Séverine Bory

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.

Origins and Dispersal of Cultivated Vanilla (Vanilla planifolia Jacks. [Orchidaceae])1

Origins and Dispersal of Cultivated Vanilla (Vanilla planifoliaJacks. [Orchidaceae]). Vanilla is a clonally propagated crop originating from Mesoamerica. Information regarding the circumstances under which vanilla cultivation began is incomplete. Presumably, the Totonac people of Papantla (north-central Veracruz, Mexico) were the earliest to cultivate vanilla; however, the oldest reports of vanilla use relate to the pre-Columbian Maya of southeastern Mexico/Central America, where vanilla was a cacao-beverage spice. We utilized Amplified Fragment Length Polymorphism (AFLP) marker diversity to infer the origins and relationships among cultivated and non-cultivated vanilla in Mesoamerica and on islands in the Indian Ocean, which comprise today’s principal production regions of vanilla. Our results suggest that, genetically, vanilla cultivated outside of Mesoamerica is most closely related to cultivated stock from Papantla; whereas unique clones of V. planifolia are found in non-cultivated and cultivated individuals from elsewhere in Mesoamerica. This is consistent with a single origin for cultivated vanilla outside of Mexico, along with multiple origins for cultivated material within Mexico. These data suggest that vestiges of pre-Columbian Maya vanilla cultivars are not found in commercial production today.

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.

Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis

In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation.

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.