Mutations in Rice yellow mottle virus Polyprotein P2a Involved in RYMV2 Gene Resistance Breakdown.

Rice yellow mottle virus (RYMV) is one of the major diseases of rice in Africa. The high resistance of the Oryza glaberrima Tog7291 accession involves a null allele of the RYMV2 gene, whose ortholog in Arabidopsis, CPR5, is a transmembrane nucleoporin involved in effector-triggered immunity. To optimize field deployment of the RYMV2 gene and improve its durability, which is often a weak point in varietal resistance, we analyzed its efficiency toward RYMV isolates representing the genetic diversity of the virus and the molecular basis of resistance breakdown. Tog7291 resistance efficiency was highly variable depending on the isolate used, with infection rates ranging from 0 to 98% of plants. Back-inoculation experiments indicated that infection cases were not due to an incomplete resistance phenotype but to the emergence of resistance-breaking (RB) variants. Interestingly, the capacity of the virus to overcome Tog7291 resistance is associated with a polymorphism at amino-acid 49 of the VPg protein which also affects capacity to overcome the previously studied RYMV1 resistance gene. This polymorphism appeared to be a main determinant of the emergence of RB variants. It acts independently of the resistance gene and rather reflects inter-species adaptation with potential consequences for the durability of resistance. RB mutations were identified by full-length or partial sequencing of the RYMV genome in infected Tog7291 plants and were validated by directed mutagenesis of an infectious viral clone. We found that Tog7291 resistance breakdown involved mutations in the putative membrane anchor domain of the polyprotein P2a. Although the precise effect of these mutations on rice/RYMV interaction is still unknown, our results offer a new perspective for the understanding of RYMV2 mediated resistance mechanisms. Interestingly, in the susceptible IR64 variety, RB variants showed low infectivity and frequent reversion to the wild-type genotype, suggesting that Tog7291 resistance breakdown is associated with a major loss of viral fitness in normally susceptible O. sativa varieties. Despite the high frequency of resistance breakdown in controlled conditions, this loss of fitness is an encouraging element with regards to RYMV2 resistance durability.

Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites.

BACKGROUND AND AIMS: The coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated. METHODS: Genetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species. KEY RESULTS: Across Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity. CONCLUSIONS: Cross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.

Site-Specific Insertion Polymorphism of the MITE Alex-1 in the Genus Coffea Suggests Interspecific Gene Flow.

Miniature Inverted-repeat Transposable Elements (MITEs) are small nonautonomous class-II transposable elements distributed throughout eukaryotic genomes. We identified a novel family of MITEs (named Alex) in the Coffea canephora genome often associated with expressed sequences. The Alex-1 element is inserted in an intron of a gene at the CcEIN4 locus. Its mobility was demonstrated by sequencing the insertion site in C. canephora accessions and Coffea species. Analysis of the insertion polymorphism of Alex-1 at this locus in Coffea species and in C. canephora showed that there was no relationship between the geographical distribution of the species, their phylogenetic relationships, and insertion polymorphism. The intraspecific distribution of C. canephora revealed an original situation within the E diversity group. These results suggest possibly greater gene flow between species than previously thought. This MITE family will enable the study of the C. canephora genome evolution, phylogenetic relationships, and possible gene flows within the Coffea genus.

An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods.

PREMISE OF THE STUDY: In this study, we developed a nuclear DNA extraction protocol for Next Generation Sequencers (NGS). METHODS AND RESULTS: We applied this extraction method to grapevines and coffee trees, which are known to contain many secondary metabolites. The nuclear DNA obtained was sequenced by the 454/GS-FLX method. We obtained excellent results, with less than 4% cytoplasmic DNA, in a similar way to a BAC (Bacterial Artificial Chromosome)-building protocol. We also compared our protocol with a classic DNA extraction using specific cytoplasmic DNA amplification. Results showed a lower cytoplasmic DNA contamination with the new protocol. CONCLUSIONS: The method presented here is fast and economical. The DNA obtained is of high quality, with a low level of cytoplasmic DNA contamination, and very efficient for the construction of sequencing libraries.

Micro-collinearity and genome evolution in the vicinity of an ethylene receptor gene of cultivated diploid and allotetraploid coffee species (Coffea).

Arabica coffee (Coffea arabica L.) is a self-compatible perennial allotetraploid species (2n=4x=44), whereas Robusta coffee (C. canephora L.) is a self-incompatible perennial diploid species (2n=2x=22). C. arabica (C(a) C(a) E(a) E(a) ) is derived from a spontaneous hybridization between two closely related diploid coffee species, C. canephora (CC) and C. eugenioides (EE). To investigate the patterns and degree of DNA sequence divergence between the Arabica and Robusta coffee genomes, we identified orthologous bacterial artificial chromosomes (BACs) from C. arabica and C. canephora, and compared their sequences to trace their evolutionary history. Although a high level of sequence similarity was found between BACs from C. arabica and C. canephora, numerous chromosomal rearrangements were detected, including inversions, deletions and insertions. DNA sequence identity between C. arabica and C. canephora orthologous BACs ranged from 93.4% (between E(a) and C(a) ) to 94.6% (between C(a) and C). Analysis of eight orthologous gene pairs resulted in estimated ages of divergence between 0.046 and 0.665 million years, indicating a recent origin of the allotetraploid species C. arabica. Analysis of transposable elements revealed differential insertion events that contributed to the size increase in the C(a) sub-genome compared to its diploid relative. In particular, we showed that insertion of a Ty1-copia LTR retrotransposon occurred specifically in C. arabica, probably shortly after allopolyploid formation. The two sub-genomes of C. arabica, C(a) and E(a) , showed sufficient sequence differences, and a whole-genome shotgun approach could be suitable for sequencing the allotetraploid genome of C. arabica.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae).

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.