Caroline Denancé

Identification of serine/threonine kinase and nucleotide-binding site–leucine-rich repeat (NBS-LRR) genes in the fire blight resistance quantitative trait locus of apple cultivar ‘Evereste’

Fire blight is the most destructive bacterial disease affecting apple (Malus×domestica) worldwide. So far, no resistance gene against fire blight has been characterized in apple, despite several resistance regions having been identified. A highly efficacious resistance quantitative trait locus (QTL) was localized on linkage group 12 (LG12) of the ornamental cultivar Evereste. A marker previously reported to be closely linked to this resistance was used to perform a chromosome landing. A bacterial artificial chromosome (BAC) clone of 189u2003kb carrying the fire blight resistance QTL was isolated and sequenced. New microsatellite markers were developed, and the genomic region containing the resistance locus was limited to 78u2003kb. A cluster of eight genes with homologies to already known resistance gene structures to bacterial diseases was identified and the corresponding gene transcription was verified. From this cluster, two genes were recognized in silico as the two most probable fire blight resistance genes showing homology with the Pto/Prf complex in tomato.

Development and validation of the Axiom®Apple480K SNP genotyping array

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom(®) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom(®) Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom(®) Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.

Genetic Diversity, Population Structure, Parentage Analysis, and Construction of Core Collections in the French Apple Germplasm Based on SSR Markers

In-depth characterization of apple genetic resources is a prerequisite for genetic improvement and for germplasm management. In this study, we fingerprinted a very large French collection of 2163 accessions with 24 SSR markers in order to evaluate its genetic diversity, population structure, and genetic relationships, to link these features with cultivar selection date or usage (old or modern, dessert or cider cultivars), and to construct core collections. Most markers were highly discriminating and powerful for varietal identification, with a probability of identity P(ID) over the 21 retained SSR loci close to 10−28. Pairwise comparisons revealed 34xa0% redundancy and 18.5xa0% putative triploids. The results showed that the germplasm is highly diverse with an expected heterozygosity He of 0.82 and observed heterozygosity Ho of 0.83. A Bayesian model-based clustering approach revealed a weak but significant structure in three subgroups (FSTu2009=u20090.014–0.048) corresponding, albeit approximately, to the three subpopulations defined beforehand (Old Dessert, Old Cider, and Modern Cultivars). Parentage analyses established already known and yet unknown relationships, notably between old cultivars, with the frequent occurrence of cultivars such as “King of Pippin” and “Calville Rouge d’Hiver” as founders. Finally, core collections based on allelic diversity were constructed. A large dessert core collection of 278 cultivars contained 90xa0% of the total dessert allelic diversity, whereas a dessert subcore collection of 48 cultivars contained 71xa0% of diversity. For cider apples, a 48-cultivar core collection contained 83xa0% of the total cider allelic diversity.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BackgroundThe amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (Northu2009+u2009East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe.ResultsA Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FSTu2009=u20090.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FSTu2009=u20090.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars.ConclusionsThe variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Erosion of quantitative host resistance in the apple × Venturia inaequalis pathosystem

Theoretical approaches predict that host quantitative resistance selects for pathogens with a high level of pathogenicity, leading to erosion of the resistance. This process of erosion has, however, rarely been experimentally demonstrated. To investigate the erosion of apple quantitative resistance to scab disease, we surveyed scab incidence over time in a network of three orchards planted with susceptible and quantitatively resistant apple genotypes. We sampled Venturiainaequalis isolates from two of these orchards at the beginning of the experiment and we tested their quantitative components of pathogenicity (i.e., global disease severity, lesion density, lesion size, latent period) under controlled conditions. The disease severity produced by the isolates on the quantitatively resistant apple genotypes differed between the sites. Our study showed that quantitative resistance may be subject to erosion and even complete breakdown, depending on the site. We observed this evolution over time for apple genotypes that combine two broad-spectrum scab resistance QTLs, F11 and F17, showing a significant synergic effect of this combination in favour of resistance (i.e., favourable epistatic effect). We showed that isolates sampled in the orchard where the resistance was inefficient presented a similar level of pathogenicity on both apple genotypes with quantitative resistance and susceptible genotypes. As a consequence, our results revealed a case where the use of quantitative resistance may result in the emergence of a generalist pathogen population that has extended its pathogenicity range by performing similarly on susceptible and resistant genotypes. This emphasizes the need to develop quantitative resistances conducive to trade-offs within the pathogen populations concerned.

A new pear scab resistance gene Rvp1 from the European pear cultivar ‘Navara’ maps in a genomic region syntenic to an apple scab resistance gene cluster on linkage group 2

Scab, caused by the ascomycete fungus Venturia pirina, leads to severe damage on European pear varieties resulting in a loss of commercial value and requiring frequent use of fungicides. Identifying scab resistance genes, developing molecular markers linked to these genes and establishing marker-assisted selection would be an effective way to improve European pear breeding for scab resistance. Most of the European pear cultivars (Pyrus communis) are currently reported to be sensitive. The pear cultivar ‘Navara’ was shown to carry a major scab resistance gene whose phenotypic expression in seedling progenies was a typical stellate necrosis symptom. The resistance gene was called Rvp1, for resistance to V. pirina, and was mapped on linkage group 2 of the pear genome close to microsatellite marker CH02b10. This genomic region is known to carry a cluster of scab resistance genes in apple indicating a first functional synteny for scab resistance between apple and pear.

Genetic mapping of Cacopsylla pyri resistance in an interspecific pear (Pyrus spp.) population

Cacopsylla pyri (pear psylla) is one of the most serious pests of pear (Pyrus spp.) in Europe. It can cause high yield losses, and its control has become difficult since it has developed resistance to a wide range of pesticides. Pear breeders are developing new cultivars resistant to pear psyllids, and Asian species, such as Pyrus ussuriensis and Pyrus × bretschneideri, are good sources of resistance. Antixenosis and antibiosis resistance to psylla were both identified in pear; they may differ in the biological mechanism and probably have different genetic backgrounds. We crossed interspecific P. × bretschneideri × Pyrus communis hybrid PEAR3, resistant to pear psylla, with the susceptible European pear cultivar ‘Moonglow’ to obtain an F1 population for the genetic mapping of the resistance. Quantitative trait locus (QTL) analysis was carried out for antibiosis by measuring the number of surviving nymphs and the nymphal development, using a novel phenotyping protocol and a saturated genetic map made of single-nucleotide polymorphism (SNP) and microsatellite (simple sequence repeats (SSR)) markers. A stable QTL was detected on linkage group (LG) 8 of PEAR3 (R2u2009=u200917.2–39.1xa0%). In addition, QTLs were detected on LG5 (R2u2009=u200910.8xa0%) of PEAR3 and on LG15 of ‘Moonglow’ (R2u2009=u200913.7xa0%).

Fine mapping of the rosy apple aphid resistance locus Dp-fl on linkage group 8 of the apple cultivar ‘Florina’

Rosy apple aphid (Dysaphis plantaginea), is one of the major insect pests of apple, causing serious physical and economic damage to fruit production. A dominant resistance gene Dp-fl was previously mapped at the bottom of linkage group LG8 from the cultivar ‘Florina’, linked to the SSR CH01h10. The development of additional genetic markers mapping closer to Dp-fl was needed to position the gene accurately and to improve the effectiveness of marker-assisted breeding (MAB). The aims of this study were to identify single nucleotide polymorphisms (SNPs) in the region of Dp-fl and to position these SNPs relative to Dp-fl. To generate a fine map of the Dp-fl interval, a total of 191 plants segregating for resistance and derived from four different populations were tested with temperature-switch PCR (TSP) markers developed for SNPs located in the region of CH01h10. All the plants were phenotypically evaluated for aphid resistance and those data compared with the genetic data. These efforts resulted in positioning the Dp-fl resistance locus in a genetic interval corresponding to a physical distance of about 330xa0kb on the ‘Golden Delicious’ genome. The new markers were tested on several apple founder cultivars in order to test the specificity of the SNPs and, thus, the best markers for the MAB were identified. Finally, the 330-kb interval was analyzed for the identification of coding sequences and putative candidate genes for D. plantaginea resistance were identified.

Polygenic inheritance of resistance to Cacopsylla pyri in a Pyrus communis × P. ussuriensis progeny is explained by three QTLs involving an epistatic interaction

Pear psylla (Cacopsylla pyri) causes severe damage on European pear cultivars, resulting in high yield losses. Its control has become difficult since it developed resistance to a wide range of pesticides, while the number of authorized molecules for pest control has decreased. Identifying pear psylla resistance factors should help breeding new resistant pear cultivars. We analyzed the quantitative resistance to psylla inherited from the genotype NY 10355 derived from Pyrus ussuriensis. Quantitative trait locus (QTL) analysis was carried out after counting the number of nymphs and estimating the nymphal development rate using a free-choice test performed on a large segregating progeny. We mapped two new loci for pear psylla resistance on linkage groups LG01 and LG04 of NY 10355 and confirmed the QTL previously detected on LG17. A strong epistatic interaction between the two QTLs detected on LG01 and LG17 appeared to be a major factor controlling the psylla infestation in the genotype NY 10355.

Slow erosion of a quantitative apple resistance to Venturia inaequalis based on an isolate-specific Quantitative Trait Locus

Quantitative plant resistance affects the aggressiveness of pathogens and is usually considered more durable than qualitative resistance. However, the efficiency of a quantitative resistance based on an isolate-specific Quantitative Trait Locus (QTL) is expected to decrease over time due to the selection of isolates with a high level of aggressiveness on resistant plants. To test this hypothesis, we surveyed scab incidence over an eight-year period in an orchard planted with susceptible and quantitatively resistant apple genotypes. We sampled 79 Venturia inaequalis isolates from this orchard at three dates and we tested their level of aggressiveness under controlled conditions. Isolates sampled on resistant genotypes triggered higher lesion density and exhibited a higher sporulation rate on apple carrying the resistance allele of the QTL T1 compared to isolates sampled on susceptible genotypes. Due to this ability to select aggressive isolates, we expected the QTL T1 to be non-durable. However, our results showed that the quantitative resistance based on the QTL T1 remained efficient in orchard over an eight-year period, with only a slow decrease in efficiency and no detectable increase of the aggressiveness of fungal isolates over time. We conclude that knowledge on the specificity of a QTL is not sufficient to evaluate its durability. Deciphering molecular mechanisms associated with resistance QTLs, genetic determinants of aggressiveness and putative trade-offs within pathogen populations is needed to help in understanding the erosion processes.