Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr

Deciphering the genome structure and paleohistory of _Theobroma cacao_

We sequenced and assembled the genome of _Theobroma cacao_, an economically important tropical fruit tree crop that is the source of chocolate. The assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of them anchored on the 10 _T. cacao_ chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example flavonoid-related genes. It also provides a major source of candidate genes for _T. cacao_ disease resistance and quality improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten _T. cacao_ chromosomes were shaped from an ancestor through eleven chromosome fusions. The _T. cacao_ genome can be considered as a simple living relic of higher plant evolution

Etude du rôle des stomates dans l interaction vigne Plasmopara viticola, agent du mildiou

Plasmopara viticola, l'agent responsable du mildiou des vitacées, utilise les stomates comme porte d'entrée pour infecter les feuilles et pour sporuler. L'observation d'un flétrissement prématuré des plantes sensibles (Vitis vinifera L. cv. Marselan) infectées en conditions d'arrosage irrégulier nous a conduit à suspecter un dysfonctionnement stomatique. Par des approches complémentaires nous montrons que les stomates des feuilles de vigne infectées sont plus ouverts à l'obscurité que ceux de feuilles de plantes saines, dès 3 jours post-inoculation, avant l'apparition des premiers symptômes. Le deuxième objectif de ce travail est, d'une part, de déterminer si la fermeture des stomates peut réduire l'infection et, d'autre part, si le complexe stomatique est le siège préférentiel de réactions de défense associées à la réduction de l'infection par Plasmopara viticola.Plasmopara viticola, the causal agent of grapevine downy mildew enters through the uses stomata to enter the plant and to sporulate. It was observed that the infected plants wilted more rapidly than healthy ones when subjected to water starvation. Complimentary approaches were used to investigate stomatal opening/closure during infection. We have shown that the oomycete deregulates guard cell functioning, causing significant water losses during the night, from 3 days post-inoculation, before appearance of the first symptom. The second objective of this study was firstly, to determine whether stomatal closure could reduce infection and secondly, to determine if stomatal complex is the site of defence reactions associated with a reduction of the infection by Plasmopara viticola.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Stomatal deregulation in plasmopara viticola-infected grapevine leaves

International audienceIn grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. • Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. • In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA)treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. • These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation

Are grapevine stomata involved in the elicitor-induced protection against downy mildew?

International audienceStomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H2O2 production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA and elicitors share common signaling events, it led us to question whether stomatal movements and H2O2 production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine–Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production

Genome-wide association studies and genomic selection assays made in a large sample of cacao (Theobroma cacao L.) germplasm reveal significant marker-trait associations and good predictive value for improving yield potential.

A genome-wide association study (GWAS) was undertaken to unravel marker-trait associations (MTAs) between SNP markers and phenotypic traits. It involved a subset of 421 cacao accessions from the large and diverse collection conserved ex situ at the International Cocoa Genebank Trinidad. A Mixed Linear Model (MLM) in TASSEL was used for the GWAS and followed by confirmatory analyses using GAPIT FarmCPU. An average linkage disequilibrium (r2) of 0.10 at 5.2 Mb was found across several chromosomes. Seventeen significant (P ≤ 8.17 × 10-5 (-log10 (p) = 4.088)) MTAs of interest, including six that pertained to yield-related traits, were identified using TASSEL MLM. The latter accounted for 5 to 17% of the phenotypic variation expressed. The highly significant association (P ≤ 8.17 × 10-5) between seed length to width ratio and TcSNP 733 on chromosome 5 was verified with FarmCPU (P ≤ 1.12 × 10-8). Fourteen MTAs were common to both the TASSEL and FarmCPU models at P ≤ 0.003. The most significant yield-related MTAs involved seed number and seed length on chromosome 7 (P ≤ 1.15 × 10-14 and P ≤ 6.75 × 10-05, respectively) and seed number on chromosome 1 (P ≤ 2.38 × 10-05), based on the TASSEL MLM. It was noteworthy that seed length, seed length to width ratio and seed number were associated with markers at different loci, indicating their polygenic nature. Approximately 40 candidate genes that encode embryo and seed development, protein synthesis, carbohydrate transport and lipid biosynthesis and transport were identified in the flanking regions of the significantly associated SNPs and in linkage disequilibrium with them. A significant association of fruit surface anthocyanin intensity co-localised with MYB-related protein 308 on chromosome 4. Testing of a genomic selection approach revealed good predictive value (genomic estimated breeding values (GEBV)) for economic traits such as seed number (GEBV = 0.611), seed length (0.6199), seed width (0.5435), seed length to width ratio (0.5503), seed/cotyledon mass (0.6014) and ovule number (0.6325). The findings of this study could facilitate genomic selection and marker-assisted breeding of cacao thereby expediting improvement in the yield potential of cacao planting material

Characterization of the model system rice-Magnaporthe for the study of nonhost resistance in cereals

The best characterized form of resistance is gene-for-gene resistance. Less well characterized is nonhost resistance in which an entire plant species is resistant to an entire pathogen species. Here, different rice genotypes were inoculated with host and nonhost strains of Magnaporthe isolated from rice, wheat and crabgrass.The different types of interactions were characterized at a cytological level using a 3,3'-diaminobenzidine (DAB) stain to investigate the occurrence of reactive oxygen intermediates or by observing the occurrence of cellular autofluorescence. Gene expression of a set of selected PR-genes was analysed using quantitative real-time polymerase chain reaction.Inoculation with the isolate from crabgrass resulted in a lack of penetration. The wheat isolate induced a hypersensitive response with varying degrees of pathogen growth inside the invaded cell according to the rice genotype. Expression analysis of our PR-gene set revealed clear differences between the different types of interactions in both kinetic and magnitude of gene induction.Our integrated study opens the way to the dissection of molecular components leading to nonhost reactions to Magnaporthe grisea in rice and points to novel sources of durable resistance to fungal plant pathogens in other cereal crops. (Résumé d'auteur

A β-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of défense responses, including HR-like cell death

International audienceSulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance

Structural characterization and mapping of functional EST-SSR markers in Theobroma cacao

International audienceTheobroma cacao L. is a major cash crop for tropical countries, providing incomes for 14 million small farmers. Establishing sustainable disease resistance and maintaining cocoa qualities are among the major objectives of breeding programs. To enrich the high-density genetic map, useful for all cocoa genetic studies, with gene-based markers, a recently produced large EST resource was mined to develop expressed sequence tag-based simple sequence repeat markers (EST-SSRs) defined in genes with a putative known function. A set of 174 polymorphic EST-SSRs was identified from a selection of 314 non-redundant EST-SSRs with a putative known function. Of them, 115 loci were mapped on the cocoa reference map. This new map contains 582 codominant markers arranged in ten linkage groups corresponding to the haploid number of chromosomes. An average interval between markers of 1.3 cM was found, with approximately one SSR every 2 cM. This new set of EST-SSRs includes 14 candidate genes for plant resistance or cocoa qualities. The percentage of polymorphic SSRs varied depending on the different gene regions from which they originated, with respectively 54%, 69%, and 82% of polymorphic EST-SSRs originating from coding sequences, and from the non-coding untranslated 5'UTR and 3'UTR regions. This new map contains a set of 384 SSR markers that are easily transferable across different mapping populations and useful for all genetic analyses in T. cacao. The new set of EST-SSRs will be a useful tool for studying the functional diversity of populations and for carrying out association mapping studies

Summary of significantly positive marker-trait associations (<i>TASSEL</i> MLM and GAPIT FarmCPU).

http://dx.doi.org/10.13140/RG.2.2.34215.21927. (XLSX)</p