homologous genomes.

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.

Phylogenetic analysis of chloroplast DNA variation in Coffea L.

The trnL-trnF intergenic spacer of cpDNA has been sequenced from 38 tree samples representing 23 Coffea taxa and the related genus Psilanthus. These sequences were used for phylogenetic reconstruction using parsimony analyses. The results suggest a radial mode of speciation and a recent origin in Africa for the genus Coffea. Phylogenetic relationships inferred from the cpDNA analysis suggest several major clades, which present a strong geographical correspondence (i.e., west Africa, central Africa, east Africa, and Madagascar). The overall results agree well with the phylogeny previously inferred from nuclear genome data. However, several inconsistencies are observed among taxa endemic to west Africa, suggesting the occurrence of introgressive hybridization. Evidence is also obtained for the genetic origin of the allotetraploid species C. arabica.

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L.

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The results were in contradiction with the present classification into two genera. Additionally, cpDNA inheritance was studied in interspecific hybrids between C. arabica and C. canephora, and in an intraspecific progeny of C. canephora, using PCR-based markers. Both studies showed exclusively maternal inheritance of cpDNA.