Dissecting the control of shoot development in grapevine: genetics and genomics identify potential regulators

Grapevine is a crop of major economic importance, yet little is known about the regulation of shoot development in grapevine or other perennial fruits crops. Here we combine genetic and genomic tools to identify candidate genes regulating shoot development in Vitis spp. An F2 population from an interspecific cross between V. vinifera and V. riparia was phenotyped for shoot development traits, and three Quantitative Trait Loci (QTLs) were identified on linkage groups (LGs) 7, 14 and 18. Around 17% of the individuals exhibited a dwarfed phenotype. A transcriptomic study identified four candidate genes that were not expressed in dwarfed individuals and located within the confidence interval of the QTL on LG7. A deletion of 84,482 bp was identified in the genome of dwarfed plants, which included these four not expressed genes. One of these genes was VviCURLY LEAF (VviCLF), an orthologue of CLF, a regulator of shoot development in Arabidopsis thaliana. The phenotype of the dwarfed grapevine plants was similar to that of clf mutants of A. thaliana and orthologues of the known targets of CLF in A. thaliana were differentially expressed in the dwarfed plants. This suggests that CLF, a major developmental regulator in A. thaliana, also controls shoot development in grapevine

Microsatellite markers for Pinus pinaster Ait.

Simple sequence repeats (SSRs) or microsatellites are valuable tools for genome mapping and population genetic studies for as they are codominant and highly polymorphic markers. Seventy-six SSR primer pairs from four Pinus species were tested to amplify microsatellites in Pinus pinaster. Twenty-six primer pairs were stemmed from a microsatellite library on P. pinaster and the other primer pairs were obtained in other species of the same genus (P. radiata, P. strobus and P. halepensis). Only three out of the 76 SSR primer pairs amplified at a single polymorphic locus in P. pinaster. The Mendelian inheritance of those three primer pairs was studied and their genetic map position was determined. The number of alleles and the level of heterozygosity were assessed in an analysis of a sample of 196 trees. The development of microsatellites in Pinus species has been reported to be a difficult task because of the size and complexity of their genome. The results of this study showed that cross-species amplification was quite unsuccessful.Marqueurs microsatellites chez Pinus pinaster Ait. Les microsatellites (SSRs) sont des outils de choix pour la cartographie génétique et les études de génétique des populations parce qu'ils sont des marqueurs codominants et très polymorphes. Soixante-seize paires d'amorces de quatre espèces de pin ont été utilisées afin d'amplifier des microsatellites chez Pinus pinaster. Vingt-neuf paires d'amorces étaient issues d'une banque enrichie en microsatellites sur P. pinaster et les autres paires d'amorces avaient été obtenues sur d'autres espèces du même genre (P. radiata, P. strobus et P. halepensis). Sur un total de 76 paires d'amorces, seulement trois ont amplifié un seul locus microsatellite polymorphe chez P. pinaster. Leur ségrégation mendélienne a été étudiée et chaque locus a été localisé sur une carte génétique. Le nombre d'allèles et l'hétérozygotie ont été ensuite évalués en analysant un échantillon de 196 arbres. Le développement de microsatellites chez les espèces du genre Pinus s'est révélée difficile en raison de la taille et de la complexité de leur génome. Les résultats de cette étude ont montré que l'amplification inter-espèces n'a rencontré que peu de succès

Allele mining of eukaryotic translation initiation factor genes in Prunus for the identification of new sources of resistance to sharka

Abstract Members of the eukaryotic translation initiation complex are co-opted in viral infection, leading to susceptibility in many crop species, including stone fruit trees (Prunus spp.). Therefore, modification of one of those eukaryotic translation initiation factors or changes in their gene expression may result in resistance. We searched the crop and wild Prunus germplasm from the Armeniaca and Amygdalus taxonomic sections for allelic variants in the eIF4E and eIFiso4E genes, to identify alleles potentially linked to resistance to Plum pox virus (PPV). Over one thousand stone fruit accessions (1397) were screened for variation in eIF4E and eIFiso4E transcript sequences which are in single copy within the diploid Prunus genome. We identified new alleles for both genes differing from haplotypes associated with PPV susceptible accessions. Overall, analyses showed that eIFiso4E is genetically more constrained since it displayed less polymorphism than eIF4E. We also demonstrated more variations at both loci in the related wild species than in crop species. As the eIFiso4E translation initiation factor was identified as indispensable for PPV infection, a selection of ten different eIFiso4E haplotypes along 13 accessions were tested by infection with PPV and eight of them displayed a range of reduced susceptibility to resistance, indicating new potential sources of resistance to sharka

Origin identification of maritime pine stands in France using chloroplast simple-sequence repeats

Des lots de graines du nord ouest de la péninsule ibérique (Portugal et Galice) ont été introduits dans les années 1950 dans le sud-ouest de la France (Aquitaine), et les peuplements issus de ces graines ont fortement souffert des gelées. Un test variétal basé sur les marqueurs terpéniques fut développé dans les années 1980 afin d'identifier l'origine géographique des peuplements adultes en Aquitaine sur lesquels des graines étaient récoltées puis commercialisées. Dans cet article nous décrivons un nouveau test qui utilise des marqueurs microsatellites chloroplastiques (cpSSRs) et simulations pour identifier l'origine des peuplements. Une étude comparative des tests biochimique (terpènes) et cpSSR a été menée sur cinq peuplements adultes. Les résultats obtenus sont identiques, mais le test ADN s'est avéré plus rapide et plus précis. L'utilisation de ce nouveau test devrait permettre de garantir l'origine géographique des peuplements de pin maritime du sud-ouest de la France (Aquitaine).Maritime pine seed-lots from north-western Iberian regions (Portugal and Galicia) were introduced in the 1950s to the south-west of France (Aquitaine region), and the stands they formed suffered considerable frost damage. In the mid 1980s, a biochemical test was developed to test the putative origin of adult stands in Aquitaine, before seeds could be distributed for commercial purposes in France. In this paper, we describe a new test employing chloroplast simple-sequence repeats (cpSSRs) to facilitate identification of stand origin based on randomisation tests. The origin of five stands of unknown origin was determined with both the cpSSR and biochemical (terpene profile analysis) tests. The results from the two tests were concordant, but the DNA-based test gave faster and more accurate results. Use of this test should help when determining the origin of maritime pine stands in the Aquitaine region of France

A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences

Pollen contamination in a maritime pine polycross seed orchard and certification of improved seeds using chloroplast microsatellites

A new concept of seed orchard was developed by Institut National de la Recherche Agronomique for the maritime pine (Pinus pinaster Ait.) breeding program: the polycross seed orchard (PSO). The expected genetic gain of the PSO can only be reached if the fathers used in the pollen mix contribute equally to the next generation (i.e., to the base material of the PSO) and if pollen contamination from the surrounding stands is limited. Using chloroplast simple-sequence repeats (cpSSR), we showed that the chloroplast DNA was unipaternally inherited in maritime pine and verified that the chloroplast haplotype composition of the megagametophyte tissue corresponded to the chloroplast haplotype of the female parent. As a practical application, a statistical test based on cpSSR markers and simulation was established to verify the PSO origin of maritime pine seed lots. As a result of the cpSSR test, it was observed that (i) departure from even distribution of the fathers in the PSO was barely significant, (ii) the minimum pollen contamination rate in the PSO was 36%, and (iii) the contamination was not evenly distributed in the PSO. As a consequence, the expected genetic gain will range between 50 and 82% of what was initially foreseen. Résumé : Dans le cadre du programme d'amélioration génétique du pin maritime (Pinus pinaster Ait.), un nouveau concept de verger à graines, le verger à graines de familles polycross (VGFP), a été développé par l'Institute National de la Recherche Agronomique. Pour installer un VGFP, des arbres élites sont croisés selon un schéma polycross, puis les descendants sont plantés à densité définitive. La variété améliorée est finalement produite par pollinisation libre entre les arbres du verger. Le gain génétique espéré d'un VGFP ne peut être réalisé que si les parents mâles contribuent également à la génération suivante (le matériel de base du verger), et si la pollution pollinique due aux arbres extérieurs au VGFP est limitée. En utilisant des marqueurs microsatellites chloroplastiques (cpSSR), nous avons tout d'abord montré que le génome chloroplastique présentait une hérédité paternelle. Dans un second temps, nous avons pu vérifier que les mégagamétophytes d'un même arbre possédaient le même haplotype chloroplastique que l'arbre mère. Un test statistique empirique construit par simulation a alors été établi pour décider de l'appartenance ou non d'un lot de graines quelconque au VGFP. Il a permi de montrer que l'écart à l'égale participation des pères dans le VGFP était tout juste significatif, et que le taux de pollution était au minimum de 36% et réparti de façon hétérogène dans le VGFP. En conséquence, le gain génétique espéré sera compris entre 50 et 82% de celui annoncé

Hereditary Angioedema-Presenting as Recurrent Abdominal Pain

Studying domesticated species and their wild relatives allows understanding of the mechanisms of population divergence and adaptation, and identifying valuable genetic resources. Apricot is an important fruit in the Northern hemisphere, where it is threatened by the Plum pox virus (PPV), causing the sharka disease. The histories of apricot domestication and of its resistance to sharka are however still poorly understood. We used 18 microsatellite markers to genotype a collection of 230 wild trees from Central Asia and 142 cultivated apricots as representatives of the worldwide cultivated apricot germplasm; we also performed experimental PPV inoculation tests. The genetic markers revealed highest levels of diversity in Central Asian and Chinese wild and cultivated apricots, confirming an origin in this region. In cultivated apricots, Chinese accessions were differentiated from more Western accessions, while cultivated apricots were differentiated from wild apricots. An approximate Bayesian approach indicated that apricots likely underwent two independent domestication events, with bottlenecks, from the same wild population. Central Asian native apricots exhibited genetic subdivision and high frequency of resistance to sharka. Altogether, our results contribute to the understanding of the domestication history of cultivated apricot and point to valuable genetic diversity in the extant genetic resources of wild apricots

A high density genetic map of maritime pine based on AFLPs

We constructed a high-density linkage map of maritime pine (Pinus pinaster Ait.) based on AFLP (Amplified Fragment Length Polymorphism) markers using a three-generation outbred pedigree. In a first step, male and female maps were established independently with test-cross markers segregating 1:1 (presence:absence of the amplified fragment in the full-sib progeny). In a second step, both maps were merged using intercross markers segregating 3:1 in the progeny. A combination of MAPMAKER and JOINMAP softwares was used for the mapping process. A consensus map was obtained and is available at URL http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. It covers 1441 cM and comprises a total of 620 AFLP markers on 12 linkage groups. The physical size of the maritime pine genome (51.5 pg/2C) was measured by flow cytometry, providing a physical/genetic size ratio of 13.78 Mb/cM. This map will be used to dissect the genetic architecture of economically (growth, wood quality) and ecologically (water-use efficiency) important traits into mendelian inherited components (QTLs: Quantitative Trait Loci). It will also provide a framework to localize more informative markers (ESTs: Expressed Sequence Tags) to be used as candidate genes in QTL detection experiments. The location of orthologous markers (ESTs and SSRs: Simple Sequence Repeats) will also allow the study of the genome structure of closely related conifer species using a comparative genome mapping approach.Établissement d'une carte génétique à haute densité du pin maritime à partir de marqueurs AFLP. Nous avons construit une carte génétique du pin maritime (Pinus pinaster Ait.) en génotypant une famille de plein-frères appartenant à la troisième génération du programme d'amélioration, avec des marqueurs AFLP. Dans un premier temps, les cartes des parents mâle et femelle ont été établies indépendamment avec des marqueurs de type “ test-cross " ségréguant dans les proportions 1:1 (présence:absence du fragment amplifié dans la famille de plein-frères). Dans un second temps ces deux cartes ont été fusionnées à l'aide de marqueurs de type “ intercross ", ségréguant dans les proportions 3:1. La construction des cartes a été réalisée à l'aide des logiciels de cartographie génétique JOINMAP et MAPMAKER. Une carte génétique consensus des deux parents comprenant 12 groupes de liaison a finalement été obtenue et est accessible à l'URL suivante : http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. Elle couvre 1441 cM et comprend 620 marqueurs. Par ailleurs, la taille physique du génome du pin maritime a été estimée par cytométrie de flux à 51.5 pg/2C, donnant un rapport taille physique/taille génétique de 13.78 Mb/cM. Cette carte sera maintenant utilisée pour étudier l'architecture génétique de caractères d'intérêt économique (croissance, qualité du bois) et écologique (efficience d'utilisation de l'eau). Il s'agira de localiser les zones du génome (QTL, Quantitative Trait Loci) impliquées dans le contrôle génétique de ces caractères complexes. La carte génétique fournira aussi un support pour localiser d'autres types de marqueurs, tels que des gènes (EST, Expressed Sequence Tags) qui seront utilisés comme marqueurs candidats pouvant correspondre aux QTL. La localisation de marqueurs orthologues (EST et SSR, Simple Sequence Repeats) permettra d'étudier en outre la structure du génome des conifères en utilisant une approche par cartographie comparée

A high density genetic map of maritime pine based on AFLPs

We constructed a high-density linkage map of maritime pine (Pinus pinaster Ait.) based on AFLP (Amplified Fragment Length Polymorphism) markers using a three-generation outbred pedigree. In a first step, male and female maps were established independently with test-cross markers segregating 1:1 (presence:absence of the amplified fragment in the full-sib progeny). In a second step, both maps were merged using intercross markers segregating 3:1 in the progeny. A combination of MAPMAKER and JOINMAP softwares was used for the mapping process. A consensus map was obtained and is available at URL http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. It covers 1441 cM and comprises a total of 620 AFLP markers on 12 linkage groups. The physical size of the maritime pine genome (51.5 pg/2C) was measured by flow cytometry, providing a physical/genetic size ratio of 13.78 Mb/cM. This map will be used to dissect the genetic architecture of economically (growth, wood quality) and ecologically (water-use efficiency) important traits into mendelian inherited components (QTLs: Quantitative Trait Loci). It will also provide a framework to localize more informative markers (ESTs: Expressed Sequence Tags) to be used as candidate genes in QTL detection experiments. The location of orthologous markers (ESTs and SSRs: Simple Sequence Repeats) will also allow the study of the genome structure of closely related conifer species using a comparative genome mapping approach.Établissement d'une carte génétique à haute densité du pin maritime à partir de marqueurs AFLP. Nous avons construit une carte génétique du pin maritime (Pinus pinaster Ait.) en génotypant une famille de plein-frères appartenant à la troisième génération du programme d'amélioration, avec des marqueurs AFLP. Dans un premier temps, les cartes des parents mâle et femelle ont été établies indépendamment avec des marqueurs de type “ test-cross " ségréguant dans les proportions 1:1 (présence:absence du fragment amplifié dans la famille de plein-frères). Dans un second temps ces deux cartes ont été fusionnées à l'aide de marqueurs de type “ intercross ", ségréguant dans les proportions 3:1. La construction des cartes a été réalisée à l'aide des logiciels de cartographie génétique JOINMAP et MAPMAKER. Une carte génétique consensus des deux parents comprenant 12 groupes de liaison a finalement été obtenue et est accessible à l'URL suivante : http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. Elle couvre 1441 cM et comprend 620 marqueurs. Par ailleurs, la taille physique du génome du pin maritime a été estimée par cytométrie de flux à 51.5 pg/2C, donnant un rapport taille physique/taille génétique de 13.78 Mb/cM. Cette carte sera maintenant utilisée pour étudier l'architecture génétique de caractères d'intérêt économique (croissance, qualité du bois) et écologique (efficience d'utilisation de l'eau). Il s'agira de localiser les zones du génome (QTL, Quantitative Trait Loci) impliquées dans le contrôle génétique de ces caractères complexes. La carte génétique fournira aussi un support pour localiser d'autres types de marqueurs, tels que des gènes (EST, Expressed Sequence Tags) qui seront utilisés comme marqueurs candidats pouvant correspondre aux QTL. La localisation de marqueurs orthologues (EST et SSR, Simple Sequence Repeats) permettra d'étudier en outre la structure du génome des conifères en utilisant une approche par cartographie comparée

Genome-wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca)

SummaryIn fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot.This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification.The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study.This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm