First Vanilla planifolia High-Density Genetic Linkage Map Provides Quantitative Trait Loci for Resistance to Fusarium oxysporum.

Fusarium oxysporum f. sp. radicis-vanillae (Forv), the causal agent of root and stem rot disease, is the main pathogen affecting vanilla production. Sources of resistance have been reported in Vanilla planifolia G. Jackson ex Andrews, the main cultivated vanilla species. In this study, we developed the first high-density genetic map in this species with 1,804 genotyping-by-sequencing (GBS)-generated single nucleotide polymorphism (SNP) markers using 125 selfed progenies of the CR0040 traditional vanilla cultivar. Sixteen linkage groups (LG) were successfully constructed, with a mean of 113 SNPs and an average length of 207 cM per LG. The map had a high density with an average of 5.45 SNP every 10 cM and an average distance of 1.85 cM between adjacent markers. The first three LG were aligned against the first assembled chromosome of CR0040, and the other 13 LG were correctly associated with the other 13 assembled chromosomes. The population was challenged with the highly pathogenic Forv strain Fo072 using the root-dip inoculation method. Five traits were mapped, and 20 QTLs were associated with resistance to Fo072. Among the genes retrieved in the CR0040 physical regions associated with QTLs, genes potentially involved in biotic resistance mechanisms, coding for kinases, E3 ubiquitin ligases, pentatricopeptide repeat-containing proteins, and one leucine-rich repeat receptor underlying the qFo72_08.1 QTL have been highlighted. This study should provide useful resources for marker-assisted selection in V. planifolia.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly.

Vanilla planifolia, the species cultivated to produce one of the world's most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

Drivers of population divergence and species differentiation in a recent group of indigenous orchids (Vanilla spp.) in Madagascar.

With over 25,000 species, orchids are among families with remarkable high rate of diversification. Since Darwin's time, major advances attributed the exceptional diversity of orchids to plant-pollinator interactions. However, unraveling the processes and factors that determine the phenotypic and genotypic variation of natural orchid populations remains a challenge. Here, we assessed genetic population structure and floral differentiation in recently diverged leafless Vanilla species in a world biodiversity hotspot, Madagascar, using seven microsatellite loci and 26 morphometric variables. Additionally, analyses were performed to test for the occurrence of any patterns of isolation by distance, isolation by environment, and isolation by adaptation and to detect possible physical barriers that might have caused genetic discontinuities between populations. Positive inbreeding coefficients detected in 22 populations were probably due to the presence of null alleles, geitonogamy and/or some admixture (sympatric species). In contrast, the only high-altitude population showed an important rate of clonality leading to heterozygote excess. Genetic diversity was maximum in western populations, suggesting a postglacial colonization to the north and south. Clustering analyses identified seven genetic groups characterized by specific floral traits that matched five botanical descriptions in the literature. A contribution of montane refugia and river barriers on population differentiation was detected. We also detected combined effects of IBD/IBE and IBE/IBA on genetic differentiation and suggested this pattern is more likely determined by ecological isolation, although pollinator-mediated divergent selection could not be ruled out for some of the species. Overall, this study provides further insights on speciation in orchids, a group for which Madagascar shows one of the world's highest level of endemism and confirms the importance of the peculiar biogeography of the island in shaping species differentiation.

Guidelines for the Choice of Sequences for Molecular Plant Taxonomy.

This chapter presents an overview of the major plant DNA sequences and molecular methods available for plant taxonomy. Guidelines are provided for the choice of sequences and methods to be used, based on the DNA compartment (nuclear, chloroplastic, mitochondrial), evolutionary mechanisms, and the level of taxonomic differentiation of the plants under survey.

Nuclear Ribosomal RNA Genes: ITS Region.

Despite possible drawbacks (intraspecific polymorphisms and possible fungal contamination), sequencing of the ITS region of the ribosomal RNA genes 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.

Genotyping-by-Sequencing Technology in Plant Taxonomy and Phylogeny.

Genotyping-by-sequencing (GBS) is a method to discover and genotype simultaneous genome-wide high-throughput single nucleotide polymorphisms (SNPs). GBS is based on reducing genome complexity with restriction enzymes. Here we describe a method developed by Elshire et al. for constructing simplified GBS libraries and recent bioinformatic approaches developed to analyze the large volume of polymorphism data generated by this method. GBS approach is suitable for population studies, taxonomic and phylogenic studies, germplasm characterization, and breeding and trait mapping for a wide range of organisms, including plants with complex genomes.

Plant DNA Barcoding Principles and Limits: A Case Study in the Genus Vanilla.

Powerful DNA barcodes have been much more difficult to define in plants than in animals. In 2009, the international Consortium for the Barcoding Of Life (CBOL) chose the combination of the chloroplast genes (rbcL + matK) as the proposed official barcode for plants. However, this system has got important limits. First, any barcode system will only be useful if there is a clear barcode gap and if species are monophyletic. Second, chloroplast and mitochondrial (COI gene used for animals) barcodes will not be usable for discriminating hybrid species. Moreover, it was also shown that, using chloroplast regions, maximum species discrimination would be around 70% and very variable among plant groups. This is why many authors have more recently advocated for the addition of the nuclear ITS region to this barcode because it reveals more variations and allows the resolution of hybrid or closely related species. We tested different chloroplast genes (rbcL, matK, psaB, psbC) and the nuclear ITS region in the genus Vanilla, a taxonomically complex group and therefore a good model to test for the efficiency of different barcode systems. We found that the CBOL official barcode system performed relatively poorly in Vanilla (76% species discrimination), and we demonstrate that adding ITS to this barcode system allows to increase resolution (for closely related species and to the subspecies level) and to identify hybrid species. The best species discrimination attained was 96.2% because of one paraphyletic species that could not be resolved.

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.

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.

Guidelines for the choice of sequences for molecular plant taxonomy.

This chapter presents an overview of the major plant DNA sequences and molecular methods available for plant taxonomy. Guidelines are provided for the choice of sequences and methods to be used, based on the DNA compartment (nuclear, chloroplastic, mitochondrial), evolutionary mechanisms, and the level of taxonomic differentiation of the plants under survey.

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.

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.

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.

Natural polyploidy in Vanilla planifolia (Orchidaceae).

Vanilla planifolia accessions cultivated in Reunion Island display important phenotypic variation, but little genetic diversity is demonstrated by AFLP and SSR markers. This study, based on analyses of flow cytometry data, Feulgen microdensitometry data, chromosome counts, and stomatal length measurements, was performed to determine whether polyploidy could be responsible for some of the intraspecific phenotypic variation observed. Vanilla planifolia exhibited an important variation in somatic chromosome number in root cells, as well as endoreplication as revealed by flow cytometry. Nevertheless, the 2C-values of the 50 accessions studied segregated into three distinct groups averaging 5.03 pg (for most accessions), 7.67 pg (for the 'Stérile' phenotypes), and 10.00 pg (for the 'Grosse Vanille' phenotypes). For the three groups, chromosome numbers varied from 16 to 32, 16 to 38, and 22 to 54 chromosomes per cell, respectively. The stomatal length showed a significant variation from 37.75 microm to 48.25 microm. Given that 2C-values, mean chromosome numbers, and stomatal lengths were positively correlated and that 'Stérile' and 'Grosse Vanille' accessions were indistinguishable from 'Classique' accessions using molecular markers, the occurrence of recent autotriploid and autotetraploid types in Reunion Island is supported. This is the first report showing evidence of a recent autopolyploidy in V. planifolia contributing to the phenotypic variation observed in this species.

Patterns of introduction and diversification of Vanilla planifolia (Orchidaceae) in Reunion Island (Indian Ocean).

The cultivated species Vanilla planifolia is a typical example of a crop introduced from its area of origin (America) to new regions where natural pollinators are absent. Although the Vanilla cultivars are exclusively vegetatively propagated, a high degree of phenotypic variation is observed among the cultivars in their introduction areas such as Reunion Island. To test several hypotheses explaining this variation-different introduction events, somatic mutations and sexual reproduction (through manual pollination)-we used AFLP markers to elucidate the patterns of introduction of V. planifolia. Most of the accessions cultivated in the world were derived from a single accession, possibly the Mexican cultivar Mansa. The patterns of diversification of this cultivated species were also studied and compared with other cultivated (V. tahitensis) and wild (V. pompona and V. bahiana) species. Except for one particular phenotype ('Aiguille'), which may come from sexual reproduction, cultivated accessions exhibit very low levels of genetic diversity. They have evolved by the accumulation of point mutations through vegetative multiplication. The genetic diversity revealed could not explain the phenotypic diversity, which may be related to epigenetics or polyploidy. This new understanding of the basis of genetic diversity of vanilla may assist to improve management of genetic resources.

Metabolism of 2-mercaptobenzothiazole by Rhodococcus rhodochrous.

2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.

Evidence of metyrapone reduction by two Mycobacterium strains shown by 1H NMR.

In situ 1H NMR monitoring of metyrapone incubations with resting-cells of two strains of Mycobacterium, Mycobacterium aurum MO1 and Mycobacterium sp. RP1, showed the biotransformation of this compound, and more precisely the carbonyl-reduction of metyrapone into the corresponding alcohol, metyrapol. This reduction produced both enantiomers. The use of inhibitors allowed us to show the multiple enzymatic activities involved in this biotransformation including carbonyl reductase (EC 1.1.1.1.84) from the short-chain dehydrogenase superfamily and aldehyde reductase (EC 1.1.1.2) from the aldo-keto reductase superfamily.

Sequential bio- and phototransformation of the herbicide methabenzthiazuron in water.

We investigated the transformation of methabenzthiazuron in water by microorganisms and solar light. This compound was very slowly phototransformed when irradiated at lambda > 290 nm, but it could be successfully oxidized into 6-hydroxymethabenzthiazuron by Aspergillus niger, as shown by nuclear magnetic resonance experiments. The toxicity of this metabolite, as determined by the standardized Microtox test, was sixfold lower than that of the parent molecule. The 6-hydroxymethabenzthiazuron was not further metabolized by A. niger but was photooxidized with ring cleavage of the aromatic ring and photodimerized on irradiation at lambda > 290 nm. In the presence of humic substances, the photodegradation was slower. We demonstrate that the transformations of methabenzthiazuron, observed either with the fungus A. niger or by the action of solar light, do not proceed via the urea chain N-dealkylation, as usually reported, but only via hydroxylation or cleavage of the benzene ring. This work shows the complementarity of both approaches, photo- and biodegradation, to study the fate of herbicides in the environment.

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity.

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.