Pascale Besse

Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F2:3 and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.

Biodiversity and preservation of vanilla: present state of knowledge

The genus Vanilla belongs to the Orchidaceae family and Vanilla planifolia, probably endemic from tropical forests in Eastern Mexico, is the main source for commercial vanilla. There has recently been an important number of publications covering Vanilla taxonomy, particularly using molecular genetics, but the taxonomy of the genus is still unclear and numerous synonyms remain. Recent studies showed that inter-specific hybridization and perhaps even polyploidization played an important role in the evolution of the genus. There has also been an important increase in the knowledge of the genetic diversity and reproductive biology of V. planifolia in natural conditions, showing that mating system diversity exists in Vanilla and that this genus could be a good model to study the role of fragrance in orchid evolution. Recent studies on the genetic consequences of V. planifolia domestication are also presented and raise major scientific questions regarding the origin of phenotypic diversity in a vegetatively propagated crop. Finally, all these studies have demonstrated the urgent need for preservation of the genetic resources of V. planifolia (primary and secondary gene pools, and cultivated resources) and current conservation efforts are presented.

Comparative genetics in sugarcane enables structured map enhancement and validation of marker-trait associations

As sugarcane is a complex polyaneuploid with many chromosomes, large numbers of markers are required to generate genetic maps with reasonable levels of genome coverage. Comparative mapping was investigated as an approach for both quantitative trait loci (QTL) validation and genetic map enhancement in sugarcane. More than 1000 SSR and AFLP markers were scored in a bi-parental Australian sugarcane population (Q3) that was segregating widely for sugar content-related traits. Two maps were constructed, one for each parent. The Q117 (female) and MQ77-340 (male) maps each contained almost 400 markers distributed onto approximately 100 linkage groups (LGs), of which nearly half could be assigned to homology groups (HGs) on the basis of SSRs. Then, using common SSR and AFLP markers, the two Q3 parental maps were aligned with the maps of the French cultivar, R570, and of the Australian cultivar, Q165A (A denotes variety covered by Australian plant breeding rights). As a result of comparative mapping, all ten HGs in the Q117 map, and all eleven HGs in the MQ77-340 map could be re-assigned to seven of the expected eight sugarcane HGs, revealing that one sugarcane HG was not covered at all in either Q3 parental map, and that other HGs were poorly represented. QTL analysis in the Q3 population identified approximately 75 marker-trait associations (MTAs) from approximately 18 chromosomal regions or putative QTL in each map for three sugar content-related traits. QTL location appeared to be consistent between the 4 maps; two of the eight HGs were observed to contain MTAs for brix in two or three maps, strongly suggesting the location of sugar content-related trait loci in these HGs.

Evidence of transoceanic dispersion of the genus #Vanilla# based on plastid DNA phylogenetic analysis

The phylogeny and the biogeographical history of the genus Vanilla was investigated using four chloroplastic genes (psbB, psbC; psaB and rbcL), on 47 accessions of Vanilla chosen from the ex situ CIRAD collection maintained in Reunion Island and additional sequences from GenBank. Bayesian methods provided a fairly well supported reconstruction of the phylogeny of the Vanilloideae sub-family and more particularly of the genus Vanilla. Three major phylogenetic groups in the genus Vanilla were differentiated, which is in disagreement with the actual classification in two sections (Foliosae and Aphyllae) based on morphological traits. Recent Bayesian relaxed molecular clock methods allowed to test the two main hypotheses of the phylogeography of the genus Vanilla. Early radiation of the Vanilla genus and diversification by vicariance consecutive to the break-up of Gondwana, 95 million years ago (Mya), was incompatible with the admitted age of origin of Angiosperm. Based on the Vanilloideae age recently estimated to 71 million years ago (Mya), we conclude that the genus Vanilla would have appeared approximately 34 Mya in South America, when continents were already separated. Nevertheless, whatever the two extreme scenarios tested, at least three long distance migration events are needed to explain the present distribution of Vanilla species in tropical areas. These transoceanic dispersions could have occurred via transoceanic passageway such as the Rio Grande Ridge and the involvement of floating vegetation mats and migratory birds.

A genetic diversity study of endangered Psiadia species endemic from Mauritius Island using PCR markers

The genus Psiadia Jacq. represents the most important indigenous genus, by the number of species present, in the Mascarene archipelago (Mauritius, Reunion, Rodrigues), and is a typical example of adaptive radiation in oceanic islands. The Mauritius species are used in traditional pharmacopoeia for their expectorant properties, and most of them are heavily threatened. Molecular genetic relationships between representatives of eight endangered endemic Psiadia species from Mauritius, conserved in Le Mondrain Reserve, and P. dentata (Cass.) DC, endemic from Reunion island, were studied. The absence of length variations of the 5s rDNA non-transcribed spacer demonstrated the recent common origin of all the species surveyed. RAPD analysis revealed a relatively high intra-specific variability in accordance with the outcrossing mode of reproduction of Psiadia species. Moreover, RAPD analysis showed the existence of four major phenetic groups: (A) P. arguta (Pers.) Voigt, P. dentata, (B) P. penninervia D. C., P. terebinthina A.J. Scott, P. lithospermifolia (Lam.) Cordem, (C) P. viscosa (Lam.) A.J. Scott, P. canescens A.J. Scott, P. cataractae A.J. Scott, and (D) P. pollicina A.J. Scott. These groups were consistent with the chemical composition of the essential oils of the species as well as with their floral characteristics, based on literature. A molecular germplasm database for Psiadia species was established, which will allow further characterisation of new samples being introduced in Le Mondrain Reserve for conservation purpose.

Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla species (Orchidaceae)

Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla was studied and demonstrated that COMT genes in Vanilla are organized with four exons and three introns. At least two to four different versions (either allelic or paralogous) of the COMT multigenic family in the genus Vanilla (in terms of intron sizes) were detected. The three introns were differentially variable, with intron-1 being the most length-polymorphic. Patterns of variations were in accordance with known phylogenetic relationships in the genus obtained with neutral markers. In particular, the genus displayed a strong Old World versus New World differentiation with American fragrant species being characterized by a specific 99bp intron-1 size-variant and a unique 226bp intron-3 variant. Conversely, leafless species of the genus displayed unexpected variations in intron lengths. Due to their role in primary (lignin) and secondary (phenolics, e.g., vanillin, alkaloids) metabolisms, COMT genes might not be neutral markers, and represent candidate functional markers for resistance, aromatic or medicinal properties of Vanilla species. Investigating the orthologous/paralogous status of the different genes revealed (in terms of intron size) will allow the evolution of the COMT genes to be studied.

Differential Responses of Vanilla Accessions to Root Rot and Colonization by Fusarium oxysporum f. sp. radicis-vanillae

Root and stem rot (RSR) disease caused by Fusarium oxysporum f. sp. radicis-vanillae (Forv) is the most damaging disease of vanilla (Vanilla planifolia and V. × tahitensis, Orchidaceae). Breeding programs aimed at developing resistant vanilla varieties are hampered by the scarcity of sources of resistance to RSR and insufficient knowledge about the histopathology of Forv. In this work we have (i) identified new genetic resources resistant to RSR including V. planifolia inbreds and vanilla relatives, (ii) thoroughly described the colonization pattern of Forv into selected vanilla accessions, confirming its necrotic non-vascular behavior in roots, and (iii) evidenced the key role played by hypodermis, and particularly lignin deposition onto hypodermal cell walls, for resistance to Forv in two highly resistant vanilla accessions. Two hundred and fifty-four vanilla accessions were evaluated in the field under natural conditions of infection and in controlled conditions using in vitro plants root-dip inoculated by the highly pathogenic isolate Fo072. For the 26 accessions evaluated in both conditions, a high correlation was observed between field evaluation and in vitro assay. The root infection process and plant response of one susceptible and two resistant accessions challenged with Fo072 were studied using wide field and multiphoton microscopy. In susceptible V. planifolia, hyphae penetrated directly into the rhizodermis in the hairy root region then invaded the cortex through the passage cells where it induced plasmolysis, but never reached the vascular region. In the case of the resistant accessions, the penetration was stopped at the hypodermal layer. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to Forv. The thickness of lignin constitutively deposited onto outer cell walls of hypodermis was highly correlated with the level of resistance for 21 accessions tested. The accumulation of p-coumaric and sinapic acids, two phenolic precursors of lignin, was observed in the resistant plants inoculated with Fo072, but not in the susceptible one. Altogether, our analyses enlightened the mechanisms at work in RSR resistant genotypes and should enhance the development of novel breeding strategies aimed at improving the genetic control of RSR of vanilla.

Nineteen polymorphic microsatellite markers from two African Vanilla species: across-species transferability and diversity in a wild population of V. humblotii from Mayotte

There is a serious lack of information on the genetic diversity and population dynamics of the 110 tropical Vanilla species, although these are keys elements to adjust conservation strategies. Nineteen polymorphic microsatellite markers were developed from two African leafless Vanilla species V. roscheri and V. humblotii to use in population genetic studies. A transferability analysis of these markers on seven Vanilla species from various geographical origins was conducted. Nine microsatellites were polymorphic in a population of 22 individuals of V. humblotii from Mayotte (Comoros Archipelago). These markers had two to eight alleles per locus and observed heterozygosity ranged from 0.23 to 0.64. Amplification parameters were calibrated to facilitate multiplexing and rapid multi-loci genotyping.

DNA Remodeling by Strict Partial Endoreplication in Orchids, an Original Process in the Plant Kingdom

DNA remodeling during endoreplication appears to be a strong developmental characteristic in orchids. In this study, we analyzed DNA content and nuclei in 41 species of orchids to further map the genome evolution in this plant family. We demonstrate that the DNA remodeling observed in 36 out of 41 orchids studied corresponds to strict partial endoreplication. Such process is developmentally regulated in each wild species studied. Cytometry data analyses allowed us to propose a model where nuclear states 2C, 4E, 8E, etc. form a series comprising a fixed proportion, the euploid genome 2C, plus 2–32 additional copies of a complementary part of the genome. The fixed proportion ranged from 89% of the genome in Vanilla mexicana down to 19% in V. pompona, the lowest value for all 148 orchids reported. Insterspecific hybridization did not suppress this phenomenon. Interestingly, this process was not observed in mass-produced epiphytes. Nucleolar volumes grow with the number of endocopies present, coherent with high transcription activity in endoreplicated nuclei. Our analyses suggest species-specific chromatin rearrangement. Towards understanding endoreplication, V. planifolia constitutes a tractable system for isolating the genomic sequences that confer an advantage via endoreplication from those that apparently suffice at diploid level.

Nuclear Ribosomal RNA Genes: ITS Region

Despite possible drawbacks (intraspecific polymorphisms and possible fungal contamination), sequencing of the ribosomal RNA gene ITS region remains one of the most popular nuclear sequences used for plant taxonomy and phylogeny. A protocol for PCR amplification and sequencing of this region using universal plant primers is provided.